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Chapter II

ANALYTICAL TECHNIQUES FOR ENVIRONMENTAL MONITORING AND CONTROL

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II.1. INTRODUCTION
Most of the analytical techniques find their applicability in environmental control, from the simplest to the most complicated one. This has increased the difficulty for the creation of this course that attempts to present over a small length and in a concise and clear manner the most important analytical techniques being applied in the mentioned domain. Regardless of the technique for obtaining the analytical data, in order to be relevant and useful in practice, they must be interpreted statistically and because of this at the beginning of this course we introduced a short chapter on basic statistics. In the following chapters a few basic notions regarding the sampling and the preparing of the sample for analysis were presented. This stage is extremely important for the analytical determination, especially when environmental samples are analyzed and, often, proper attention is not paid to it. The fact is known that generally the sampling and sample preparing for analysis generate the largest errors in environmental analysis. After that, the material was structured in two big chapters as follows: analytical techniques used for environmental analyses and methods for the monitoring of the most important pollutants. For the presentation of the analytical techniques a fairly large importance was given to the “classical” methods like gravimetry and volumetry, these techniques maintaining a certain importance in almost all the environmental control laboratories. Afterwards, the most important instrumental analysis techniques were presented in a concise manner (in our opinion) with applications for environmental control as follows: spectrophotometric methods in UV and VIS, methods based on the atomic emission and absorption, electrochemical methods, chromatographic methods, mass spectrometry and the hyphenated GC-MS technique. Due to their increasing importance, a chapter regarding the immunoanalytical techniques was included. Unfortunately, due to the fact that the size of the work was limited, in the presentation some important instrumental techniques such as like the radiometric and radiochemical analysis techniques were not included, and the description of most of the analytical techniques was done without extensive information. The course ends with the presentation of the monitoring methods for some important environmental pollutants: phenols, nitrogen compounds, heavy metals, pesticides and polychlorinated biphenyl compounds. We hope this presentation includes the major environmental pollutants, even though we are convinced that some important pollutants were not included, this being due to the limited printing space available to us.

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II.2. ELEMENTARY STATISTICS
Mihaela BADEA, Mihaela-Carmen CHEREGI In an analysis, the collection of the data is followed by the data handling. Statistics is necessary to understand the significance of the collected data and therefore to set up limitations on each step of analysis. The design of the experiments (including size of sample required, accuracy of measurements required, and number of analyses needed) is determined by a proper understanding of what the data represent.

II.2.1. ACCURACY AND PRECISION
Accuracy is the degree of agreement between the measured value and the true value. Precision is defined as the degree of agreement between replicate measurements of the same quantity, or in other words precision is how close the shots are to one another. It is impossible to have good accuracy without good precision, but good precision does not guarantee a good accuracy. For example, in the case of a systematic error in the analysis, the error does not affect the precision, but it does affect the accuracy. Since all real analyses are unknown, the higher the degree of precision, the greater the chance of obtaining the true value.

II.2.2. ERRORS AND WAYS OF EXPRESSING ACCURACY
Two main classes of errors can affect the accuracy or precision of a measured quantity. Determinate (systematic) errors are those that are determinable and that presumably can be either avoided or corrected. Some common determinate errors are: instrumental errors (faulty equipment, uncalibrated weights and glassware, etc), operative errors (personal errors in manipulations, mathematical errors in calculations, prejudice in estimating measurements). Indeterminate errors, often called accidental or random errors, represent the experimental uncertainty that occurs in any measurement. These errors are revealed by small differences in successive measurements made by the same analyst under virtually identical conditions, and they cannot be predicted or estimated. Mathematical laws of probability can be applied to understand these errors. The indeterminate errors should follow a normal distribution, or Gaussian curve (Figure II.2.1). μ is the true value and s represents the standard deviation of a population measurements, and this measure of precision defines the spread of the normal population distribution. It is apparent that there should be few large errors and that there should be an equal number of positive and negative errors.

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y

s

s

x

µ − 3s

µ −2s

µ−s

µ

µ+s

µ + 2s

µ + 3s

Figure II.2.1. Normal distribution for a population of measurements.

Indeterminate errors come from the limited ability of the analyst to control or to make corrections for external conditions, or the inability to recognize the appearance of factors that will result in errors. There are various ways and units in which the accuracy of a measurement can be expressed, but to calculate an error, the ‘true’ value, xt must be known. The difference between the true value and the measured value, with the regard to the sign, represents the absolute error, e, and is reported in the same units as the measurement.
e = xi − xt

If the measured value is the average of several measurements, the error is called the mean error. The absolute or mean error expressed as percentage of the true value is the relative error.

xi − xt ⋅ 100 xt In very accurate, the relative errors are less than 1 %. er =

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V. The standard deviation is calculated with formula: s= ∑ (x i =1 n i − x) 2 n −1 The quantity ( xi − x ) is called the ‘residual’ or the ‘deviation from the mean’ for each measurement. ± one standard deviation encompasses 68 % of the measurement and ± two standard deviation encompasses 96% of the measurement. The RSD is 82 . The advantage of using s to quote uncertainty in a result is that it has the same units as the mean value. which is called the variance. for example a set of n replicate measurements x1. The most widely useful measure of precision is the standard deviation. μ. the square of the standard deviation. Under a normal distribution. The mean of this population. …xn. V = s2 = ∑ (x i =1 n i − x) 2 n −1 The relative standard deviation (RSD) is useful for comparing the uncertainty between different measurements of varying absolute magnitude. MEASURES OF PRECISION Some basic definitions from statistics need to be introduced to express the idea of precision. For a very large population of replicate measurements is used s2 or σ2. x is the arithmetic average and defined by the equation: x= ∑x i =1 n i n where n is the number of measurements and xi is each individual measurement. The quantity (n-1) is called the ‘degrees of freedom’ for the measurement. but n must be at least 20 measurements. x is sometimes called sample mean to differentiate it from the true or population mean. The formula for μ is the same as above.II. A population is any complete set of data. x2.3.2. symbolized by s or σ and is used for small populations The standard deviation is a statistical measure of the precision for a series of repetitive measurements.

84 4. He calculated extensive values of a factor t by which the estimated standard evaluation of the mean of small population could be multiplied to obtain confidence limits for the related population of means.92 2.90 1. The confidence limits are calculated from the standard deviation using the formula: µ =x± t ⋅s n Table II.58 The t term is taken from tables calculated by Student for various numbers of degrees of freedom and degrees of confidence. The term ‘confidence interval’ is also used. The confidence limits are another statistical measure of the precision for a series of repetitive measurements.13 1. Student developed the statistics necessary to define the confidence limits.95 2.71 3. and represents the span between the confidence limits. A brief set of values for Student’s t factor is given in Table II.01 1.2.75 1.37 2.18 2.65 95 % 12. Degree of freedom (n-1) t 1 2 3 4 5 6 7 15 ∞ 90 % 6.45 2.71 4.13 2.03 3.96 99 % 63.35 2. s. and is commonly expresses as percentage (%): RSD (%) = s ⋅ 100 x The RSD (%) is also called the ‘coefficient of variance’ (CV).66 9.60 4.1.50 2.31 2. In case of small populations consisting of the usual two to four replicate laboratory measurements.94 1.57 2. Values of Student’s t.78 2.93 5. 83 .1.2.30 3.calculated from the standard deviation.

then this sample must be representative of the entire body. then a selective sample is acquired. If it is thought that a bulk system is homogeneous for a particular component. In other words. sample tracking and sample preparation processes. or assumed. The way the sample is obtained. SAMPLE AND SAMPLING A sample is a small portion of a larger body of material that is obtained for laboratory analysis. This would be just one sample taken from one location at random in the bulk system. SAMPLE AND SAMPLING AND PREPARATION Mihaela BADEA Because laboratories process a large volume of samples and must maintain a high level of quality. In the case in which the carbon monoxide level in the immediate vicinity of the leak is important.1. handled. A selective sample is a sample that is obtained from a particular part of the bulk system that is known. Other 84 . including the sampling.II. a series of samples could be obtained from different parts of the bulk system and then combined into one sample. II. the air next to a leaking gas furnace exhaust would have a higher level of carbon monoxide than the air in a room elsewhere in a building. The results of an analysis can only be useful if the sample really does represent what it is intended to represent .3. the processes and procedures in the laboratory must be well defined and rigorously followed. to have a different composition. then a random sample is taken. prepared. that is also then taken to be the concentration level in the system. as it usually always is. Each step must be documented in a permanent record. Having no reason to assume a greater concentration level in one bottle or box compared to another. Another method for solving the problem of non-homogeneity of a bulk system is to take a selective sample. A representative sample is therefore a sample that possesses all the characteristics of a larger bulk system in exactly the same concentration levels as in the system. An example would be when determining the level of active ingredient in a pharmaceutical product stored in boxes with individual bottles in a warehouse. For example. a sample is chosen at random. is very important in an analytical laboratory. If the purpose of such analysis is to present the concentration of a component in the entire body of material. etc. it represents the system and whatever concentration level is found for a given component of a sample. This kind of sample is called a composite sample. In situations in which it is likely that the substance to be determined is not homogeneously distributed throughout the entire system.3.

But the handling of the sample between the 85 . For example. or both) must be increased. from which a smaller sample is acquired by pouring into a vial to be taken into the laboratory (secondary sample. secondary sample.designations for samples are bulk sample. II. subsample. The correct procedure is to perform the analysis many times and deal with the variances with statistics. such a result cannot be considered reliable. If both the sampling variance and the lab analysis variance are high.2. If variances in the sampling and lab work are both insignificant. However. How to obtain the sample and what to do with it once it reaches the laboratory are obviously important factors. and reports the answer for this one analysis as the analysis results. before a portion is finally carefully measured into a flask (test sample) and diluted to make the sample solution. or laboratory sample). due to possible large variances in both the sampling and the lab work. If the lab analysis variance is high but the sampling variance is low. Chemists want to have as low a variance (or standard deviation) as possible for the greatest accuracy. If the sampling variance is high but the lab analysis variance is low. then one must measure 16 samples each 16 times. a water sample from a well may be collected in a large bottle (bulk sample or primary sample).1. analyzes it one time in the laboratory.1. For example. Sample Handling The importance of a high-quality representative sample has already been underlined.3. then one must perform the analysis 16 times. The fact that sampling introduces a second statistics problem means that one must also consider taking a large number of samples and dealing with the results with statistics just as one performs a laboratory analysis a large number of times and deals with those results with statistics. then poured into a beaker (another secondary sample or sub-sample). One might think that a lab analyst obtains a single sample from a bulk system. before actually being used in an analysis. These terms are used when a sample of a bulk system is divided. then the number of measurements (either the number of samples. these results may be valid. and test sample.1. then one must measure one sample 16 times. If it is not possible to have a low enough standard deviation to suit the need. II. the number of lab analyses. then one must measure 16 samples each one time. it can be shown that if a measurement system generates data with a standard deviation of 10 ppm and one needs to know an average concentration to ±5 ppm with 95% confidence. possibly a number of times. primary sample. Statistics of Sampling A consideration of statistics is required in a discussion of sampling because of the randomness with which samples are acquired. sub-sample.3. laboratory sample. The sampling is similar to a laboratory analysis — the results vary randomly and are affected by random errors that cannot be compensated.

Extraction techniques for aqueous samples include liquid-liquid (separation funnel or continuous) and solid-phase. glass vs. the methods include Soxhlett.g. dilution. Sample Extraction The extraction processes are used more often for the organic analytes that are extracted to bring them into the appropriate solvent prior to analysis. and sonication.3. leaching. Organic pollutants in potable or non-potable waters.3. by preventing contamination using a particular material for the storage container (e.1.the sampling technician. the shipping/receiving clerk. These can be achieved by adding a preservative if required. the driver. II. sediments. this sample is in the hands of five different handlers . The most important processes include extraction and cleanup.3. A sample can have a number of custodians along the way to the laboratory. Each should maintain documentation of his/her activity and duties and copies of the chain of custody should be filed. Depending on the sample matrix. supercritical fluid. by refrigerating the sample if is necessary this for the integrity maintaining. The extraction method varies depending on whether the sample is liquid or solid. other procedures such as grinding and chemical manipulations may be required. The key concept is that the sample’s integrity must be strictly maintained and preserved.3. soils. It is very important to document who has handled the sample and what responsibility each handler has at various junctures between the sampling site and the laboratory. etc. II. and filtering.sampling site and the laboratory is often something that doesn’t receive the adequate consideration. sludge. solid wastes. digestion. and other matrices must be brought into an appropriate organic solvent for their injection into the gas or liquid chromatographic column. and the laboratory technician. Along the way. Some extraction processes are repeated multiple times (such as three) to improve the efficiency of extraction. In other words. The preparation process varies depending on the sample matrix. Such extraction also enables the increase the concentration of analytes in samples by several orders of 86 .. the subordinate. by looking up to the specified holding time. A sample of lake water may be taken by a sampling technician at the site. and the analytical method. the material to be analyzed. the chain of custody must be maintained and documented. For solid samples. plastic) when is necessary. SAMPLE PREPARATION Most samples must be prepared before analysis. A shipping/receiving clerk may log in the sample and give it to a subordinate who takes it to the laboratory. The sampling technician may give it to a driver who transports it to the analysis site.

Certain widely used solvents such as diethyl ether or methylene chloride are highly volatile. but constant at any given temperature. the greater the amount of solvent the more of the solute would dissolve in it. when extraction is performed in a glass vial. Before extraction. It is important to vent out the excess pressure. they partition or distribute in the aqueous and the solvent phases and. In addition. The partition or distribution coefficient. In other words. fast. is equal to the ratio of the concentration of the solute in the solvent to that in the water. The selection of organic solvent must meet the following criteria: 1. especially after the first shaking of the sample with the solvent. Upon mixing the aqueous sample with the solvent. The method requires a measured volume of the aqueous sample to be passed through a 87 . repeated extractions using smaller portions in equal amounts will give greater extraction efficiency than a single-step extraction. the solvent should be less dense than water. A measured volume of the liquid sample is repeatedly extracted with an immiscible organic solvent. and cost effective in comparison to liquid-liquid extraction. In other words. Depending on the nature of the sample matrices.magnitude for their detection at ppb or ppt level. Excess pressure build-up may cause rupture of the separation funnel. it must be immiscible with water. A glass container that has even a slight crack should not be used for extraction. 2. the organic pollutants should be soluble in the solvent (their solubility must be greater in this solvent than in water) 3. P. various extraction techniques may be effectively applied for accurate and low level detection of organics. a detection level comparable to the liquid-liquid extraction column could be readily attained. rinse the separation funnel with a few milliliters of the solvent. the density of the solvent should be greater than water when the extraction is carried out in a separation funnel or a continuous liquid-liquid extractor. on the other hand. For any given volume of solvent. Solid Phase Extraction Organic substances can be extracted from aqueous samples by solid-liquid (known as solid phase) extraction. at equilibrium. The process is simple. increasing the volume of the solvent will cause more dissolution of the solute into the solvent. the concentration ratio of the solute in both the phases is constant. By using a suitable capillary column. the pollutants dissolve more in the latter because they are more soluble in the solvent. Liquid-Liquid Extraction The aqueous samples are commonly extracted by the liquid-liquid extraction technique. the analysis can be carried out using a smaller volume of sample. Since P is independent of volume ratio.

cartridge tube packed with a suitable solid adsorbent material. The organic pollutants in the sample are adsorbed onto the solid surface from which they are eluted by a properly selected solvent. The sample is applied at the top of the tube and drawn through the bed by a syringe or vacuum, maintaining a flow rate of 1 to 2 drops/sec. Alternatively, particles with larger pore size may be used to allow fast flow rates for large volume samples. The tube is washed with a non-polar solvent for polar analytes and polar solvent for non-polar analytes. Finally, the analytes are eluted out of column by a suitable solvent. Polar solvents should be used for polar analytes and non-polar solvents for non-polar analytes. The sample extracts may be concentrated further by evaporation of the solvent. The selection of the adsorbent packing material is based on the polarity of pollutants to be analyzed. The non-polar hydrophobic adsorbents retain the non-polar analytes and allow the polar substances to pass through the column. The hydrophilic adsorbents adsorb the polar components, allowing the non-polar materials to pass through. Various stationary phases for solid phase extraction are used, for example: octadecyl (C-18) bonded silica, octyl (C-8) bonded silica, silica, florisil, silica gel, etc. Soxhlet Extraction Semi-volatile and non-volatile organic pollutants from solid samples may be extracted by Soxhlet extraction. The sample is placed in a porous extraction thimble and immersed in the solvent. The extraction comprises a series of batch processes involving distillation and condensation of the solvent along with periodic fill-in and siphoning of the solvent in and out of the extraction chamber. This causes an intimate mixing of the sample with the solvent. Soxhlet extraction using a fluorocarbon solvent is commonly employed to leach out petroleum hydrocarbons from the soil. Other than this use, its application in environmental analysis is limited, because it is slow, taking up several hours to complete. The extraction also requires a relatively large quantity of solvent and usually a pre-concentration step is necessary. Supercritical Fluid Extraction A supercritical fluid is defined as a substance that is above its critical temperature and pressure. It exhibits remarkable liquid-like solvent properties and, therefore, high extraction efficiency. Such common gases as carbon dioxide and nitrous oxide have been successfully employed as supercritical fluids in the extraction of organics from solid matrices. The solid sample is placed in an extraction vessel into which the pressurized supercritical fluid is pumped. The organic analytes dissolve in the supercritical fluid and are swept out of the extraction chamber into a collection vessel. The pressure is released at the valve attached to the collection device where it drops down to atmospheric pressure. The supercritical fluid then returns to its gaseous state and escapes out, leaving the analytes in the collection vessel in an appropriate solvent such as methylene chloride. The extraction efficiency of supercritical fluids may be enhanced by mixing into it a small amount of a co-solvent such as acetone or

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methanol. Supercritical fluid extraction offers certain advantages over other extraction processes: 1. It is relatively a fast process with greater extraction efficiency; 2. Sample concentration steps may be eliminated; 3. Unlike liquid-liquid extraction or Soxhlet extraction, a large amount of organic solvents is not required. II.3.3.2. Sample Cleanup Samples may undergo a cleanup process to improve the analysis process and generate more reliable results. The sample extracts may be purified by one or more of the following techniques: 1. Partitioning between immiscible solvents 2. Adsorption chromatography 3. Gel permeation chromatography 4. Destruction of interfering substances with acids, alkalis, and oxidizing agents 5. Distillation The cleanup procedures presented in table II.3.1. may be applied for different classes of organic substances. Table II.3.1. Cleanup methods for organic extracts. Analyte group Organochlorine pesticides Polychlorinated biphenyls (PCBs) Organophosphoric pesticides Chlorinated herbicides Chlorinated hydrocarbons Polynuclear aromatic hydrocarbons Cyclic ketones Nitrosamines Phenols Phthalate esters GPC – Gel permeation chromatography Cleanup method Florisil, GPC, sulfur Florisil, GPC, sulfur, KMnO4 – H2SO4 Florisil, GPC Acid-base Florisil, GPC Alumina, silica gel, GPC Florisil, GPC Alumina, florisil, GPC GPC, acid-base, silica gel Alumina, florisil, GPC

Acid-Base Partitioning This is applied to separate acidic or basic organic compounds from neutral organics. The solvent extract is shaken with water that is highly basic. The acidic organics partition into the aqueous layer, whereas the basic and neutral compounds stay in the organic solvent and separate out. After this, the aqueous layer is acidified to a pH below 2, and then extracted with methylene chloride. The organic layer now

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contains the acid fraction. Phenols, chlorophenoxy acid, herbicides, and semi-volatile organic pollutants are cleaned up by the procedure described above. Alumina Column Cleanup Highly porous and granular aluminum oxide - available in three pH ranges (acidic, neutral and basic) - is used in column chromatography. Analytes are separated from the interfering compounds based on their different chemical polarity. The column is packed with alumina, and then covered under anhydrous Na 2SO4. The extract is then loaded on it. A suitable solvent is selected to elute the analytes. The interfering compound is left adsorbed onto the column. The eluate is then concentrated further. Alumina can be prepared in various activity grades by adding water to Grade I (prepared by heating at >400°C until no more water is lost). Among the common pollutants, phthalate esters and nitrosamines are separated. Basic alumina (pH 9 to 10) is most active in separating basic and neutral compounds: alkali, alkaloids, steroids, alcohols, and pigments. Certain solvents such as acetone or ethyl acetate cannot be used. This form of alumina can cause polymerization, dehydration, and condensation reactions. The neutral form is less active than the basic grade and is used to separate aldehydes, ketones, esters, and lactones, etc. The acidic form (pH 4 to 5) is used to separate strong acids and acidic pigments. The alumina column cleanup is also used to separate petroleum wastes. Silica Gel Cleanup Silica gel is a form of amorphous silica with weak acidic properties. It is made by treating H2SO4 with sodium silicate when used for cleanup purposes. Interfering compounds of different polarity are absorbed onto and retained on the column. There are two types of silica gel: activated and deactivated. The former is prepared by heating silica gel for several hours at 150°C. It is used to separate hydrocarbons. The deactivated form contains 10 to 20% water and is used to separate plasticizers, steroids, terpenoids, alkaloids, glycosides, dyes, lipids, sugar, esters, and alkali metal cations. In environmental analysis, silica gel is used to clean up sample extracts containing single component pesticides, PCBs, polynuclear aromatic hydrocarbons, and phenol derivatives. Methanol and ethanol decrease adsorbent activity. Florisil Column Cleanup Florisil is a form of magnesium silicate with acidic properties. It is used for clean up of sample extracts containing the following types of analytes: nochlorine pesticides, organophosphoric pesticides, phthalate esters, nitrosamines, haloethers, nitroaromatics, and chlorinated hydrocarbons. Florisil is also used to separate aromatic compounds from aliphatic-aromatic mixtures, as well as to separate esters, ketones, glycerides, steroids, alkaloids, and some carbohydrates. It also separates out nitrogen compounds from hydrocarbons.

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Gel-Permeation Cleanup This separation is based on the size of the porous, hydrophobic gels. The pore size must be greater than the pore size of the molecules to be separated. Gelpermeation cleanup (GPC) is used for cleaning sample extracts from synthetic macromolecules, polymers, proteins, lipids, steroids, viruses, natural resins, and other high molecular weight compounds. Methylene chloride is used as the solvent for separation. Elution is carried out using a suitable solvent, and the eluate is concentrated for analysis. Sulfur Cleanup Sulfur is found in many industrial wastes, marine algae, and sediment samples. Sulfur may mask a region of the chromatogram, overlapping with peaks of interest. For example, in pesticides analysis, sulfur can mask over many pesticides such as lindane, aldrin, and heptachlor. Sulfur has the solubility similar to the organochlorine and organophosphoric pesticides and it cannot be separated by Florisil cleanup method. The removal of sulfur is achieved by treating the extract with one of the following three substances: copper, mercury, or tetrabutyl ammonium-sodium sulfite reagent. The sample extract is vigorously shaken with one of the above reagents. The clean extract, free from sulfur, is then separated from the reagent. Permanganate - Sulfuric Acid Cleanup Interfering substances in the sample extract may often be destroyed by treating the extract with a strong oxidizing agent, such as KMnO 4 or a strong acid like conc. H2SO4, or a combination of both. In such a case, the analyte should be chemically stable to these reagents. For example, interfering substances in the sample extract for the analysis of polychlorinated biphenyls can be effectively destroyed by treatment with a small quantity of KMnO 4 - H2SO4 mixture. PCBs are chemically stable under the condition of treatment, and do not react with the acid-permanganate mixture at such a short contact time. II.3.3.3. Digestion Samples analyzed for metals are usually digested. The digestion process uses strong acids and heat to increase the precision and accuracy of the measurement by providing a homogeneous solution for analysis by removing metals adsorbed to particles and breaking down metal complexes. Different digestion techniques are used depending on the analytical method and target accuracy levels. II.3.3.4. Dilution

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2000. Stanley E. Seventh Edition. Greenberg. This information should also be noted with the result. the reported result based on the detection limit will be increased proportionately to the dilution. Ltd. Manahan. 1998. West Sussex. Sampling for Analytical Purposes. A record of the dilution factor should be kept with the result. John Wiley & Sons. while if it is filtered it is considered a dissolved result. Arnold E. Commonly.. 1998. Soil and Solid Wastes. Inc. Chemical Pollutants in Air. or other substances in the sample may interfere with the analysis (matrix interference). Baltimore.5. Clesceri. For filtered samples. Boca Raton. The reasons for this may be that the concentration of the analyte is outside the concentration range where the analytical technique is linear.Sometimes it is necessary to dilute the sample prior to analysis. Boca Raton. USA. Eaton. Water. Pierre Gy. II. England.3. Standard Methods for the Examination of Water and Wastewater. Florida. 1997. once a sample has been filtered it is preserved. Pradyot Patnaik. Inc. Inc. and this needs to be considered in interpreting the results. REFERENCES 1. Florida. United Book Press... USA 4. the size of the openings in the filter (such as 1 micron) should be included with the result. USA. Ed Leonore S. Filtering The sample may or may not be filtered. CRC Press. 3. Environmental Chemistry. 92 . For non-detected results.3. Handbook of Environmental Analysis. 20 th Edition. 2. the resulting measurement is referred to as a total measurement. Dilution affects the result itself as well as the detection limit for the result. either in the field or in the laboratory.. CRC Press. Andrew D. If the sample is not filtered.

ANALYTICAL TECHNIQUES USED IN ENVIRONMENTAL ANALYSIS II. GRAVIMETRIC METHODS José MARTÍNEZ CALATAYUD Gravimetric analysis. is the most common type used in environmental control engineering. in addition. (d) chemical precipitation. Its most important application in the environmental field is with the analysis of sulfate or sulfite. The mass of the analyte present in the sample is determined from the mass of the precipitate.4. Changes in the sample mass when heated are recorded. (b) thermogravimetry.g. (c) electrogravimetry or electro-deposition which usually involves the electrochemical reduction and simultaneous deposition of metal ions at a cathode.II.4. samples may have to be extensively treated to remove interfering substances. total suspended solids).. the most “classical” of all quantitative methods (among the oldest of analytical techniques). the most common in a “classical” sense. Based on the preparation of the sample before weighing the analyte compound. It relies on a chemical reaction to transform the solved analyte in a very low soluble precipitate. Since weight can be measured with greater accuracy than any other fundamental property. gravimetric analysis was ( it is yet) one of the most accurate analytical methods. for the analysis of volatile solids. there are four fundamental types of gravimetric analysis: (a) physical gravimetry. 93 . relays on some final determination of weight. The compound of interest (analyte) is precipitated in a solid compound of known composition. It involves the physical separation and classification of matter based on volatility and particle size (e. and. Gravimetric procedures are lengthy and tedious compared against instrumental methods.1. As a result. only a very few gravimetric methods are currently used in environmental analysis.

1. Whatman #934AH) are the total dissolved solids (TDS) and those retained on the filter are the total suspended solids (TSS). Most of the impurities in potable waters are inorganic salts that resulted in the dissolved state. it is not lost by evaporation and drying at 180 o C for one hour.. the parameters TS (total solids) and TDS (total dissolved solids) have a relevant importance.4. Physical Gravimetry Solids As the operational definition of Total Solids (TS) content has been adopted through years of use the following “all matter that remains as residue upon evaporation and drying at 180 oC for one hour”. Thus.g. and then. 1. According to this operational definition and depending on certain empirical parameters the result may be smaller or greater than the amount of solids present in the sample.1. This is an operational definition because solids in a water or wastewater sample are a diverse collection of dissolved and particulate matter rather a specific chemical compound.4. Gravimetric methods in environmental analysis control Type of gravimetry Physical Analyte Total Solids Suspended Solids Dissolved Solids Oil & Grease Surfactants Thermal Precipitative Volatile Solids Volatile Solids Volatile Suspended Solids Mg 2+ Na + Silica SO4 2II.1. An operational criteria serves for a classification of total solids.5 microns.Table II. Sources of drinking water containing high concentrations of inorganic salts are not suitable (more than 1000 mg/L TDS are Procedure Evaporation Filtration Filtration + Evaporation Extraction with C2Cl3F3 + distillation of solvent Extraction into ethylacetate + evaporation Evaporation + 550oC for 15 min Evaporation + 550oC for 15 min Filtration + 550oC for 15 min With Diammonium hydrogen phosphate and final pyrolysis With zinc uranyl acetate Precipitation/ ignition/ volatilization (with HF) With Ba2+ 94 . this definition establishes the following: all solids passing through a filter paper of a certain pore size (e. Some authors prefer to define the total dissolved solids as all matter that is not retained by a #934AH filter.

because such materials are often difficult to remove in the treatmentplants.ecs. Figure II. It is very important to avoid unrepresentative sampling or particle break-up. which results as an useful parameter to assess either the need for “softening” and the corrosivity of a water.html) 95 .umass. Relationship Between Sediment Size and Sampling Bias (reproduced from: http://www. this should be considered either at the point of sampling and at laboratory subsampling. These errors are high for big particles.1. Figure II. The sampling errors are associated to the sample size. This requires that sample intake velocities should be similar to the flow velocities at the point of mixing. Waters of this type are even unsuitable for agricultural uses due to the negative effect on plants of the high ionic concentrations. or at least. and as a function of particle size. A recommended caution is to face the sampling device intake into the stream flow at 20 degrees. In natural water samples.4.unacceptable).4. the TDS (total dissolved solids) usually correlates well with the total hardness (expressed as total [Ca] + [Mg]). the disturbance should be minimized.edu/cee/reckhow/courses/572/572bk15/572BK15.1 depicts the percent error expected at different ratios intake velocity/stream velocity. The former topic means not disturbing the flow pattern of the sample stream.

html) For a general discussion on sampling techniques see reference Handbook for Sampling and Sample Preservation of Water and Wastewater (USEPA. 1982).umass. analysis should be conducted as soon as possible after sampling. the mixing at the point of sampling is sufficient and depth-integrated sampling is not required. The obtaining of a representative sample in the laboratory (laboratory subsampling) is best accomplished by shaking and a quick pouring the aliquot into the receiving beaker (as depicted in Figure II.ecs. Recovery vs particle size during sub-sampling with different mixing techniques. Collection and save of samples must be performed in clean glasses or highdensity polyethylene flasks.2) Figure II.When the presence of settle able solids is suspected and for producing a homogeneous cross-section of settle able solids.4. and.4. 96 .reproduced from: http://www.edu/cee/reckhow/courses/572/572 bk15/572BK15.2.

4. The procedure can be developed on a steam bath instead of the oven. A special attention should be paid to evaporating dishes while hot or in use. due to the high price of platinum. Preheat the evaporating dish (100 mL recommended capacity) for 1 hour at 550± 50 oC. However. Procedure: 97 . then. vycor should be used. This approach is preferred for most waters than the indirect method of subtracting dissolved solids from total solids. in a drying oven or in the open air for a period of 15 . they must be safely stored in a desiccator to avoid the collection of dust and absorption of moisture while it is not in use. The identified filters should be weighed.g. Procedures A.20 minutes. This operation should be repeated up to constant weight. Keep into a desiccator down to room temperature and weigh. On the other hand. 3. Measure a sample aliquot (about 75 mL or the required volume to obtain 200 mg TS) and then evaporate to dryness at 98 ºC on the pre-weighed dish in a drying oven at 98 oC. Suspended Solids (or non-filterable residue). Platinum is the most inert material and it is preferred over vycor as the material for evaporating dishes. heated and dried while remaining in small aluminum pans. Procedure: 1. Keep the dish in a desiccator to achieve room temperature and weight with a repeating system to obtain constant weight. Total solids are the final weight ratio of a dried sample (total weight minus tare) versus the sample volume. Directly measured by drying and weighing of the solids retained during filtration (Whatman #934AH glass fiber filters with a nominal pore size of 1. and. high contents in alkaline salts leach metal ions from the glass. to avoid the absorption of moisture or even the collection of dust. porcelain is avoided due to the difficulty on obtaining a constant weight. TS (total solids) or Total Residue.Some care must be present in sampling and storing the collected samples: e. Estimated precision: ±4 mg or ±5% and for settled wastewater is possible to reach ±1 mg. cool it protected from room dust. Additional drying for 1 hour at 103 – 105 oC is highly recommended. 2. air or solved oxygen oxidize iron and manganese ions and then the precipitation of their basic salts occurs. B.4 microns).

3. Analyze the filtrate in accordance with the reported TDS procedure. 2. The filter (934AH glass fibber) should be previously washed by rinsing it three times with 20 mL of distilled water. 4. or an indirect alternative is subtracting from the total solids the suspended solids. The filter (#934AH glass fibber) should be previously washed by rinsing it three times with 20 mL of distilled water. or highly colored waters should be treated at the higher temperature. respectively.200 mg of suspended solids and dry for 1 hour at 103-105 oC. The drying temperature has a relevant importance: (a) At 103-105 ºC deviation occurs from the physically occluded water due to crystal irregularities and the presence of water of crystallization.g. The presence of large amounts of residue (over 200 mg) exacerbates the problem of occluded water. some water of crystallization may remain. Final drying for 1 hour may be conducted at either 103-105 o C or o 180 ± 2 C. carbonate. 4.. Repeat the drying and weighing to achieve a constant weigh (± 0. Keep into a desiccator to cool and weigh. Procedure: 1. (d) In general. dry by heating at 103-105 o C for 1 hour. chloride or sulfate. Maintain suction until the filter is dry and then. (e) Drying to 180± 2 º C gives TDS results closest to the calculated ones from a complete chemical analysis. However. nitrites and chlorides). 3. magnesium.5 mg). (b) A negative bias should be done by the volatilization of CO 2 from the conversion of bicarbonate. This positive bias could happen with sample waters with high contents in calcium. These parameters can be directly obtained by the weight of total solids from the filtered sample. alkaline waters. Maintain suction until the filter is dry and then. 6. 98 .1. 5. Estimated precision ±5 mg/L and ±20 mg/L for low and large (up to 200 mg/L or more) concentrations. Dissolved Solids (D S or filterable residue). especially in the presence of sulfates. This high temperature can be recommended for total loss of occluded water. Keep the filter into a desiccator to reach the room temperature and then weight it. 103-105 or 180 may be used for waters of low color and low alkalinity. However. An operational definition is: the theoretical dissolved solids are “all that is not water”. (c) At 180± 2o C will occur additional volatilization of some inorganic salts (e. 2. the hard sample waters. Filter the sample aliquot required yielding 50 . C.

Be careful not to have fat in the grindings Sample taking: . these can settle by centrifugation for 15 minutes at 3000 RPM. 500 milliliters). in some cases. • Emulsions formation can be avoided during the extraction acidifying until pH 1 or adding sodium chloride.D. acidifying the sample only after the first extraction.the complete content of the bottle of the sample taking goes to a movement separation funnel (1000 milliliter). before the extraction. they are washed with petroleum gasoline and they are dried. detergents. Interferences and previous treatment: • Some members of the organic substances from the before mentioned group are waxes. emulsion producers.. • All glass materials used are cleaned carefully before the sample taking or after the determination. If the extract contains finely dispersed substances. In order to continue treating the extract. filling to the bottle until a certain annular signal (p. the aqueous phase is left in a second separation funnel (1000 milliliters) and the organic phase is joined in a separation funnel of 100 milliliters. is filtered through a soft filter paper with the surface free of fat. In their determination are also included. centrifuged if necessary. Then the sample is shaken intensely for about two minutes. the bottle is washed once or twice with 10 milliliters of petroleum gasoline every time and the dissolvent is added to the water sample. Technique: 1. other organic substances which can be extracted with petroleum gasoline and whose boiling point is over 200 oC approximately. in total concentrations > 1 mg/L. Next. If they must be detected in fatty acid form. After separating the layers.The best method is to use a glass bottle with glass cork. In case of low impure waters . sulfuric acid is added to the sample until pH is lower than 2. and the filtrate goes to an evaporation flask of 100 milliliters. Oil and Grease (OG). having been washed itself previously with petroleum gasoline. • Fatty acids can be determined separately in a second extraction. the petroleum gasoline volume is completed to 25 milliliters. is used a vacuum rotating evaporator. The extract.. The separation funnels used for the extraction are washed again with about 5 or 10 milliliters of petroleum gasoline and the dissolvent and the extract are joined in the separation funnel of 100 milliliters. 99 .e. After that. The filter is washed twice with 5 or 10 milliliters of petroleum gasoline. • Fatty acid salts (soaps) are not determined. The extraction is repeated two more times with 15 milliliters of petroleum gasoline every time. Low boiling point hydrocarbons (gasoline) are not included in the determination.

the capsule is washed with petroleum gasoline. results in 100 . it is dried outside for 60 minutes in a desiccator on silica-gel. in fact. whose temperature is regulated with a thermostat to 20±1 oC. the flask is separated carefully. for a medium number of flask revolutions. After disconnecting the vacuum and the rotation. The extraction takes place during 3 or 4 hours with petroleum gasoline. we start the evaporation for about five minutes at about 25 Torricelli (if it is possible to about 2 Torricelli). changing the position of a key with three steps.2.cleared values to 1 mg L-1.we evaporate to dryness 500 milliliters or less of water sample or mud in a porcelain capsule with 30 g of sea sand in the water bath. the extraction flask is connected to the vacuum rotating evaporator and the dissolvent evaporates until about 20 milliliters remain. We start a vacuum oil pump and we connect it. After 24 hours on the surface of water.oil and grease total or extract of petroleum gasoline in mg/L = (a*1000)/b a = mg of the extraction remainder (weight) b = milliliter used of the test of water or mud Results indication . to a led distillation at 280 °C.. the crude oil practically has lost by evaporation all hydrocarbons with a number of carbon atoms lower than 14. as described in 1. Later. which can be associated with a polymerization. with the apparatus. in stages – in case it is necessary – we filter it and it goes to a calibrated evaporation flask connected to a vacuum rotating evaporator. After that. until the pressure has lowered until about 50 Torricelli and the residual volume of the extract is about 0. 4. 3. Apart from evaporation and certain processes of dissolution. The extract is centrifuged.5 milliliters. Calculation . − aromatic hydrocarbons. The remainder goes quantitatively to a Soxhlet extraction cartridge. Characterization and Determination of Hydrocarbons The great complexity of the oil can be classified according to the three following groups: − saturated hydrocarbons or alkanes. and it is weighted In case of strongly impure waters or mud with large water content . what corresponds. The apparatus is evacuated. The oxidation. Next. − heavy products (aromatic compounds with high molecular weight containing heteroatoms such as sulfur. which is put in a Soxhlet extraction apparatus. oxygen or nitrogen). with a water tube. we fix the flask with a NS clamp (pay attention standardized grinding) to the tube for steam circulation of the vacuum rotating evaporator and suspended it in a water bath. petroleum products undergo chemical and bacterial degradations.

we use either the carbon tetrachloride. scoops. If we prefer the direct injection of water. It is convenient there to carry out sample taking in a turbulent column or using a special apparatus. then a residue weighing after solvent evaporation. In case of a pollution accident. solenoid winnow. hexane. In this case. while being extractable. The use of gas chromatography makes it possible to reach a good sensitivity for the determination of the hydrocarbons whose boiling point is located between 150 and 450 °C. this method cannot be used for hydrocarbons whose boiling point is higher than the one of the employed solvent. Solvent evaporation can lead to azeotropic entailed wastage. The automatic sample taking is done by a special apparatus (paddle wheels. etc. or with a preliminary extraction with a solvent. this method is very appropriate then for the determination of light hydrocarbons. The possible evaporation or microbial action wastages limits the storage times and imposes very quick extraction after sample taking. so this method is 101 . The residues obtained after boiling at 300 °C are less sensitive to these phenomena. at a medium depth. are not hydrocarbons. From a practical point of view. It also makes it possible to specify the importance of N-alkanes compared to other present hydrocarbons. it is possible to practice an organic solvent extraction. ether oil or chloroform.) respecting certain obligations. and it can be manual or automatic. The determination can be carried out either by injecting water directly in the apparatus. . the hydrocarbons are presented in a surface film form or in droplets. they can also be emulsified in water or adhere to the suspended particles. For the manual sample taking is used a glass bottle with ground or screwed stopper whose capacity is determined by weighing. A certain number of determination methods were developed.a rise in viscosity and density of the recovered product. This type of not very significant methods applies badly to routine controls because of the evaporation wastages or the interferences whose elements can cause and which. recovery percentages can be different according to the used solvent. it is advisable to multiply the sample taking and to consider an average value of the obtained results.The first ones include gravimetric or volumetric methods based on the solvent extraction and those based on the density or the optical properties of the elements to determine. See greases and oils determination method in wastewater. Indeed. Of course. Because of the homogeneity errors. in pressure or depression air. hydrocarbon quantity is important.The second group are much more significant methods. Sample taking is carried out in a turbulent zone. and they can be classified in two main categories: . Infrared spectrometry allows under certain conditions the simultaneous determination of hydrocarbons and phenols but its sensitivity varies with hydrocarbons found. or pentane. because of the peaks interference on the chromatograms. the sample taking after decantation corresponds in fact to a preconcentration. it is difficult to carry out homogenous sample taking. we can use a separation pre-column. We can employ carbon tetrachloride.

1. volatile solids. The lost volatile portion upon ignition is generally assumed to be equivalent to the organic fraction and the residual portion is considered the inorganic fraction. but not lost upon drying at 103-105 oC for 1 hour”. most of this is calcium carbonate (for samples over the interval from of moderate to high hardness). VSS. TSS. this procedure leads to the oxidation and/or volatilization of some of the sample compounds. TSS. If the recovered quantity is sufficient. liquid chromatography allows the saturated or aromatic hydrocarbons determination. fixed suspended solids. After this process. and. used for volatile solids analysis in environmental engineering (thermogravimetric procedure). TS. Combining the fractions obtained from ignition and filtration procedures. fixed dissolved solids. total solids. The operational definition for volatile solids would be: “all matter lost upon ignition at 550 oC for 15 minutes. and fixed solids are all those substances that remain as residue.E. (b) (b) Alternatively trapping the evolved gases and weighing the trap is used in many fields for the determination of total carbon and hydrogen in solids (this is known as combustion analysis). Positive bias should be due to incomplete oxidation of organic compounds or loss of recalcitrant water of crystallization. two different procedures could be applied: (a) Measuring the loss of sample weight. 102 . volatile dissolved solids. Volatile Solids and Fixed Solids After ignition of the sample for 15 minutes at 550 oC. total dissolved solids.satisfactory only for hydrocarbons having more than 12 carbon atoms by molecule (P. TDS. A steady increase in apparent weight indicates this problem. higher than 220 °C approximately).4. fixed solids. Negative errors can occur due to the decomposition of certain inorganic compounds. Temperatures over 600 oC could damage the glass fibber filters by melting and a significant loss of weight can occur. and VSS. TDS. TS. volatile suspended solids. loss of ammonium salts (NH 4HCO3 → NH3 + H2O + CO2) or CO2 release from magnesium carbonate (MgCO 3 → MgO + CO2). the lost weight of material is called the volatile solids. Thermogravimetry Thermogravimetry and/or combustion analysis involves the sample treatment at 500 oC or more.2. II. But usually.g. which decomposes only at temperatures over 800 oC. only four of these are used: namely. and the gas chromatographic method is used to specify the compounds composition. there are 9 separate groups: namely. e. Remember to cool the sample to room temperature before weighing or the method accuracy will suffer severely due to convection currents around the balance pan created by the differences in temperature. total suspended solids.

103 . The third tube is placed for protecting the two former traps from backflow of atmospheric water and carbon dioxide. Vessels. With the aid of a catalyst (a transition metal). respectively. Two successive traps. Enlarge ignition period up to 1 hour for sludge. The total carbon and hydrogen contents in a solid sample can be determined by combustion in the presence of pure and dry oxygen (Figure II. NaOH + CO2 → NaHCO3. soil and sediment samples.3. A special attention should be paid to occluded water. serve to retain all water and carbon dioxide released. sediments. This type of analysis is sometimes applied to soils. filters. at least 1-hour ignition interval is recommended. 2. After the sample combustion both tubes are weighed to determine the increase. Procedure: 1. Ignite the sample in a furnace for 15 – 20 min at 550 ± 50 ºC.). sludge and soils. Cool in open air for 15 min and then keep it in a desiccator down to room temperature Figure II. 3.Additional problems appear in the analysis of sediments. P2O5 is an efficient desiccant (it is deliquescent) and the sodium hydroxideimpregnated asbestos (ascarite) will trap all of the carbon dioxide through the chemical reaction. dried sludge and extracted aquatic organic matter. Combustion (or thermogravimetric) analysis for determination of carbon and hydrogen. evaporating dish and any other material should be dry and weighed to a constant weigh.4. P2O5 and ascarite.3.4. the combustion process releases only CO 2 and H2O.

Heating the solutions and adding the precipitant reagent slowly with rapid mixing to decrease the super-saturation degree will help. when unwanted ions or molecules are retained (physically trapped) in the precipitate due to: Inclusion. removed from the mother solution by filtration and weighing. 3.4. To avoid it. This calcination procedure (1 hour at 800 – 900 ºC) should be performed on the porcelain crucible used for this operation. Crystal purity increases by re-dissolving the precipitate and then repeat the precipitation. add a drop of sulfuric acid and repeat the calcination. Occlusion..II. to obtain a non-filterable precipitate. it also means yield losses due to solubility. the physical trapping of impurities from mother liquor within crystal irregularities. Final weight. total minus tare (crucible). Precipitative Gravimetry Gravimetric Determination of Ionic Contents An ion gravimetric determination requires the conversion of the analyte into an insoluble compound of a known stoichiometry. if not tiny micro-crystals are formed rather than a few large ones and micro-crystals behave as colloids and pass through the filtering devices. Co-precipitation. Ba+2 + SO4-2 = BaSO4 (precipitated) 1. First of all. a single substitution in the crystal lattice by an ion of similar size. crystal growth should occur faster than crystal nucleation. Sulfates. The formation of high purity and non-filterable crystals implies to allow reaction to continue for at least 2 hours at temperature over 80 . A. During this process the reducing character of the filter paper can originate barium sulfur. Low pH is required to avoid the co-precipitation of barium carbonates and/or phosphates. Sulfate. 104 .3. 1985). 4. BaSO4 + C → BaS + 4 CO. Finally it is weighed at room temperature (keep it into a desiccator). Filter the precipitate by decantation on a filter paper and the resulting solid mass is washed and dried in an oven for 1 hour at 800 oC. Barium sulfate precipitates quantitatively by adding excess of ion Ba2+ under acidic conditions (5x10-2 mol L-1 in hydrochloric acid).1.90 oC. 2. Sulfites and Barium Methods based on the weight of precipitated BaSO4. This process (digestion of the precipitate) minimizes the formation of filterable BaSO 4 crystals from the initial colloidal particles. The historical method of choice for sulfate in waters and wastewaters is the gravimetry with barium (APHA et al.

Chloride. To avoid the precipitation of reduced sulfur ions it is advisable to add hydrogen peroxide to transform into sulfate ions on the other group. Dry at 110 ºC and then calcinate the precipitate with the aid of Pt crucible. and reduced sulfur species like sulfite. The homogeneous precipitation techniques are able to avoid this erroneous effect. However. the precipitating reagent is generated slowly and homogeneously by slow hydrolysis of sulfamic acid in boiling aqueous solution: NH2SO2OH + H2O → NH4HSO4 Determination of barium as oxalate Procedure: Add 10 mL of solution concentrated ammonium chloride to 200 mL of sample and then one drop of 1% heliantine. It could be gravimetrically determined by precipitation with silver. The precipitation is not carried out by slow addition of precipitant to the analyte containing solution. classical gravimetric analysis has been implemented in continuous flow-systems (precipitation weighing included). the usually recommended procedure for water and wastewater samples (APHA et al. In this way. sulfide. The air stream also functions to dry the retained precipitate on the filter prior to weighing. constancy in the amount of moisture retained by 105 . relatively impure solid particles results. Let it settle for 24 hours at room temperature and protected from dust. Filter on paper (no ashes material) and wash with a lot of boiling water up to no acidic residues can be detected in the filtrate. two fixed volumes of sample and reagent are inserted into two channels. Interferences come from the precipitation of different silver salts with a lot of present anions namely: bromide. This effect cannot be entirely eliminated and is a source of error. In fact. iodide. which is accommodated on the balance pan. Cool to room temperature into a desiccator and the weigh. Such a procedure tends to produce conditions of super-saturation and a consequent rapid formation of small. For this purpose. where they react to form a precipitate. Determination of barium as BaSO4. Add excess of acetic acid (clearly acidic medium) and 25 mL of the sodium oxalate solution. cyanide (high contents of these ions is not usual). The precipitate is retained on a filter inside the flow-cell. 1985) is the titration by precipitation with potassium chromate (red precipitate of silver chromate) as titrant. Filter to separate any precipitate if formed. Adjust the suitable medium by dropping hydrochloric acid up to red color and immediately the required ammonium up to the change into yellow. and thiosulfate. FIA-gravimetric procedures The best testimony to the high flexibility of FIA for adaptation to all types of procedures is probably the fact that the analytical balance has been used as a detector in FIA manifolds.. A stream of dry air propels both solutions to a merging point.

192. Separations analyte-interferences can be accomplished through selective dissolution of the retained precipitate.. According to the authors “classical gravimetric analysis. The analysis of up to 30 samples h -I was possible. Linear calibration graphs were obtained over the range 0. A method for the total content is reserved for samples presenting high contents (several tens of mg L-1). E.A.G. Silica The gravimetric method for determination of silica in water should be selected according to the form that is present in the sample. Arruda.”for routine analyses. To avoid silica adsorption on the glass walls (high silica content samples) use polyethylene. Jacintho. Zagatto and B. In the former purpose filtration separation is required and is accomplished by placing the filter at a strategic part of the flow assembly. Gravimetric procedure. Reis. as ionic or colloidal state.03% (w /v) Ba for a signal-to-noise ratio of three.each sample is ensured. n = 5). there are very few published procedures on the FIA-gravimetric analysis. total loss of water molecules is completed through a 106 . remains important mainly for accuracy assessment and the evaluation of standards for instrumental calibrations” …“gravimetry is seldom applied to large-scale analysis. washed and leaded to the flow-cell of the detector. the favorable characteristics of gravimetry could be better exploited after automation”. 129 – 133). After weighing.999994. the analyte is precipitated. The linearity of the calibration graph revealed that the retained water was proportional to the amount of precipitate formed. B.40 -1. For small amounts (les than 10 mg L -1) is suitable the spectrophotometric method to the o-phosphate ionic form. One devoted to calcium determination is reported (as references O. “the feasibility of gravimetry in flow analysis is demonstrated with the determination of barium as oxalate ”.A.60% (w /v) (R = 0. as the drying step was only partial. because it involves a number of prolonged operations usually to be carried out by skilled analysts”…. Reactions of precipitation have been proposed in flow systems with the goal of the separation and/or pre-concentration of the analyte or turbidimetric determinations. In this way. which means the humidity of the crystals was constant. The silica is precipitated partially de-hydrated by concentrated hydrochloric acid. A slight variation under < 5% of the slope of the calibration equations was observed. Unlike the usual gravimetric procedures that are based on calculations on the obtained stoichiometric precipitate. At the present. the flow procedure requires calibration graphs.F. a throughput of up to 30 samples/h can be achieved.Z. When the goal is the preconcentration. M. According to authors carryover effects were not relevant. The detection limit associated was 0. . ACA 258. sometimes regarded as obsolete. If only selectivity enhancement is needed. the precipitate is removed by dissolution with an injection of an appropriate solvent. retained in the filter.

weighed as magnesium pyrophosphate (MgP2O7). Calcination of the two filters at 1200 ºC. 5. 8. Any used reagent (pure water included) should be silica exempted and the hydrofluoric acid gives a non-volatile residue. 6. 9. 107 . Dry completely the residue. warm gently for 1 min and filter. If a slow precipitation is observed. Wash the residue with 0. 3. 4. 5. Determination of Lithium Only recommended for high contents like (thermo-mineral waters). Add two drops of HCl and 20 mL of 20 % ammonium phosphate and warm to boil. place it into the furnace at 110 ºC and repeat the above reported procedure by using a second filter. Determination of Magnesium The gravimetric procedure should be applied for contents over 10 mg L -1 and it is performed on the filtrate from the separation of calcium. Add 10 mL of HF and two drops of 50 % sulfuric acid. Evaporate the acetic filtrate down to 150 mL.2 mg L-1. rub the beaker walls with the aid of a glass rod. Estimated precision ±0. After addition of 50 mL of water. 2. 2. D. Dry and calcinate at 1200 ºC up to constant weigh. Procedure: 1.5 mL l -1 HCl and rinse with pure water to complete elimination of any acidic residue by testing that the washing water is without presence of chloride (AgNO3 reaction). 4. Add ammonia to about neutral point plus concentrated ammonia to 1/5 volume. Dry and calcinate with a weighed crucible.calcination step. Procedure 1. 7. Keep the residue for 1 hour at 105 – 110 ºC. Keep at room temperature for a 12 h interval. To 1 – 2 liters of sample add 10 mL of hydrochloric acid. C. 6. Filter by using a free ashes paper and wash with 50 % ammonia. Add 5 mL of HCl and keep for 5 min protected from dust. Dry to residue (Pt capsule is preferred) by means of a water bath 3. Addition of hydrofluoric acid converts the precipitate into a volatile fluorsaliclic acid and after new calcinations the weigh loss corresponds with the pure silica content. and then. Weigh.

76.D. Pre-wash and dry into a desiccator (at void) a filter crucible of pore average 10 -20 μm.Guy. E. II. An Application of the Uranyl Zinc Acetate Method for the Determination of Sodium in Biological Material. & Norman V. Dunot edit. 6. mL of sample. F. pp.APHA. eau de mer . Rapid precipitation of barium sulphate.Hem. 30-32. 4. 71-75. 16th edition.WANDENBUCKLE. 3.Hem. 90-98. (1939). T. J. 1959. (1948). Bull. Liaison Labo. The dry process will end into a desiccator up to constant weigh. or 14th edition.H. M..fold) with 1 mL of ethyl oxide. Determination of Sodium The gravimetric alternative to the flame photometric estimation of sodium is the procedure based on the weigh of the precipitate obtained with reaction with zinc uranyl acetate. M. AWWA. Comptes rendus Acad. 1923. Anal.67. E.93171. APHA. USGS Water Supply Paper #1473: 1st ed. sept-oct. Manuel de chimie analytique. 89-98. F. 34-36. TUTHILL.. previously saturated with zinc and sodium uranyl acetate. 11. Washington. Paris. 108 . FISCHER. Chapter C2. BORDAS. 7è édition. 8.D. p. II. P. Des Sciences.1977. 2nd ed.B. pp. F. Book 3. Field Methods for Measurement of Fluvial Sediment.P. Anal.95 V (being M.M.Chem. The sodium amount (mg L-1) is = M x 14. Des Sciences.Biol. 1924.TYNER.RHINIHAMMER.564. 1970. 32-34. Volume 1. CLEMENT. BOLL. Sur la détermination de la silice dans les eaux. 9. 67.P.176. 92-100..DIENERT... Jean RODIER. R. 7. Dunot edit.WANDENBUCKLE. F.P. 20. Chem.DIENERT. Minear & Keith eds. 70-73. 10.TREADWELL.W. Une étude de la sílice colloïdale. BUTLER. P. Dunod. 1931. F. Paris 1984. (1953). K. pp. 76-80. Paris. eliminate the ethyl remaining by aspiration. 91. 13.. Chapt. F. Techniques of Water-Resources Investigations of the United States Geological Survey. 12. Chem. 4 in Water Analysis. Then wash (3 . Manuel de chimie analytique.1478. Comptes rendus Acad. 2. and. 3. WPCF (1985) Standard Methods for the Examination of Water and Wastewater. BOLL. L´Analyse de l´eau-Eaux naturelles. REFERENCES 1. eaux résiduaires. 1544.178. H. J.TREADWELL. mg of precipitate and V.. 5. (1939). Procedure: 1. RANCHET.E. Filter the precipitate and wash (5-fold) with 2 mL of ethylic alcohol at 96 º C.B. 25. 73. Contribution à l´amélioration du dosage des hydrocarbures dans les eaux. J. J. or 15th edition. 2. et C.

EMSL.1. 74-82. “end-point”. 178-179. C. V. D. etc. The latter is based on the addition of an intermediate reagent having the excess (not reacted with the analyte) is titrated.L. 243-245.A. 16. September.ISO ICS 13 Environment. 18.2. 284-285. McCarty (1978) Chemistry for Environmental Engineers. John Wiley & Sons. 1982. (1987) Chemical Analysis. a) Ba rule: ISO 548:1981 b) Sulfates: ISO 2480:1972.Docs. Fundamentals of Titrimetry Volumetric or titrimetric analysis is a classic quantitative method which employs an exactly measured volume of a standard solution containing a known concentration of reagent "A".Sawyer..Snoeyink.2.14. pp.Rubinson.L. Safety (Excludes ISO 14000) en http://webstore. the process proceeds until a perceptible change is produced (chemical indicator).org/ansidocstore/dept. Health Protection. direct titration (as above reported) and back titration. ISO 2997:1974 II. (1980) Water Chemistry. EPA-600/4-82-029 [Gov. & P. This theoretical point is known as the “equivalencepoint”. Alternatively. If the A additions to the sample proceeds far away from the equivalence point. aluminum. this reagent A is added step by step (from a burette) to the unknown concentration of the analyte “B” in the sample. 15. where the number of added A equivalents to the sample solution equals the number of equivalents of "B" originally present.).. 17. Brown & Co.4. a “physical indicators” in which a physical property (conductivity. absorbance. should be produced as close as possible to the complete reaction A + B in an “stoichiometric completion”.. McGraw Hill Publ.) is continuously measured.23/5:600/4-82-029]. a non-perceptible change could be also used. Publ..asp?dept_id=220 19. Little.USEPA (1982) Handbook for Sampling and Sample Preservation of Water and Wastewater. Two basic modalities are normally used: namely. pp. New York. etc. EP1.Gravimetric procedures (from web-site of ISO) are 107 references for metallic samples (silica. Boston. pp. etc. K. Accuracy is related to the coincidence among end-point (or indicator point) and the equivalence-point. 109 . VOLUMETRIC METHODS José MARTÍNEZ CALATAYUD II. further calculations allow to obtain the endpoint. 65-69.1%. Publ.ansi.4. US EPA. The change. 454-462. soils. & Jenkins. Titrimetric methods reach a precision of up to 0.N.

the titrant must be standardized. where the titrant A is introduced continuously. (c) An indicator (external or internal) to identify the endpoint. Several advantages of these pumps are the simplicity of operation. allow to work in a wide range of flow rates (for the titrant transfer. simple and reliable control of flow-rate.1 mL/min). There are very relevant factors as stirring speed. At present. (b) A standard titrant solution of accurately known concentration of A to react with the analyte with a well-known and repeatable stoichiometric procedure. However and due to the changing properties of the flexible tubing they need to be periodically calibrated.Several basic requirements can be reported for a well-defined titrimetry: (a) A quick and quantitative reaction A + B is necessary. (c) Suction-stroke piston pumps or metering pumps are more precise and require less calibration. (c) enable application of elaborate techniques for analyzing the data by computerized systems. etc. Of paramount importance are the kinetics of the volumetric reaction and the response of the indicator system. easily automated. There are commercially available acid and solvent resistant tubing. or automatically. (d) When the chemical for the titrant solution is not available in a kinetically stable form of pure and well-defined composition. (b) Peristaltic pumps highly versatile and reliable. The random and systematic error resulting from the empirical estimation of the endpoint may be estimated by conducting a blank titration. (f) It should be interesting if possible to have an A + B reaction sufficiently selective to avoid a previous sample treatment to remove interferences. This operation is an independent titration against stable. For titrations in small volumes low flow rates are used (0. 110 . a relevant parameter to be optimized).1 . in analytical chemistry automation is of paramount importance for the following reasons: (a) convenience and speed. they enjoy a similar degree of versatility. highly concentrated mineral acids (not in titrimetry) and organic solvents. pure chemical known as a primary standard. configuration of the cell. For automatic transfer of the titrant to the analyte solution are proposed several instrumental devices: (a) Piston burettes are highly reliable and do not require calibration. Titrations can be performed manually step by step (or point by point). Other empirical limitation of the pumps is the use of corrosive reagents. (e) Accurate measurements of the sample and added titrant volumes. input of titration. (b) performing analysis without supervision.

. expressed as carbonates Winkler Permanganate I3. I(I-) oxalate I(I-) I(I-) 111 .S2O32DPD Ferrous K2Cr2O7 Sample pretreatment Volumetric reaction OH.2.S2O32I3.deep blue Eriochrome Black T intense red .Table II.deep blue pH 12 Ca-EDTA 2Direct titration As Ca and Mg total amounts. Analyte (A) Standardized Chemical Indicator titrant solution (internal) (B) Observed change Neutralization (acid-base) Alkalinity HCl Acidity NaOH Nitrogen (III) H2SO4 Volatile Acids NaOH Precipitation Chloride AgNO3 Chloride Hg2+ nitrate Complex formation Ca2+ EDTA Hardness EDTA methyl red phenolphthalein potassium chromate yellow-precipitate Diphenylcarbazone Eriochrome Blue Black R intense red .4.+ H+ H+ + OH – digest/distillatio Macro-Kjeldahl n NH3 & Acidimetric distillation distillation Oxidation/Reduction Dissolved Na2S2O3 O2(DO) Ca2+ KMnO4 Chlorine/ClO2 Na2S2O3 SO32Na2S2O3 Chlorine/ClO2 FeSO4 Concentration Fe(NH4)2(SO4)2 of dissolved oxygen..Titrimetric methods for environmental water analysis. COD Starch Deep blue – colorless (white turbidity) auto Starch Deep blue – colorless (white turbidity) Starch Deep blue – colorless (white turbidity) DPD ferroin Mn2+.

and any other with a negligible concentration. These may include two relevant concentrations. must bind the metal less strongly than EDTA does. Hardness. The complete complexation of the metal is quick as the stability constants are quite large and the formed complexes show a welldefined stoichiometry. (b) by the counter ions. To calculate the chemical requirements for this softening. calcium-hardness and magnesiumhardness. The carbonate hardness means the hardness that could be precipitated as carbonates. Hardness may be classified according to: (a) the constituent anions: namely. which binds very strongly to many metals. the indicator-analyte complex at the suitable pH for the procedure must not be affected by other metal ions in the sample and obviously. or. it is necessary to establish both the total hardness. 1:1. Ca (II) and Mg (II). This former and “classic” method employs an specific dye of the metallochromic type. and the calcium hardness. The end-point can be estimated either by a chelating metal chemical (internal indicator) or an ion-selective electrode sensitive to the analyte. Ba (II). a chelating ligand which gives a solution change in color in going from the complexed to the uncomplexed indicator-analyte form. The EDTA molecule is: [HOOCCH2]2NHCH2CH2NH[CH2COOH]2 This is a hexadentate ligand. When hardness is less than alkalinity. The term “total hardness” means the Ca-hardness plus the Mg-hardness or the sum carbonate hardness plus non-carbonate hardness. Definitions and Environmental Significance Hardness means the total concentration of divalent cations in the water sample. like carbonate or non-carbonate hardness. like Sr (II). precipitation that occurs when the sample is heated. To be effective. the carbonate hardness is similar to the total hardness and non-carbonate hardness is equal to zero. Hardness is commonly minimized in drinking water treatment plants by precipitate softening or cation exchange. A. Carbonate hardness could be equal to the alkalinity (in mg L -1 as CaCO3) when the alkalinity is smaller than the total hardness.Complexation Titrations In a complexometric titrations commonly a metal is the analyte and the most usual titrant is the sodium salt of the ethylenediaminetetraacetic acid (Na 2-EDTA). 112 . The non-carbonate hardness is also known as “remaining hardness”. Fe (II) and Mn (II). evaporated or if the pH was raised. and its stability is highly dependent of the pH.

The order of reaction is as follows: Ca (II) and Mg (II) present in the sample are complexed by the addition of the indicator. At this pH many metal ions will hydrolyze and precipitate. To ensure this way. Hardness is originated by limestone and related minerals.Hardness is considered a problem in drinking waters and sometimes in industrial process waters and agricultural waters due to several considerations. from complex Mg (II) . a controversial point is that hardness has been associated with lower incidences of cardiovascular health problems. 113 . More important is the fact that Na 2-EDTA is highly effective at high pH for calcium and magnesium. especially when the water is heated as it happens in water-heaters and industrial boilers. inhibiting its action. at this pH the color change experienced by the indicator is most easily perceptible by the human operator. the presence in the solution of an auxiliary complexing agent is need. B. It is also possible to perform the back titration. Fundamentals of the Determination Eriochrome black T forms a deep red complex with calcium and magnesium when the indicator is added to the sample with adjusted pH (at about 10).3 and 11. due to that. bearing in mind that the titrant metals bind less strongly to Na2-EDTA than the analyte. these pH values allow the complex formation due to a partially de-protonated form that is effective as a chelating agent (pKs. with a different metallic standard solution. On the other hand. the order and extent is in according to the stability constant and the status of the ion in solution. first the EDTA complexes the Ca (II) present as cation. Due to that. and in addition. the excess of Na2-EDTA is titrated.6). this amount is in excess. hardness has historically been expressed as a mass concentration of equivalent calcium carbonate. then the Mg (II) and finally Ca (II) and the Mg (II) (with this order) complexed with the indicator. As calcium is the dominant hardness species and carbonate-bicarbonate is the most prevalent counterions. are 6. A known amount of Na 2EDTA is added. Divalent cations will complex with the carboxyl groups in soap and cause its precipitation. The titration with Na 2-EDTA forms complexes with metallic cations (here Ca and Mg). for total hardness triethanolamine and ammonia are used. a high excess of both remain in the cationic form. Also useful might be citrate or tartrate. may cause severe hydraulic problems in pipes by precipitating as metal carbonates. from intense red to deep blue. In addition. A complexing agent which competes successfully against hydroxide binding preventing precipitation but with no enough strength to interfere with the complexing reaction of Na2-EDTA or the metallochromic indicator. The indicator passes to a “free” ionic form and the solution suffers the change of color. a small amount of Mg – EDTA should be added with the buffer. the last coloration gives the end-point.indicator to the “free” indicator. Then.

Add 1.2'.9 g ammonium chloride in 143 mL conc.011 mol L -1) .Some compounds. Transfer quantitatively to a 1-liter volumetric flask and dilute to the "mark" with distilled water. Second. Finally. Add 200 mL distilled water and boil for a few minutes to expel dissolved CO 2. ammonium hydroxide. The reader must bear in mind several modifications. 5 – 6. the standard calcium solution. The complexometric procedure is quite similar to the above reported for total hardness.5005 g anhydrous calcium carbonate (primary standard grade) and carefully add small amounts of 6 M HCl (approx 50% of conc. of the indicator. respectively. The resulting solution is titrated vs. dissolve 16.01mol L-1) .005 mmol L-1) .2''-nitrilotriethanol). An inter-laboratory study resulted in 114 . Cool. (c) EDTA Titrant Solution (approx.9% and 0. binding the metallic cations.723 g Na2-EDTA (sodium ethylenediaminetetraacetate dihydrate) in 1000 mL distilled water. (b) Indicator Solution (approx. First. C.5 g of Eriochrome Black T (1-(1-hydroxy-2-naphthylazo)-5-nitro-2-naphthol-4-sulfonic acid) in 100 g triethanolamine (2.8%. (d) Standard Calcium Solution (0. For best results the titration should be completed within 5 minutes. Procedure: Place exactly 50 mL of the sample into a 125 mL Erlenmeyer flask and add 1-2 mL of the buffer solution and several drops. the EDTA-complexometric method.Dissolve 3.Dissolve 0. Due to the larger stability constant of the EDTA-Ca over the corresponding magnesium. or by a redox method or the most usual. can interfere as cyanide. these samples require a previous addition of a suitable inhibitor. by titrimetry.Weigh 0.25 g Mg-EDTA and dilute to 250 mL with distilled water. use a metallochromic indicator that only complexes with calcium as the Eriochrome blue black R. Calcium Calcium can be determined by atomic absorption spectrophotometry (AAS). 0. titrate with the EDTA solution until all reddish coloration disappears. Reagents: (a) To prepare the buffer solution. 0.) until CaCO 3 just dissolves. add a few drops of methyl red indicator and adjust to the "intermediate" orange color by adding 3N NH4OH or 6M HCl as needed. and hydroxylamine. to avoid the magnesium analytical activity and make the analysis specific for this calcium-hardness determination. sulfide. Maintain constant stirring during this operation. EDTA binds preferentially with Ca. magnesium is partially removed by using a higher pH (about 12) which results in precipitation as Mg(OH) 2 and calcium hydroxide does not precipitate (less acidic cation). An inter-laboratory study by using a synthetic sample containing 610 mg L -1 CaCO3 found a relative standard deviation and a relative bias of 2.

add 3 mL of NaOH solution or the required amount if the pH is still below 12.EDTA solution with continuous stirring. The reaction with dichloramine requires larger amounts of iodide and the reaction is slower. Finally. titrate with the Na2 . monochloramine reacts quickly to form tri-iodide. Reagents: (a) Standard 0. and in addition. Cerium (IV) and permanganate (MnO 4 -). the presence of HgCl2 apparently inhibits this reaction maybe by forming an unreactive complex Hg(II)-monochloramine.N-Diethyl-p-phenylene (also known as DPD) reacts with chlorine or tri-iodide to form an intense red coloration due to a free radical. Residual Chlorine by the N.2%. (d) Standard calcium solution. DPO reacts with a faster kinetics with chlorine.9% for synthetic water samples containing 108 mg/L calcium and 82 mg/L magnesium. ferrocyanide (Fe(CN)6-4). and a relative bias of 1. ferrous (Fe+2) and sulfite (SO3-2).5005 g anhydrous calcium carbonate (primary standard grade) and proceeds as above reported Redox Titrations Redox titrations are based on oxidation-reduction reactions between the analyte and the titrant. (b) 1 mol L-1 sodium hydroxide solution. However. iodate (IO3-).01 mol L-1 Na2-EDTA Titrant Solution prepared as above. Add solid Eriochrome Blue Black R indicator (0. After addition of iodide. Procedure: Place exactly 50 mL of the sample into a 125-ml Erlenmeyer flask: then. The DPD solutions are kept in an acidified media to avoid the oxidation by atmospheric oxygen. Reaction with monochloramine is slower depending on its concentration. Common strong oxidants used as titrant include dichromate (Cr2O7-2). iodine (I3-).1-0.2 g). solve 40 g L-1 of NaOH (c) Eriochrome Blue Black R. Periodically replacing of 115 . grind in a mortar 200 mg powdered dye [sodium-1-(2hydroxyl-1-naphthylazo)-2-naphthol-4-sulfonic acid] together with 100 g NaCl to about 40-50 mesh. this is titrated (back titration) with ferrous ion producing a colorless solution.N-Diethyl-p-Phenylene Diamine (DPD) Method The N. For a routine analysis the accurate weighing and level is enough. which then develops the red color. and their concentration (titer) must be checked regularly against the corresponding standard. only permanganate solution should be protected from room light and dust. the reducing titrant agents can be affected by oxidation by atmospheric oxygen. reaction catalyzed by a basic medium. by reacting with the DPD. weigh 0. arsenite (AsO3-3). until the color changes from red to royal blue. The most used reducing agents are thiosulfate (S2O3-2). Most of the oxidizing (titrant) agents are stable.a standard deviation of 9.

like hydrogen peroxide and oxidizing species of manganese and persulfate. Make up to 1 liter and the resulting solution must be stored in a brown glass-tight closed bottle. Continue titration until the red color again disappears (volume B). Reagents: (a) Phosphate buffer solution: Dissolve 24 g anhydrous Na 2HPO4 plus 46 g anhydrous KH2PO4 in pure water. After two minutes continue titrating until the red color is again discharged (volume C). H2SO4 and fill up to 1 L. after adding 20 mg HgCl 2 to inhibit biological growth. Ozone (iodometric method) A portion of the gas stream is directed to a gas bubbler filled with 2% KI solution for a well-known period of time. Fill to the mark (1 L) with water.N-diethyl-p-phenylenediamine oxalate in water containing 2 mL conc.106 g Fe(NH 4)2(SO4)2.6H20 in water containing 1/4 mL of conc. Environmental Significance 116 . (c). The neutral buffer for the reaction chlorine or tri-iodide with DPD should be added in the moment of the reaction. interfere seriously by oxidizing iodide.+ 2K+ The iodine formed is then titrated with sodium thiosulfate using starch. The detection limit is 18 μg L-1. Procedure: Place 5 mL of the buffer reagent and the DPD indicator solution and then. (d) Solid potassium iodide. For low expected amount off dichloramine concentrations can be used half the reported amount of potassium iodide. allow 2 minutes standing time if color driftback indicates incomplete reaction. Add to this solution 100 mL distilled water in which 800 mg Na2EDTA have been dissolved. H2SO4 and 200 mg Na2-EDTA dihydrate. Dichloramine (DCA): Add about 1 g of KI to the solution titrated in step (b) and mix.the solution is recommended (about one month). The presence of oxidants. For high dichloramine concentrations. Dissolved Oxygen A. add 100 mL sample and mix. (b) For monochloramine (MCA): Add a small amount of potassium iodide to solution from step (a) and mix. (a) For free residual chlorine (FRC): Titrate rapidly with ferrous ammonium sulfate until the red color disappears (volume A). (c) Ferrous ammonium sulfate as titrant: Dissolve 1. The stoichiometric reaction is: O3 + 2KI + H2O → I2 + O2 + 2OH. (b) Dissolve 1 g N.

O.) can be summarized with the following points: (a) is required for the survival of aquatic life. its solubility ranges from 14. (b) is very important in biological treatment processes. The titration is performed at pH under 5. discharges of organic wastes may depress the dissolved oxygen concentration. Fundamentals in the determination of D. smaller pH media produce a relevant decomposition of the thiosulfate. Dissolved oxygen may be determined by the Winkler titrimetry and the Membrane Electrode method. is used in the assessment of oxidation state in ground-waters and sediments. B. O. the D. The reaction sequence is: Mn (II) + 2 OH. Its solubility also varies with atmospheric pressure and salinity. concentrations indicate when aerobic and anaerobic organisms will predominate.6 mg L -1 at 0oC down to about 7 mg L-1 at 35oC. oxidizes iodide to triiodide (formed iodine combines with excess of iodide) and the resulting tri-iodide is titrated as usual with sodium thiosulfate with the aid of the starch for better end-point. (c) the parameter D.O.→ Mn (OH)2 4 Mn (OH)2 + O2 + 2 H2O → 4 Mn (OH)3 117 . The activity coefficient relates both parameters. The oxygen performs a quick oxidation of manganese ions [from Mn (II) to Mn (IV)] at high pH (see the diagrams E – pH).O. In addition. In the assessment of the oxidation state in sediments (collected into aquatic sediments) and deep ground-water samples (collected deeper into the surface) the D. The paramount importance of the dissolved Oxygen (D. dissolved oxygen determinations establish the suitability of oxygen transfer systems to aerobic suspended culture operations such as activated sludge. At low pHs. The larval stages of certain coldwater fishes are quite sensitive to the oxygen concentration. (d) is used in the assessment of the strength of a wastewater through either the Biochemical Oxygen Demand (BOD) or respirometric studies. As it is required for the survival of aquatic life it is considered an important water quality parameter for natural aquatic systems. It may also be used to indicate the suitability for the growth of such sensitive organisms such as the nitrifying bacteria. Commonly.Oxygen is a rather insoluble gas. mainly due to the microbial-mediated oxidation of the waste upon discharge of organic compounds. The difference between both methods is that the Winkler method measures dissolved oxygen concentration where the potentiometric electrode method measures the activity.O. Eventually the concentration level is low enough to allow the anaerobic process Thinking on biological treatment processes and in a general point of view. often drops.

The reaction with dichromate is not as quick as iodate and in addition the green coloration from Cr (III) may interfere with the endpoint.+ 3 SO42I3. the alkali azide iodide reagent also contains sodium azide.→ S4O62.+ 6H2O Cr2O7-2 + 9I.→ 2 Mn2+ + I3. Thiosulfate is not a primary standard reagent due to an ill-defined number of hydration water molecules. high pH will retard downward pH drift from absorption of atmospheric carbon dioxide and minimize the conversion of thiosulfate into sulfate and elemental sulfur. NaN3. The azide reacts with it to release nitrogen gas and nitrous oxide. O is obviously very sensitive to sample contact with air.+ 3I. it should be limited to avoid relevant increases in D.2 Mn (OH)3 + 3 H2SO4 → Mn2(SO4) + 6 H2O Mn2(SO4)3 + 3 I.+ 12H+ → 6I3. Sampling This parameter D.+ H+ → N2 + N2O + H2O The nitrite interferes by raising the apparent concentration of dissolved oxygen according to the following reaction. its solution should be necessarily standardized against potassium dichromate or potassium iodate.+ 14H+ → 2Cr+3 + 3I3. Thiosulfate solutions should be preserved from the growth of sulfur bacteria (oxidize the reagent to sulfate) by adding a small amount of NaOH. NaN3 + H+ → HN3 + Na+ HN3 + NO2. sulfite. 2IO3. to an excess of iodide is added a known amount of the primary standard. 2NO2. In addition. C.+ 7H2O These oxidants treated with an excess of iodide release stoichiometric amounts of tri-iodide.O. thiosulfate and aldehydes. which then reacts with the thiosulfate. Specialized sampling techniques 118 .+ 4H+ → I3. neither of which interferes. 2 NO + 1/2O2 + H2O → 2NO2.+ 2 S2O32.+ 2H+ Other interferences include reducing agents as ferrous iron. In either case.+ 3 ITo eliminate the interferences from nitrite.+2 NO + 2H2O Note that the produced nitric oxide can then scavenge any oxygen introduced during titration to produce nitrite and start the cycle.+ 16I.

The alkali-iodide-azide reagent is prepared by dissolving 500 g NaOH and 150 g KI in water. This solution should be standardized with Potassium Iodate or Potassium Dichromate with the aid of starch for a clear endpoint. Analytical Procedure Procedure: To a 300 mL BOD bottle filled with sample add 1 mL of manganous sulfate solution and 1 mL of the mixture alkali-iodide-azide: Cap avoiding the trapping of any air bubble. Titrate (200 mL of sample) with the 0.2H2O in pure water and dilute to 1 liter.are required to minimize the close contact atmospheric air-water sample. then shake.2g of salicylic acid (recommended as solution preservative) in 100 mL of hot pure water. Allow the precipitate to settle to about half the height of the bottle and add 1 mL of concentrated sulfuric acid. add some few drops of the starch solution and continues the titration until the deep blue color disappears. add a small amount of distilled water and re-cap. COD The chemical oxygen demand is expressed as the amount of potassium dichromate reduced by the sample during 2 h of reflux in a medium of boiling. (a) (b) (c) (a) (b) (c) (d) Chemical Oxygen Demand. then level to 1 liter. Specialized samplers for this parameter are commercially available. containing 5O% H2SO4 and Ag2SO4 as catalyst.5H2O in water and add 0. The titration proceeds until a very pale yellow color. stop the titration. Reagents: Dissolve 400 g MnSO4. then add 10 g NaN3 in 40 mL water and dilute to 1 liter 0.4 g NaOH. The starch solution is prepared by dissolving 2g of soluble starch and 0. The reaction between dichromate (add an excess) and organic matter is as follows: CxHyOn + R Cr2O72-→ x CO2 + 2R Cr3+ + [7R+n-2x] H2O (a) reduction of dichromate: 6e.33 volts 119 . If any air bubble has been trapped.205 g of Na 2S2O3.+ 14H+ + Cr2O7-2 → 2Cr3+ + 7H2O Eo = 1.Dissolve 6. D.025 mol L -1 sodium thiosulfate solution without reaching the colorless end-point. Re-cap and mix.025 mol L-1 Sodium Thiosulfate . A white turbidity remains.

Analytical Chemistry. pp. Flaschka. Sci.H. C.edu/cee/reckhow/courses/572/572bk16/572BK16.V.10-phenanthroline. 168-188. Jenkins.N. Minear & L. 24-29. D. L. Wiley. 3. 8. (1982) "Alkalinity and Acidity".A. McGRAW Hill. 2003. McCarty (1978) Chemistry for Environmental Engineering. (1983) Environ.A. G. pp. 343-376. 4.85135.77 volts The endpoint of this titration of dichromate can be determined colorimetrically by using 1. New York. 6. APHA. 2000. Fe+2 → Fe+3 + eEo = -0. Schwarzenbach.). EDTA Titrations. Chapter 3 in Water Analysis: Volume 1.L. Inorganic Species.http://people. the correction is established through a blank with pure water. Christian. G.(b) after oxidation of the organic matter is complete. and P. & Technol. Washington (16th ed. Sawyer.tau. APHA. Pergamon Press. R.ac. 7. Volumetric Methods 120 . 3rd Edition. ISBN 0-07237547.edu/fs/r. McQuaker et al. Flaschka (1969) Complexometric Titrations.http://www. Also it can be precipitated as AgCl The addition of HgCl2 serves to remove the free chlorine.. Keith editors.umass. 17:431-435.R.htm Titrimetric methods of analysis 12. The presence of organic impurities can be oxidized by Cr2O7-2 resulting in an excess of Cr 3+. A. G & H. REFERENCES 1.. Academic Press. Modern Analytical Chemistry. McGraw-Hill Publ. 9. 11. http://www. and WPCF. New York. J. 5. 1959.ecs.il/~advanal/TitrimetricMethodsOfAnalysis. Snoeyink and D. 1980. Harvey. the presence of Ag 2SO4 (used as catalysts) avoids this precise oxidation and the chloride is subjected to an unpredictable oxidation procedure.morehead-st.+ 14H+ + -2 Cr2O7 → 3Cl2 + 2Cr3+ + 7H2O). Standard Methods for the Examination of Water and Wastewater. 10. H. The chloride ion interferes by being oxidized to chlorine (6Cl . London. Water Chemistry.doc. the excess dichromate is titrated by the reducing agent Fe+2. Kramer.html 2. Methuen. AWWA. At the endpoint the color changes from blue-green to reddish brown (Fe(II) -phenanthroline). which forms a colored complex with the resulting Fe (II) ions.hunt/360-04vol. Wiley.

An electromagnetic wave. Only electric component is active in ordinary energy transfer interactions with matter.4.3. Electric Vector Wavelength Electric Component Amplitude Direction of Propagation Magnetic Component Magnetic Vector Figure II. ν. The wavelength and frequency are related to the velocity of light by expression: 121 . The units of frequency are cycles per second or sec -1. Some phenomena such as: refraction.4. wavelength. On the other hand photoelectric effect suggests particle properties of the electromagnetic radiation.4. λ.3. UV-VIS SPECTROMETRY Andrei Florin DĂNEŢ II. The “particle-wave” duality explains the behavior and the nature of electromagnetic radiation. In Figure II.4.4 an electromagnetic wave has an electric component and a magnetic component oscillating in planes perpendicular to each other. Nature of Electromagnetic Radiation Electromagnetic radiation is a form of radiant energy which exhibits both wave and particle properties. As indicated in Figure II.4.3. is the distance between two corresponding point on the wave.1. Wave properties.4. Another important property of an electromagnetic wave is its frequency. reflection and rotation of plane-polarized light are examples of wave properties.II.

The wavelength of the maximum of “the broad band” correspond to the electronic transition and the width of it to the superimposed vibrational and rotational transitions 122 .2) where E = the energy of photon in ergs.5). At longer wavelengths than visible light (about 800 nm) there is the infrared (IR) region.4. Molecular Absorption of Electromagnetic Radiation The total energy state of a molecule includes electronic. Thus for each electronic level of a molecule. The 10200 nm region is the vacuum ultraviolet region and need special instruments for measurements. Therefore. The absorption of UV or visible radiation generates a transition between electronic levels of the molecule. the energy (the photon) can be absorbed. The ultraviolet and visible part of the spectrum is a very small part of the total range of possible (and detectable) frequencies of electromagnetic radiation (see Figure II.4. Particle properties. The energy of each photon is proportional to the frequency of the radiation and is given by the relationship: E = hν = hc/nλ (II. Photons cannot lose just part of their energy through normal absorption.3. The UV region is most useful for analytical purpose is 200-400 nm. The individual photon energy is the basis for the phenomenon of light absorption. To describe how electromagnetic radiation interacts with matter.λν = c/n (II.sec. ν = the frequency of the electromagnetic radiation in cycles per second and h = Planck’s constant. there are also superimposed vibrational and rotational states as illustrated in Figure II. The photon energy must be equal to an allowed energy transition in the absorbing species.4.4.624 x 10-27 erg. for an electronic transition we observe a “broad band” absorption spectrum and that is typical of most absorptions of ultraviolet and visible radiation. a light beam is considered as a train of photons. vibrational and rotational components.6. 6. The energy difference between molecular electronic levels is much greater than that between vibrational states. II. All of these energy components are quantified.2.4. When the photon energy matches an allowed energy transition within the material through which the photon is passing. The entire range of radiation is commonly referred to as the electromagnetic spectrum. and at frequencies higher than blue light there is the ultraviolet (UV) region (10-400 nm).1) where c = the velocity of light in a vacuum and n is the refractive index.

V3 E1 V2 V1 V0 Energy V3 V2 E0 V1 V0 r3 r2 r1 r0 r0 to r3 are rotational energy levels ∆E1 ∆E2 ∆E3 E0 and E1 are electronic energy levels with E0 = ground state electronic level V0 to V3 are vibrational energy levels with V0 = lowest vibrational level in given electronic level Figure II.5. UV-Vis domain of the spectrum. vibrational and rotational levels.4. 123 . Hz 3x10 16 3x10 15 3 3x10 3x10 3x10 3x10 14 4 13 5 12 6 11 Figure II. Schematic representation of molecular electronic.4.Electromagnetic radiation γ rays X rays Ultraviolet Visible λ.6. nm 10 10 10 Infrared 10 10 10 Microwaves Radiowaves 2 ν.

The loss in radiation intensity. which normally consists of the solvent plus sample constituents other than the principal absorbing species. is directly proportional to radiation intensity in that point: .. the transmitted intensity of the radiation ( It) represents the incident intensity of the radiation minus that lost by scattering. Radiation absorption process.3) where k is a proportionality constant. -dI represents the decrease in radiation intensity in an infinitesimally small layer. a beam of radiation is directed at a sample and the intensity of the radiation that is transmitted is measured. With this “blank” solution in the cell.7. Referring to figure II. reflection and any absorption by the other constituents (normally quite small). absorption may occur. dl. gives the loss in radiation intensity due to absorption by the sample. Using the differential notation of calculus. the negative sign is introduced because the radiation intensity decrease as dl increases. Integrating the equation II.4.3. i. I (I – dl) I0 dl l It Figure II.4.4.dI = k I dl (II.3 over the entire cell length.4.3. Quantitative Law of Radiation Absorption In quantitative absorption studies. l. Let us consider a monochromatic radiation. Consider the changes in radiation intensity that occurs as monochromatic radiation passes through the absorption cell in Figure II. the amount of radiation absorbed in this layer.e. let us consider what happens to the radiation as it passes through the sample.7. 124 . We first fill the cell with a “blank” solution.7. If the photons that strike the sample posses an energy equal to that required to cause a quantized energy change.4. We denote this radiation intensity as I0.II.4. -dI.

) A is the absorbance. ln I0/It = kl.4.It I0 ∫ dI = −k I ∫dl 0 1 (II. that is defined as the molar absorptivity (commonly called the molar absorption coefficient).4) Solving. so we can write: k’ = ac (II.7) where a is denoted the specific absorptivity (the concentration is given in grams/litre).4. The value of ε is characteristic of the absorbing molecule or ion in a particular solvent and at a particular wavelength.4. Beer (a German physicist) established that if the radiation absorption is due to a dissolved specie.4.4.4343 k. Therefore from equation (II. the proportionality constant k ’ is dependent of that concentration (c).6) It = I0e-kl (II. If the concentration is given in moles/litre a is replaced by ε. The percent transmittance is defined as 100 x T. The value of ε is independent of concentration and the path length of the radiation.8) Equation (II.5) where k’ = 0.6) we obtain: A = log I0/I = εcl.8): log T = . If we replace k' with εc in equation (II. In equation (II. That is the Lambert law.9) 125 .4. I = I010-εcl (II.4. which is the fraction of the incident radiation that is transmitted by the sample.8) has been referred to as the Lambert-Beer law. The term I/I0 is defined as the transmittance (symbol T). Very often the transmitted radiation intensity is denoted I (without subscript t) denomination that we shall use in what follows.4.εcl or -log T = εcl = A (II.4. Converting from natural logarithms to base 10 logarithms (designated by log) we obtain: log I0/I = k' l or I = I010-k'l (II.4.8.

Multiple Component Systems.8. it is assumed that the species act independently of one another and that their absorbances are additives. if there are n components.8 0. takes the form: Aλ = ∑ A λ = l ∑ε λ c n n n n n (II.0 0. The relationships between absorbance.6 0.4 0.10) In principle. n absorbance measurements at n different wavelength are required to determine the concentration of n components in a mixture. Absorbance and transmittance vs. concentration at a given wavelength and cell pathlength.4. the total absorbance expression at any wavelength. (2) the absorbing species act independently of each other in the absorption process and (3) the absorption occurs in a volume of uniform cross section. In general.2 40 20 Concentration Figure II. T% 100 80 60 A 1 1. This provides n independent simultaneous equations in n unknowns. 126 .In the derivation of the Lambert-Beer law it is assumed that: (1) the incident radiation is monochromatic.4. transmittance and concentration at a given wavelength are illustrated graphically in Figure II.8.4. When systems that contain more than one absorbing component are studied. λ.

the absorbance measured is an “average” absorbance over the band. at the absorption maximum the change in absorbance with concentration is at a maximum.4. True deviations from Lambert-Beer’s law occur only in systems where the concentration of the absorbing species is so high that the index of refraction for the absorbed radiation is changed. this yields greater sensitivity and higher accuracy. Second. The best wavelength for quantitative analysis is λ1 for two reasons. however. 127 . Such behavior is called a “deviation from Lambert-Beer’s law”.9 demonstrates that the shape of the calibration curve often depends on the bandwidth. Figure II. Unless the molar absorptivity is invariant within the wavelength band used. absorbance varies in a non-linear way with respect to concentration. Another instrumental cause of deviation is the necessity of working with a band of wavelengths rather than truly monochromatic radiation. Apparent deviations from Lambert-Beer’s law may be due both to limitations of instrumentation and to effect of non-symmetrical chemical equilibrium. For some systems. within this band the molar absorptivity is relatively constant and a linear calibration curve is obtained as in Figure II. First. The greater the slope of the absorption curve through the wavelength band is. the greater the deviation. Instrumentation Limitations Indeterminate instrumental variations which cause apparent deviations include: (1) stray radiation reaching the detector (reflected within the instrument). (2) sensitivity changes in the detector. a curved calibration plot may be prepared with samples of different concentrations.9. Due to the logarithmic nature of absorbance. In order to treat these systems. Two wavelength bands of equal width are designated λ 1 and λ2. this is not a true average.Deviations from Lambert-Beer’s Law Many absorbing systems in dilute solution follow Lambert-Beer’s law rather closely. and (3) power fluctuations of radiation source and the detector amplification system.4. Double beam operation tends to cancel out most of the random causes of deviation.

includes both forms: CHA = (HA) + (A-).is non-absorbing at λ1. Effects of finite bandwidth.9. The analytical concentration.λ1 C λ2 A C/2 dλ λ→ (a) dλ A at λ1 A at λ2 Concentration (b) Concentration (c) Figure II. HA is involved in the equilibrium: HA + H2O = H3 O+ + AFor which we have the acidity constant Ka. But this ratio 128 . (b) linear curve at λ1. Lambert-Beer’s law is valid when using either (HA) or C HA. An example of such system is presented below.. Let us consider an aqueous solution of a weak acid HA which has an absorption maximum at λ1. CHA. As long as the ratio (HA)/CHA remains constant. (c) Non-linear calibration curve at λ2. The anion of the acid A. Non-Symmetrical Chemical Equilibrium An absorbing species that is involved in non-symmetrical chemical equilibrium may exhibit “apparent deviations” from Beer’s law.4. (a) One component at two concentrations.

Such a system will show an apparent deviation from Lambert-Beer’s law if CHA is used as the concentration.4. rapid. the acid exists primarily as HA. This is the principle of photometric. above 350 nm. In the case of colorimetric analysis the measurements were carried out with white light without any optical instrument. Visual comparison of the sample color with that of reference solution of known concentration was performed. and there is no problem. spectrophotometric or colorimetric analysis.3. C HA. the fraction dissociated does not vary with total concentration and absorbance is proportional to CHA. Light Sources Two light sources are commonly used in the UV-VIS domain: an incandescent lamp made from a tungsten filament housed in a glass envelope is used for the visible portion of the spectrum. The first two methods of analysis use more or less narrow spectral bands obtained with filters or monochromators.5. At a constant pH in the intermediate region (buffered solution). a medium pressure deuterium arc lamp is used for the ultraviolet portion of the spectrum. II. it becomes possible to quantify a chemical species that has no significant absorption.3. In figure II. However. reproducible and has to yield a UV-VIS absorbing derivative that is stable in solution. Instrumentation for UV-VIS Spectrometry A UV-VIS spectrophotometer consists of three main components: the source.4.4. II. Measurements are based on the Lambert-Beer law. If (H3O+) >> Ka. The sample can be placed in the optical path before or after the dispersive system and the recorded spectra can be treated using a number of different computer algorithms. The derivatization (a chemical transformation) that has to be specific. in un-buffered solutions the fraction dissociated changes with pH. It is not necessary that the compound contain a chromophore as long as derivatization is carried out before measurement to ensure absorption of the light. the dispersive system (combined in a monochromator) and a detector. If + (H3 O ) << Ka the acid is highly dissociated with a correspondingly small absorbance.depends on the pH of the solution: fraction un-dissociated = (HA)/ C HA = (HA) / [(HA) + (A-)] = (HA) / {[(HA) + Ka(HA)]/(H3O+)} = 1/[1 + Ka/(H3O+)].4. total.10 are presented three mechanisms for transmitting illumination through a sample solution. 129 . which in turn is a function of total concentration. Quantitative Analysis in the UV-VIS The spectral domain of the UV/VIS is well known because it includes the visible part of the spectrum and is widely used in quantitative analysis. Through derivatization.

For simple devices as dispersive system is used a wideband. The detector measures the light signal at a given wavelength. which are integrated into an assembly called a monochromator. Light emitted by the source is dispersed by prisms or gratings.4. Three common methods of transmitting the probe illumination through a sample solution. Single Beam Spectrophotometers Many routine measurements are conducted at fixed wavelengths using simple photocolorimeters equipped with wideband. Sample Window Lens To detector Light source Light source Transparent sample container Lens Optical fibers Waste Lens Window To detector Light source Thickness traversed Sample container Figure II. An 130 .10. The monochromator extracts a narrow spectral band of the spectrum.Dispersive system. It converts the light intensity selected by the monochromator exit into an electrical signal. interchangeable color filter. interchangeable color filters.g. Detectors Two types of detectors exist: photomultiplier tubes and semiconductors (e. silicon photodiodes and charge transfer devices (CCD/CID)).

analytical blank (containing the solvent and reagents for the analysis.L. Two rotating mirrors. 131 .4. New York. John Wiley and Sons Inc. Double Beam Optical Spectrophotometers (Scanning Type) In Figure II.. Christian. and then is replaced by the solution to be analyzed. One of the beams passes through the sample while the other passes through the reference.11. Gary D. 2004. Pecsok and L. Shields. 1968. Analytical chemistry (6th edition). New York. Double-beam instrument employing two choppers. allow the comparison of transmitted light at the detector of the two beams with the same wavelength. London. which are synchronized with the displacement of the grating. Wiley. R.11 is presented the scheme of a double beam spectrophotometer. Blank Mirror Mirror Rotating mirror (detail) Sample Monochromator Lamp Chopper 1 Chopper 2 Detector Amplifier Display/ Printer ADC Computer Figure II. Amplification of the modulated signal allows the elimination of the stray light. without the sample to be measured) is first placed in the optical path. They are preferable to single beam instruments for measurements in problematic solutions.D. 2. Double beam spectrophotometers allow differential measurements to be made between the sample and the analytical blank.4. Modern Methods of Chemical Analysis. REFERENCES 1. called choppers.

Strobel.M. M. M. Heineman. Dăneţ. 9. perhaps the oldest instrumental techniques now widely used.. Rouessac. Because the transitions are quantized.3. liberate the elements and transform them into a gaseous atomic state. and Rouessac. New York. Merritt. H. 1995.4. 8. New York. 9. metalloids and non-metals) in environmental samples. 2000. Van Nostrand Company. 2000. J. Analytical Chemistry. Chemical Analysis. Vol. 1995. London. only electronic transitions can occur when energy is absorbed.R. H. F. 7. Bucureşti. 5297-5353. Wiley-VCH. Metode Instrumentale de Analiză Chimică. ed.. The sample solution is heated in the instrument to a temperature of between 2000 or 3000 degrees Celsius to break chemical bonds. Oxford University Press. the total concentration of the element is measured without distinguishing the chemical structures present in the cold sample. ATOMIC ABSORPTION AND EMISSION Mihaela Carmen CHEREGI II.A. W. 132 .1. atomic absorption spectrometry (AAS). 1998.Lynne and J. M.. 1981. Widmer Eds. A.. F. pp. 4.4. 5th Ed. L. Modern Instrumentation Methods and Techniques.. Since atoms cannot rotate or vibrate as molecules do. Dean. There are various ways to obtain free atoms and to measure the absorption or emission of radiation by these. Kellner. Instrumental Methods of Analysis. II. Introduction Atomic absorption and emission spectroscopy. Academic Press. line spectra are observed. 2nd Edition. Ltd. Spectrophotometry and spectrofluorimetry. Editura Ştiinţifică. in which the amount of the radiation absorbed by ground state atoms in a flame or in a small electrical oven (graphite furnace) is measured. John Wiley and Sons. Encyclopedia of Analytical Science. 3rd Ed. Mermet. are two methods of quantitative analysis that can be used to measure approximately 70 elements (metals.A. Oxford. H. 5. The principle behind these methods of elemental analysis depends on measurements made on an analyte that is transformed into free atoms. 6. Otto. H.4. Gore. New York. This chapter deals with the spectroscopy of atoms. Willard.4. Weinheim. Thus. A. Chicester. Chemical Instrumentation: A Systematic Approach. R. 1989. The principal techniques described in this chapter are: a. Wiley.

aspiration of sample into the flame (or graphite furnace). extraction of the quantitative information from the registered signal. and concentrations in the mg/L (ppm) range or lower can be accessed.4. in which atoms are excited in a flame. Many models of instruments allow measurements to be conducted by these techniques. inductively coupled plasma. conversion of the sample into free atoms and the gaseous atomic state formation (atomic cloud). AAS is identical in principle to absorption spectrophotometry described in a previous chapter and the Lambert-Beer’s law is followed in this technique. The instrument yields the absorbance by rationing the transmitted intensities in the presence (I) and absence (I0) of the sample: A = . 4. the cathode made from the same element as that being determined. spark. 2. The main steps in AAS measurements are: 1. 5.logT = log(I0/I) 133 . II. too: A=k⋅C where A is the absorbance . The absorption depends on the number of ground state atoms of atomic cloud in the flame and on the path length in the flame. atomic emission spectrometry (AES). the wavelengths of radiation given off by the source are the same as those absorbed by the atoms in the flame. electrical arc. Both of these variables are difficult to determine. 3.b. Therefore. which rely on different principles. The monochromatic light is given off by hallow cathode lamp.2. C is the concentration of the element and k is a coefficient unique to each element at a given wavelength. and the radiation intensity emitted by a fraction of excited atoms that return to their ground-state is measured.4. Atomic Absorption Spectrometry Principles The law that governs the absorption of light by the atoms is Kirchoff’s law and it states that incandescent gases can absorb the same radiation that they can emit in certain conditions. measurement of the transmitted radiation intensity. but the path length can be held constant and the concentration of the atomic cloud is directly proportional to the concentration of the analyte in the solution being aspirated. absorption of the monochromatic radiation by the ground state atoms from the atomic cloud.

The tube is filled with an inert gas (argon or neon) at a reduced pressure. The beam-light emitted by the source ( I0). A higher voltage (300V) is applied between the electrodes. the basic components for AAS are a light source. The monochromator’s role is to select a very narrow band of wavelengths and to eliminate extraneous light resulted from the atomization device. Sources The key element in AAS is the source. Instrumentation Similarly to absorption spectrophotometry. a cell (the flame). The procedure used involves classical protocols: prepare the calibration curve or standard additions. located after the atomization device. I0 is the full intensity of the beam-light emitted by the source and perceived by the detector in the absence of the sample. and I is the attenuated intensity of the beam-light perceived by the detector when the sample is present. it was consider that the line bandwidth emitted by the source is equal to the line bandwidth absorbed by the atoms. Inside of the lamp. The detector measures the ratio I0/I and the logarithm of the ratio is displayed. forming a cloud of atoms. In order to simplify the graph. a monochromator and a detector.4. as long as the range of concentrations stays within the linear conditions of absorbance. depending on the wavelengths emitted by the cathode (e. HCL is a glass tube with a borosilicate or quartz window.Measurements are made by comparing the unknown to standard solutions.g.13. The various components of an atomic absorption spectrometer are described as follows. A. The optical scheme and the working principle of an atomic absorption spectrometer are presented in Figure II. which must emit a sharp-line because the width of the absorption line is very narrow. a cylindrical hollow cathode made of the element to be determined (a cathode of lead is used for lead determination) and a tungsten or zirconium anode are enclosed. most metallic compounds are decomposed and the metal is reduced to the elemental state.12. A sharp-line source that emits monochromatic and specific wavelengths and used almost exclusively in AAS is the hollow cathode lamp (HCL). passes through the atomization device (flame or graphite furnace). The positive ions are accelerated toward the negative cathode 134 . The atoms absorb a part of I0 that is proportional to the analyte concentration in the sample. around 10 -3 nm. quartz is used for lines in the UV region). causing the inert gas atoms to be ionized at the anode. The attenuated beam-light (I) is then focused on the entrance slit of the monochromator.4. In the atomization device. The main disadvantage of making measurements in AAS is that a different source is required for each element. The optical path ends at the entrance slit of the detector (photomultiplier tube). which must be a very narrow band characteristic of the analyte metal. The diagrams of a HCL and of the excitation of the cathode atoms are presented in Figure II.

the characteristic emission spectrum of elements is obtained. which form.and some of them possess enough energy to strip atoms from the cathode. 135 . (a) (b) Figure II. at the surface. When the excited cathode atoms return to their ground state.12. M(G)* is the element in its excite atomic state and M(G) is the element in its atomic state. Schematic diagrams of an atomic absorption instrument (a) and of its working principle (b). an atomic gas. The emission steps may be represented as follows: Ne M (C )    →M (G ) * →M (G ) + hν + where M(C) is the element in its metallic state in cathode. The atomic gas is excited to higher electronic level by collisions with the high-energy inert gas atoms.4.

13. (a) Schematic diagram of HCL. The flame is a mixture of a fuel and an oxidant gas and it has a rectangular base of about 10 cm by 1 mm. due to the selective volatilization of one of the elements form the cathode and its condensation on the walls of the lamp. and then classical lamps using metallic vapor are used instead. HCLs cannot be used for mercury and sodium (their boiling points are too low). may be preferred. Se or P. These lines are passed through the atomization device. The width of the emitted lines of the cathode-excited atoms depends on the Doppler. Hg. a brighter source such as microwave-excited or electrodeless discharge lamp composed of the element to be measured.Screen Atomic vapor Metal + Cathode Anode - Ne * Quartz window - Ne Ne + Ne Ne + Excited metal atom Ne Ne Ne Electron Inert gas (Ne. Ar) hν (a) (b) Figure II. Electrode-less lamps with very intense emission use a radio-frequency emitter to excite the metallic vapor and are especially used for elements such as As. For certain sample types multi-element hallow cathode lamps are attractive. and certain ones are absorbed by the analyte atoms because they possess the right energy to result in the discrete electronic transitions. the cathode is an alloy of several elements and the lines of all the elements are emitted. Stark (ionization) and Lorentz (pressure) effects but even then it is narrower than the corresponding absorption band of the sample atoms. It is aligned with the optical axis of the instrument. In some instances. the entire source line-width is absorbed. There are more then 100 types of HCLs made in pure elements.4. The sample solution is aspirated by the nebulizer and is introduced in the flame as a fine spray. The most strongly absorbed line is the one corresponding to the electronic transition from the ground state to the first excited state. In the flame. B. known as the resonance line. Devices for sample atomization Flame atomization. (b) Diagram of cathode atoms excitation under impact with inert gas ions. and therefore. They may exhibit shorter lifetimes than the single-element lamps. the analyte is transformed in an atomic cloud 136 .

) was the second development in the sample module for AAS. It is a flameless device without nebulization and consists of a hollow graphite rod or cup that can hold a precise volume/quantity of the sample (a few µL or mg deposited with an automated syringe).4. This rod of 8 mm inner diameter and 40 mm length is oriented parallel to the 137 . For the lowest possible detection limit and greater sensitivity the electrothermal or resistive furnace atomizer (Figure II. The most popular flames in AAS are acetylene–air and acetylene–N 2O (for refractory elements). Free radicals present in the flame have absorption and emission spectra in the near UV and they can interfere with the measurement of the element. Collisions with high-energy flame gas atoms are principally responsible for atomization. ions reduction to gaseous atoms.14. It is used for arsenic and selenium when they are separated from the sample by volatilization as their hydrides (AsH3. Recommended flame type and their temperature for analytical determinations by flame AAS. Not all the parts of a flame are valuable for atomization or for observation of the atomic cloud spectrometrically. In Table II. Thus. dissociation of the salt into free ions. For sensitive measurements atomization should be as complete as possible.4. the hotter the flame.4. Table II.following the processes: solvent evaporation. Combustible mixture H2 – air H2 – O2 CH4 – O2 C2H2 – air C3H8 – air C3H8 – O2 C4H10 – air C2H2 – O2 C2H2 – N2O H2 – Ar – entrained air Tmax (° K) 2300 2950 2950 2450 1998 3173 2200 3400 3200 1850 Flames must also be stable and something must be known about the background levels of chemical species derived from the flame gases as well as those derived from the sample. Electrothermal atomization. H2Se) and passage of these gases into the flame.2. Thus. the observation flame height must be adjusted for some elements.2 are listed some analytically useful flames and their temperature. The hydrogen– argon–entrained air flame is preferred for wavelengths below 200 nm where the acetylene–air flame absorbs a large fraction of the radiation. the more effective is the process.

therefore the detection limits are often 100 to 1000 times improved over those of flame aspiration. A coil-shaped tungsten filament (W-coil) serves for low-cost.optical axis and it is surrounded by a double sleeve containing an inert gas to protect it from oxidation and allow circulating water to cool the device. and then the rod is heated resistively by passing an electrical current. electrically conducting substances can also be adapted as atomizers. Transverse hole for introducing sample Hollow graphite rod Inert gas (Ar) Sample HCL beam-light Cooling water To power source Figure II. nickel.14. the sample is dried at a low temperature for a few seconds (~100 to 200 °C) 2. the smoke from pyrolysis in flushed out by flowing argon gas. To avoid splashing.4. Certain metals such as molybdenum. A small volume of sample (1–5 µL) is placed in the graphite rod through a hole in the top. pyrolysis at 500 to 1400 °C to destroy organic matter that produces smoke and scatters the light source during measurement. the absorption of the metal atoms in the hollow portion of the rod is measured and a sharp peak of absorbance versus time is recorded as the light path passes through the atomic cloud. compact and 138 . many metals being determined at concentrations of 1 µg/L. vanadium. and finally the sample is rapidly thermally atomized at a high temperature up to 3500 °C. The heating is done in an inert atmosphere to prevent oxidation of the graphite at high temperature and also to prevent the refractory metal oxides.1 % from flame atomization. Other high melting. 3. and barium react with the graphite at high temperature forming carbides and to prevent this pyrolytically coated tubes are used. Electrothermal atomizers have a conversion efficiency of 100 % compared to 0. the temperature is gradually increased according to a three-step cycle: 1. Schematic diagram of electrothermal atomizer.

the bound mercury is reduced by a reducing agent such as tin chloride (SnCl2) in acid solution. decomposed by heat. and enchanting the 139 . Hollow cathode lamps are available for all and microwave electrode-less lamps for many. For hydride preparation sodium boronhydride is added to an acidified sample. Liquids with high salt content offer problems when the solution is evaporated. arsenic. will react with the reducing agent in a separate vessel to form the volatile hydride.portable instrumentation for environmental and clinical analyses. bismuth. precision can be better then ±1 %. the hydride is carried into a quartz cell placed in the flame by a further flushing. while the linear dynamic ranges of concentrations remain unaffected. Because chemical atomization is a selective reaction. Sample preparation. the detection limits are even better than or within a factor of 10 of those of graphite furnace. For instance. Other distinctive modes of atomization are the conversion of the compounds of metalloids and many low-activity metals to hydrides. and requires specialized instruments. The elemental mercury is formed instead of hydride and it may be volatilized easily by bubbling an inert gas through the solution and passing the gas through a special cell. Quantitative Determinations in Atomic Absorption Spectrometry AAS is widely applicable as a generally sensitive technique for quantitative determination of elements (about 70). The generator vessel must be flushed with Ar or N2 to remove oxygen. antimony or selenium. which are difficult to reduce in a flame when they are in higher oxidation states. tin. A way to remove these difficulties may be the complexation of the analyte in the sample solution and then the extraction of the complexed species. these metalloids and low-activity metals are also concentrated by removal from the original matrix. and the interfering substances masking. This is called “cold vapor” method. Encrustation of salts around the slot of the burner leads to unsteadiness of the sample flow and of fuel and oxidant gases and introduces serious errors. with a strong oxidizing agent. which does not need to be put into the flame. The determination of these elements in the presence of considerable amounts of organic matter often requires the organic material digestion. For over 20 elements that have been determined using W-coil AAS. AAS is capable of a precision of ± 2 % and when a double-beam procedure and a background correction are employed. High heating temperatures up to 3000 °C can be achieved by using a simple power supply such as a car battery. and measured by atomic absorption. Chemical vaporization. lead. In the majority of cases samples are present in solution and for samples that requires dissolution. When hydride formation is complete. For mercury. eliminating the interferences. acid digestion or solvent extraction may be the first step of analysis. In electrothermal atomization salts rise the smoke and high background correction. The extraction also offers the advantage of concentrating the analyte.

Atomic Emission Spectrometry Principles The main steps in AES measurement are: 1.4. The linear curve obtained is extended through the concentration axis and the distance from the point of its intersection to the origin is equal to the concentration of the analyte in the sample. In spectrophotometric measurements the precision is best when the absorbance varies in the range of 0. 4. 8-hydroxyquinoline with ethyl acetate or butanol and ethylenediaminotetraacetic acid. the sample solution is introduced into the excitation device as a fine spray. the use of a less sensitive line of the element. a plot of absorbance versus concentration for each analyte. 3. at least three different concentrations. The salt is dissociated into free gaseous atoms in the ground state and then a certain fraction of these atoms can be raised to an excited electronic state. Analytical signal readings are quantified by reference to a working curve.4. Many metals. Analyte determination using standard addition method is attractive only if the concentration of the analyte in the sample is very low. When the concentration range of an element in a sample exceeds the optimum absorbance values.15 to 1. Measurement of the emission wavelengths and intensities. the dilution of the sample.3.sensitivity of the measurements if an organic extractant is used. II. where the solvent evaporates leaving the dehydrated salt.0 units. to equal volumes of blank solution and sample aliquots. especially in the case of organic solvents. Excitation of the atoms and their ions to higher electronic states. Ultra-pure reagents and solvents should be used. These excited atoms return to their ground state emitting photons 140 . Standard addition method. respectively. Some common complexing agents for metals and solvents are: ammonium pyrrolidine dithiocarbamate used with methyl isobutyl ketone. several options are open: the variation of the optical path length through the flame by a rotating burner. Conversion of a sample to free-atom gas. The standard solutions must be prepared to be nearly identical in composition with the samples as possible in order to minimize the errors. The absorbance for all these solutions is measured and then it is plotted versus concentration. 2. by the effect of high temperature. the absorbance range can be extended with a good precision. Extraction of qualitative and quantitative information from these signals. Basically. Calibration curves and working conditions. The method involves spiking an equal volume of standard solutions. If an internal standard is used in a dual-channel instrument. AES is a well-established technique for determining inorganic constituents in various types of samples. metalloids and low-activity metals are most sensitively determined by chemical conversion to hydrides.

High-voltage arc and spark are used for solid samples and they are mostly used in semi-quantitative analysis in industry. Hence. thousands of different spectral lines can be observed. 5. A. The excitation sources must fulfill the following criteria: 1. the emission of radiation can occur from either excited or ionized atoms. Only the alkaline and alkaline earth metals are determined by flame emission spectrometry. a simple interference filter may be sufficed. in recent years prism spectrometers have become relatively rare. It should be noted that the next generation will be represented by the grating spectrometers. the instrument being programmed to identify and quantify the elements in a real sample. The monochromator can be replaced by a polychromator – a double module in which the exit slit of monochromator has been replaced by a multi-channel detector mounted in the focal plane. Nowadays. Some of these lines could be more intense than those of the analyte. The intensity of the emitted radiation is proportional to the concentration of analyte in the sample. The main excitation sources are flame. as there are fewer lines. one or several specific spectral lines are monitored for each analyte.or polychromator that represent the heart of the apparatus. Flame is a lower-energy source and the emission spectrum is much simpler. Excitation sources The excitation source is the most critical module because it must volatilize and atomize the sample as uniformly as possible since the concentration of atoms in the atomic cloud should be representative of that in a sample. inductively coupled plasma and glow discharge. ease of use. a high performance monochromator is required. 3. and a microcomputer that controls the instrument. Instrumentation The atomic emission spectrometer consists of three principal components: the device responsible for bringing the sample to a sufficient temperature. spectrometers able to solve many interferences and problems related to the matrix effects are used. 2. and. Flame photometric analysis is much simpler and it is not always necessary to have a high-resolution monochromator. the optics including a mono. which can be present at ultra-trace levels. 141 . reproducibility in sample introduction and energy transfer.(hν) of characteristic wavelength. high-voltage arc or spark. 4. In AES. for this reason. high energy flux. stability of excitation. It should also excite atoms. high sensitivity.

). the high temperature of plasma eliminates many chemical interferences present in flame because molecules of compounds formed completely dissociate. the temperature reaching 9000 to 10. the external tube having at its superior part two water-cooled copper tubes. At this high temperature. flows upward through the quartz tubes. The plasma is well suited for refractory elements (boron.15. the temperature increases considerable. the chemical interference in ICP-AES is very low. As this environment becomes more and more conductive. The plasma is an incandescent ionized gas (argon) heated inductively by radiofrequency energy at 4–50 MHz and 2–5 kW (Figure II. This induces a circulating eddy current in a gas. Argon gas.Inductively coupled plasma (ICP) emission spectrometry is used for rapid multi-element determinations in environmental samples. The plasma is isolated from the tubes by a gaseous sheath of flowing. ionizes and excites producing emission spectra. Different from AAS. 142 .000 K. non-ionized argon injected through an external tube concentric to the first one. phosphorus. etc. which in turn heats it. the sample atomizes. Also. which is ionized by a discharge of a Tesla coil or a pilot spark. cadmium). The copper tubes are connected to the radiofrequency generator creating a variable magnetic field in the flowing gas inside the coil.4.) and difficult to excited elements (zinc. uranium. The sample aerosol generated in the nebulizer and spay chamber is introduced into the ICP via a third tube with a diameter of 1–2 mm.

Wavelength scanning instruments (monochromator type) have the dispersive system movable in order to focus each wavelength on the fixed exit slit. Quantitative Determinations in Emission Absorption Spectrometry AES is described as a very sensitive (detection limits of few ppt) and rapid quantitative elemental analysis. each application may be considered as an individual case.5 mL of sample solution. minor (≈1 %). or stray light can occur that may alter the net signal intensity. This order-separating device allows simultaneous detection over the whole spectral range. Spectral interferences from ion–atom recombination. and trace (<0. Wavelength Separator This is the device that separates specific emission lines of the analyte using planar. 30 elements can be determined simultaneously. The dynamic range of emission methods is so large that concentration levels are identified by terms major (>10 %). each corresponding to an analytical line or measurement channel. The calibration curves are used for quantitative determinations.15. Their sensitivity and spectral response allows simultaneous measurements of lines. Certain instruments better tolerate dominating matrices such as soil or mud. Predefined elements. spectral line overlaps. These can be avoided by selecting alternate analytical wavelengths and making background corrections. characterized by precise spectral lines. Fe and Al. Several units of this type can be placed in series. multi-element analysis can be accomplished in 30 seconds consuming 0. molecular bad emission. in both the horizontal (due to the grating) and vertical (due to the prism) dimensions. constituting double or triple monochromators of high performance. A fixed optic arrangement using echelle grating in association with a focusing prism produces a double dispersion of the lines. The advantages of using an ICP include the possibility to identify and quantify all the elements excepting argon. working curves covering 5 decades of concentrations (1 ppb–100 ppm) and the detection limits are mostly in ppb range.Figure II. A well selected set of standards employed to obtain plots of intensity ( Ia) of the analytical lines versus concentration (C): 143 . However. are detected by as many photomultiplier tubes as there are secondary slits. There are also instruments with dispersion surfaces compatible with two-dimensional sensors. where there are high concentrations of elements such as Si. Schematic diagram of ICP used for AES. concave or echelle gratings. in concentration from ultra-trace levels to major components.01 %).4.

emission by analyte (AF) 2. lower flame temperature Ensure constant solvent composition. (EF less important for AF) Method of reducing error Use hotter or fuel-rich flame. A more correct equation for the working curve than may be: Ia – I b = m ⋅ C Accuracy and precision are dependent on the regulation of excitation conditions. Absorption and light scattering by molecular species in flame (AF) Ionization of analytes (AF. including the selection of an appropriate integration time.4. Many sources fluctuate and times of volatilization and excitation of elements will vary. EF) Emissionn or absorption of another atomic species or its oxide within waveband passed. a background correction Ib should be made if background is greater than the order of magnitude of acceptable error. In every case. and other methods. EF) Flame temperature change with solvent composition or high analyte concentration appears. Correct for background by deuterium arc.4. Table II. avoid certain anions. Physical and Chemical Interferences in AAS and AES The effectiveness of flame spectrometric methods for a particular element depends only in part on external variables such as fuel gas pressure and width of the burner slot. use solvent extraction or ion exchange to effect prior separation of offending ion. chelate the analyte 1. EF) 1. less important in AF). 2. Type of error Chemical (cation or anion interference) Background interference Cause of error Technique affected Analyte stable compounds formation that atomized with difficulty in flame (AF.Ia = m ⋅ C where m is the slope of the curve. modulate source emission for AF and selectively detect analytical signal. between 190 and 175 nm use separate flame with Ar shielding. reduce concentrations Ionization interference Excitation interference Spectral interference Use narrower slit or select another intense analytical line in a spectral region free of such interference 144 . It is also determined by processes called interferences or inter-element (matrix) effects. II. possible encrustation of solids on burner (AF. Sources of interference and error associated with flame atomization*. Zeeman. Emission by flame gases (EF. wait for steady state. Change flame or use other analytical line for EF.4. Add easily ionized non-analyte.3.4.

Annick Rouessac. Boca Raton: CRC Press LLC.. K. 72. Enke. Jackson. A. New York. “Analytical Chemistry”. Ezer. electrochemical sensors and detectors are very attractive for on-site monitoring of priority pollutants. 72. 17. J.1. Introduction Electroanalytical techniques represent the most effective answer to the increasing worldwide demand of reliable and rapid determinations of the widest variety of analytes in complex matrices.* It is assumed that instrumental variables such as spectral slit width and gas pressure will be set appropriately. Boca Raton: CRC Press LLC. Such devices satisfy many of the requirements for on-site environmental analysis. Christian. ELECTROCHEMICAL METHODS OF ANALYSIS Giuseppe PALLESCHI. Levine. Fifth Edition 1994.M.. Alina Steluţa LUPU II.B. Karl B. B. Cooke. 4. Stanley E.P. Sci. 3. 7.E. K. Charles A.3. In particular. The Art and Science of Chemical Analysis 2001. Anal. 6. “Air Pollution Control Technology Handbook” 2002.G. Broekaert.T. Bings.C. Schnelle. Seventh Edition 2000. 5. 8.4. “Environmental Chemistry”. John Wiley & Sons. A.W. 2. N. Salido. J. 10. John Wiley & Sons. 175-180. Chem. Bogaerts. “Chemical Analysis – Modern Instrumental Methods and Techniques” 2003. Stanley E. 2000. REFERENCES 1. 169R-188R. 2002.4. “Fundamentals of Environmental Chemistry” 2001. Chem. Manahan. Brown.. CRC Press LLC. AF . Francis Rouessac. Elwood. Manahan..4. Hou. 2691-2712. X. 159R-167R. 2001. EF – atomic emission flame spectrometry. New York. Anal Chem. Anal. 74. The main types of interferences and general ways of combating them are presented in the Table II. They are inherently sensitive and 145 . M.5. Gary D. II. as well as for addressing other environmental needs.5. New York.H. S. Jones. Anal. John Wiley & Sons. C. Simeonsson .– atomic absorption flame spectrometry. 9. 2000.A. Electroanalytical chemistry can play a very important role in the protection of our environment.

particularly in ionselective electrodes. In voltammetric sensors and at some electrode materials. Potentiometric Methods 146 . and hence probing of speciation. II. Each chemical species as well as each element or oxidation state has an associated potential for oxidation and reduction.5. 3. such as current. 4. particularly with controlled potential. II. 6. possibly battery-powered. potential and charge. There is the possibility of furnishing not only the results but also treated data in real time or close to real time. for application in situations where other probes may not be usable. Miniaturized sensors. associated with voltammetric sensors. Portable sensors with dedicated instrumentation. in an environmental context are: 1. that can be used outside the laboratory.3. Such specificity is not possible with most other analytical techniques. however. Conductimetric Methods The concentration of charge is obtained through measurement of solution resistance and is therefore not species-selective.selective towards electroactive species. 2. can be useful in situations where it is necessary to ascertain. An applied potential in voltammetric sensors can lead to high selectivity and specificity. The choice of electrode material can lead to selectivity.4. since complex applied potential programs can be used together with accumulation of the species to be measured at the electrode surface. The potential benefits of electrochemical monitoring. Conductimetric detectors. so that interference problems may be resolved in this way. The three types of electroanalytical measurements that can be performed offer different degrees of selectivity. whether the total ion concentration is below a certain permissible maximum level or for use as an on-line detector after separation of a mixture of ions by ion chromatography.5. 5. certain species do not react. portable and inexpensive. namely measurements of electrical quantities. Modern electrochemical instrumentation. leads to high sensitivity and low detection limits. The electroanalytical techniques are concerned with the interplay between electricity and chemistry. using computerized control and particularly in flow systems for on-line monitoring.4. An obvious example is the high over-potential for hydrogen evolution at mercury electrodes. for example. and their relationship to chemical parameters. fast and accurate. compact.2.

A filling solution. must not contain the ion to be analyzed. The sensing electrode could be a solid state. i. in many cases. a glass electrode. In ionselective electrodes. 147 . or the most familiar type.e. a potential develops across the surface of its membrane. When a sensing electrode is immersed in a solution containing the same ion to which it is selective. (To be more precise. The reference electrode should be either a single junction or a double junction type electrode containing a freely flowing filling solution and should produce a stable potential. Nevertheless. a function of all species present in solution and their concentrations. Detection limits of the order of 10 -7 mol/L of the total concentration of the ion present in a particular oxidation state. commonly used in the reference electrodes. which include both metals and anions. ammonia.since pH is defined as the negative logarithm of the hydrogen ion concentration. The difference between activity and concentration is explained in more detail later. in order to avoid confusion. pH Electrode The pH electrode is the most well-known and simplest member of this group and can be used to illustrate the basic principles of ISEs. dissolved gases. This potential is measured as voltage and is proportional to the concentration of the ion in the solution. but it may be noted here that in dilute solutions they are essentially the same. could be analyzed by this technique using a gas sensor electrode. careful choice of the electrode material can give good selectivity to one particular species. This is a device for measuring the concentration of hydrogen ions and hence the degree of acidity of a solution .e. which are a sensing electrode and a reference electrode. i. are KCl and KNO 3.The equilibrium potential of an indicator electrode is measured against a selected reference electrode using a high-impedance voltmeter. A selective-ion electrode system constitutes the two half-cells. effectively at zero current.. and oxides of nitrogen. the current path between the two electrodes can be highly resistive. the more familiar term of concentration will be used in this section. The filling solutions. and a solution containing the specific ion to be analyzed. Thus. At an inert redox indicator electrode such as platinum the potential measured is a mixed potential. the term ‘concentration’ should really be replaced by ‘activity’ or ‘effective concentration’. with only minimal interference from other ions. however. although down to 10 -11 mol/L differences in concentration can be measured. A filling solution completes the electrical circuit between the sample and the internal cell. such as oxygen. Many ions. a liquid membrane. The voltage caused by the sensing electrode is compared against a stable potential produced by a reference electrode. In addition. or a gas-sensing electrode. This is an important factor in ISE measurements. may be analyzed rapidly and with a good degree of accuracy by ion-selective electrodes. carbon dioxide. a readout meter. pH=7 means a concentration of 1x10 -7 moles per liter.

In order to measure the electrode potential developed at the ion-selective membrane the ISE/pH electrode must be immersed in the test solution together with a separate reference system and the two must be connected via a millivolt measuring system. When the electrode is immersed in a test solution containing hydrogen ions the external ions interact with the external side of the membrane and a variation of the membrane potential occurs between the external and internal concentrations.500 coulombs). log(A) = the logarithm of the activity of the measured ion. A sensitive. which is proportional to the number of hydrogen ions in the external solution. The potential difference developed across the membrane is in fact directly proportional to the logarithm of the ionic concentration in the external solution. T = the absolute Temperature. R = the gas constant (8.log [H +]) then read off the unknown pH from the measured voltage. 2. n = the charge on the ion (with sign). there is a build up of charge on the inside of the membrane. high impedance millivoltmeter or digital measuring system must be used to measure this potential difference accurately.303 = the conversion factor from natural to base 10 logarithm. glass membrane sensitive to hydrogen ions. Because of the need for equilibrium conditions there is very little current flow and so this potential difference can only be measured relative to a separate and stable reference system which is also in contact with the test solution. At constant temperature this should be a constant depending on the valence of the ion 148 . it is only necessary to measure the potential difference in two standard solutions of known pH. This causes a positive or negative deviation from the original stable reference voltage that is registered on the external measuring system. F = the Faraday constant (96. in order to determine the pH of an unknown solution. The factor 2. but is unaffected by it (see later for a discussion of reference electrodes). Thus. At equilibrium.303RT/nF is known as the slope of the electrode (from the straight line plot of E versus log(A) which is the basis of ISE calibration graphs). E0 = is a constant which is characteristic of the particular ISE/reference pair. The relationship between the ionic concentration (activity) and the electrode potential is given by the Nernst equation: E = E0 + (2. (It is the sum of all the liquid junction potentials in the electrochemical cell). but not to other ionic species. Thus.The most essential component of a pH electrode is a special.314 joules/degree/mole).303RT/ nF) · Log(A) where E = the total potential (in mV) developed between the sensing and reference electrodes. construct a straight line calibration graph by plotting millivolts versus pH (= . the electrons added or removed from the solution by the ISE membrane (depending on whether it is cation or anion sensitive) are balanced by an equal and opposite charge at the reference interface.

e. for low concentration samples. thus causing the problem of ionic interference. when measuring other ions. by high-pressure liquid chromatography or capillary electrophoresis. after the voltammetric profile has been investigated.being measured. This means that. ii) The calculation of ionic concentration is more dependent on a precise value for the potential difference than is the pH value.g. Several species that react at different applied potentials can be determined almost simultaneously in the same experiment without the need for prior separation.4. a potential difference of about 55 mV can be expected for every decade change in concentration. amperometric sensors at fixed potential can be employed.4. when measuring samples. Differences Between pH and Other Ion-Selective Electrodes i) In contrast to the pH membrane.. equivalent to 1 pH unit. iv) It is more usual to plot a calibration graph using the ionic concentration with a logarithmic scale on the X-axis rather than the pX factor (analogous to pH) on a linear axis. Voltammetric Methods Where the current is registered as a function of applied potential. and variable ionic conduction across the ion-selective membrane. but only a 10 -3 V error will cause a 4% error in the calculated concentration of a monovalent ion and an 8% error for a divalent ion. i. other ion-selective membranes are not highly selective and sensitive to some of the other ions that may be present in the test solution. Thus. Many show a curved calibration line in the region 10 -7 to 10-5 mol/L and very few can be used to determine concentrations below 1x10 -7 mol/L. e. In many practical sensors or detectors used after separation.1 pH units. Under normal operating conditions. the slope is seen to vary between 50 and 60 mV for mono-valent ions (25 to 30 mV for divalent ions) because of variations in temperature. II. Thus.5. For example. it would take an error of more than 5 millivolts to cause a change of 0. it is essential to take extra precautions to minimize any drift in the value and any errors in the measurement of the electrode potential. The collective number of voltammetry stands for a number of different electroanalytical methods for the determination and identification of inorganic and 149 . Very low detection limits of down to the 10-12 mol/L level can be reached using state-of-the-art instrumentation and pre-concentration of the analyte on the electrode surface. iii) Most ISEs have a much lower linear range and higher detection limit than the pH electrode. v) Some ISEs will only work effectively over a narrow pH range. it may be necessary to construct a calibration graph with several points in order to define the curve more precisely in the non-linear range. deviations from "ideal" behavior. or in detectors in continuous flow. more information and lower detection limits can usually be gained.

g. hanging mercury drop electrode (HMDE). a correct voltage and time for accumulation and applying a convenient mode of current-voltage measurement. They are usually made of an inert material that is not affected by electrode reactions. recording units like x/t or x/y recorders. the species of interest are accumulated at an appropriate working electrode by applying a suited voltage during a defined time under constant agitation of the solution in the electrochemical cell. The electronics consist of a very stabilized voltage source (potentiostat). Various monographs on polarography and voltammetry have been published [2. and printer. or CSV). In modern instruments. amplifier for the measurement of current in the range of picoamperes to milliamperes. 150 . the reference electrode. and the electrochemical cell. must be prevented or suppressed as far as possible. by forming an insoluble compound with the material of the electrode. After this. the auxiliary electrode. capacitive currents (iC). devices for the superimposition of different pulses and waves. all other kinds of interfering currents. The instrumentation for voltammetric measurements consists of two main parts: the electronics. As only the Faraday currents evolved by the electrode reactions of the species to be determined are of interest. or by adsorption of very stable complexes of the metal ion to the electrode. e. visible display unit (VDU).organic substances by the measurement and interpretation of currents in dependence on the applied voltage to an electrode. a scrupulous choice of the supporting electrolyte. The electrochemical cell consists of a vessel of appropriate size (from a few μL to several mL) containing a mixture of the supporting electrolyte and the solution to be analyzed in which the working electrode. Working Electrodes (WE) Working electrodes (WEs) are those at which the electrode processes occur. In voltammetric measurements. HgSe. the voltages are mostly given relative to the applied reference electrode. above all Faraday currents from accompanying substances (iFR). and migration currents (iM). In published voltammetric methods. mostly a polarograph. the use of microprocessors and computers has resulted not only in simplification of the voltammetric measurement but also in an increase in the accuracy and precision of the analyses. or ASV) or to more negative values to reduce species accumulated in a reducible form (cathodic stripping voltammetry. a gas inlet tube. Accumulation may occur by reduction of the species to the element and forming an amalgam with the working electrode. and a stirring device are immersed.g. e. Ni(DMG) 2. The maximum of the cathodic or anodic current (i P) flowing at distinct voltages is directly representative for the quantity of the species whereas the voltage at the maximum of the current (Up) is indicative of the kind of species. agitation is stopped and the voltage at the working electrode is continuously changed to more positive values in order to re-oxidize the reduced species during accumulation (anodic stripping voltammetry.g. e. timer for synchronization.3].. This can be achieved by a suitable sample preparation.

i. The whole surface of the electrode is electroactive. Besides this. mercury has a great over-voltage against hydrogen.g.... The other type uses a microvalve (needle valve or flat valve) instead of the micrometer rod to determine the drop size by the time the valve is opened (usually a few msec). Glassy carbon has many advantages compared with other electrode materials (e. This mercury film is formed in situ during the accumulation of the metal ion to be determined by addition of a certain concentration of a soluble mercury salt into the electrolyte (e. An important feature in electrochemical applications is its very low chemical reactivity.e.Different kinds of working electrodes can be used for voltammetric analyses. For the determination of most metal ions it is not as versatile as mercury because the application of negative voltages is limited by the lack of over-voltage against hydrogen. noble metals) because its surface is practically gas-tight and cap easily be renewed by polishing. The drop size is determined by the screw thread of the micrometer and the rotation [1].e. Plane or cylindrical electrodes of very different sizes are disposable. Hanging Mercury Drop Electrode (HMDE) The HMDE is the most frequently used WE for voltammetric analyses. The main application of glassy carbon in the determination of metal ions is its use as carrier material for the mercury film electrode. B. One type consists of a capillary joint to a displacement vessel into which a micrometer gauge rod is screwed. on the kind of accumulation to be used. C. a rather negative voltage can be applied without reduction of H+ to H2. The surface area of this electrode may be selected from fractions of l mm 2 to several mm2. l mg Hg(NO 3)2/Litre). A. Though the MFE is distinguished by its 151 . Glassy Carbon Electrode (GCE) Glassy carbon electrodes (GCEs) are mainly used for anodic oxidations at stationary and rotating disk electrodes above all in electrochemical detectors in highperformance liquid chromatography (HPLC) and as auxiliary electrodes in voltammetric cells [7]. The choice of the electrode depends on the metal to be determined. Mercury Film Electrode The mercury film electrode (MFE) offers some advantages in the determination of amalgam-forming metals by stripping voltammetry in the concentration range below 0. each measurement can be made with a completely new electrode. Its main advantage is the ease of replacing the electrode with high reproducibility and very constant surface area (1-2%). The MFE is formed by electrodeposition of a thin mercury film (10-50 nm thick) on an inert carrier material-mostly glassy carbon.g. The application of a positive voltage is limited by the dissolution of Hg above all by the formation of insoluble or undissociated compounds of Hg with substances in the electrolyte. Nowadays there are two main types of stationary mercury electrodes. i.5 μg/Litre [1]. as well as on the voltage range to be applied..

high sensitivity, this electrode has some disadvantages. After every determination step the surface of the electrode must be thoroughly cleaned and the reproduction of a further identical mercury film is not guaranteed because by the cleaning the active sites of the carrier material may have changed. The mercury film is seldom uniform and depends also on the composition of the electrolyte. The MFE is somewhat difficult to use. D. Gold Electrode As the surface reactions in the anodic region are much simpler for gold than for platinum, gold electrodes are often preferred to platinum electrodes in electrochemical measurements. The rotating gold electrode has been proven to be very useful in the determination of traces of mercury in biological materials [1]. The surface of gold electrodes is very susceptible to oxidation in the presence of coordinating anions at applied positive voltage. This behavior can be used for cleaning and polishing of the electrode surface. Reference Electrodes (RE) In a voltammetric cell, the voltage applied to a working electrode is measured relative to a reference electrode (RE). The potential of the reference electrode, URE, should be constant and independent of the electrolyte in the cell. It should not be changed by the passage of a small current, i.e., the electrode must be unpolarizable. These types of electrodes normally consist of a metal in contact with its sparingly soluble salt and a solution containing the respective anion in a relatively high concentration. The RE is joined to the electrolyte in the cell by a liquid conducting connection. At the boundary between the liquid junction and the electrolyte within the cell, a junction potential occurs which is included in the voltage of the reference electrode. This diffusion potential may cause interferences if the solution in the reference electrode and the analyte are very different. The REs usually used in potentiometry are also suitable for voltammetry. The most used REs with their voltages (URE) at 25°C relative to the normal hydrogen electrode (NHE) are: Hg/Hg2Cl2 (sat. KC1): + 0.244 V Ag/AgCl (sat. KC1): + 0.198 V Ag/AgCl (3 M KC1): + 0.207 V Ag/AgCl (sat. KNO3): + 0.467 V Auxiliary Electrodes (AE) Of the three electrodes arranged in the electrochemical cell, the auxiliary electrode (AE) is the current carrying counter electrode to the working electrode. By its use the RE is protected against the flux of current and by this no polarization causing a shift of its potential occurs. The AE must be made of very good conducting material that is chemically and electrochemically as inert as possible. Rods of

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platinum and glassy carbon have proven to be the electrodes of choice for voltammetric measurements. If Pt wires sealed in glass tubes are used, attention must be given to possibly serious contamination with lead. Pt can only be sealed in soft Pbcontaining glasses that can be leached out by the electrolyte. II.4.5.5. Modes of Current-Voltage Measurements There are different currents governing a voltage-current curve (voltammogram). Only the Faraday current (iP) resulting from the electrode reaction of the species to be determined is of analytical interest. While most interfering currents can be suppressed or eliminated by different provisions, the capacitive current (i C) can only be diminished or eliminated by the application of appropriate measurement methods. This capacitive current arises from the electrical double layer on each electrode, which behaves as a capacitor that is recharged by every change of voltage. The charge required for this appears in the voltammogram as a current that is not specific to the species to be determined. The ratio i C/iF determines to a great part the detection limit of voltammetric methods. Different modes of measurement have been developed to overcome this problem and to enhance sensitivity. Direct Current Mode (DC) Measurements in the direct current (DC) mode are performed by continuously changing the voltage applied to the working electrode (in cathodic or anodic direction) after the accumulation step. At the voltage of the reduction or oxidation of the accumulated species an asymmetrical current peak is obtained. The baseline is a sloping curve. The current at the peak maximum (i P) is representative for the concentration of the species, whereas the corresponding voltage (Up) is indicative for the kind of species. In this measurement mode the i C is not compensated and the evaluation of the voltammogram is rendered difficult by the form of the baseline. This ancient method is seldom applied to the determination of trace metals in biological materials. Normal Pulse Mode (NP) In the normal pulse mode (NP) short square-wave DC pulses (duration 50-60 msec) with continuously increasing amplitudes are superimposed to a constant base voltage at the working electrode. These pulses must be exactly synchronized with the current measurement. After the application of a pulse the i C induced by the pulse voltage decreases exponentially with time whereas the interesting i F occurring at a particular voltage, depending on the kind of the species to be analyzed, decreases with the square root of the duration of the pulse. In order to eliminate the influence of the i C after a delay of 30-40 msec from the start of the pulse the iF is measured during 15-20 msec and stored until the next measurement. These currents may be recorded directly in function of the applied pulse

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amplitude. In this case a curve with a shape analogous to the DC mode is obtained, but the iC is largely suppressed and the sensitivity increased. Mostly, the current of the preceding pulse is subtracted from that of the following pulse and the difference is plotted function of the voltage. A differentiated curve results. In both cases the current maximum (iP) is directly proportional to the concentration of the species and the respective voltage (Up) indicative of its kind. This measurement mode is particularly suitable for solid working electrodes, e.g., in electrochemical detectors for ion chromatography and HPLC. By using an HMDE, the maximum applicable pulse amplitude is limited (∆U < 500 mV) because by the application of greater pulses the mercury drop may be disturbed and fall off. Differential Pulse Mode (DP) In principle, this method is a combination of the DC and the NP mode. Square-wave DC pulses of small and constant amplitude (∆U= 5-100 mV) during 4060 msec are superimposed on the continuously changing DC voltage. The application of the pulses and the measurements of the currents must be correctly synchronized. For every pulse the current is measured twice during precisely defined identical intervals (e.g., 20 msec). The first measurement ends just before the start of the pulse, the second with the end of the pulse. The current of the first measurement is subtracted from that of the second and the resulting derivative ∆i/∆U is plotted in function of the DC voltage ramp. The shape of the curve shows rather sharp peaks on a smooth baseline. As both measurements take place within a short time interval, the ramp DC voltage will be almost the same. With the pulse amplitude being small and the measurement of the current occurring toward the end of the pulse, the respective i C has completely decayed. Any residual iC is eliminated still further by subtracting the two currents. The DC ramp voltage at the current maximum (Up) is indicative of the kind of species; the maximum current (i P) is directly proportional to the concentration of the species. The DP mode provides the most sensitive measurement method in many applications. Because evaluation of the voltammogram is rather simple, this mode of measurement has become the most widely used. Alternating Current Mode (AC) Alternating current (AC) measurement is in principle a DC measurement where a sinusoidal AC voltage with a small constant amplitude (10-100 mV) and a moderate frequency (30-100 Hz) is superimposed on the continuously changing DC voltage ramp. This superimposed AC voltage causes an AC component in the cell current which depends on the applied DC voltage. After filtering out the DC component the AC component is measured selectively. Plotting the AC current as function of the DC voltage gives a peak (not totally symmetrical) in the DC voltage region where the reaction at the working electrode occurs. The Up is indicative to the

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kind of species. According to Matsuda [1], the maximum current i P, representative of the concentration, can be estimated: ip = constant ⋅ n2 ν1/2 ∆U C where n = number of electrons involved in the electrode reaction; ν = frequency of superimposed AC voltage; ∆U = amplitude of superimposed AC voltage; C = concentration of the species. The relationship ip = constant ⋅ ν1/2 can only be used within certain limits. The measured AC current consists of two components: the Faraday AC current (i F) caused by the electrode reaction, which is of interest, and the capacitive AC current (i d resulting from the electrical double layer. Whereas the i F is proportional to ν1/2, the iC is directly proportional to ν. Thus the relation iF/iC decreases with increasing frequency. For quantitative determinations lower frequencies (10-100 Hz) should be used. The phase of the iF is shifted by π/4 relative to the applied AC voltage, whereas the interfering iC is shifted by π/2. By use of a phase-selective detector the ratio i F/iC and hence the sensitivity of the method can be increased. In the AC mode, above all, species with reversible electrode reactions can be evaluated quantitatively. The i P decreases rapidly with increasing irreversibility. This insensitivity to irreversible reactions can sometimes be an advantage, because many interfering substances (e. g., organic species) react irreversibly at the working electrode. Chronoamperometry Chronoamperometry involves stepping the potential or the working electrode from a value at which no Faraday reaction occurs to a potential at which the surface concentration or the electroactive species is effectively zero. A stationary working electrode and unstirred solution are used. The resulting current-time dependence is monitored. As mass transport under these conditions is solely by diffusion, the current-time curve reflects the change in the concentration gradient in the vicinity of the surface. This involves a gradual expansion of the diffusion layer associated with the depletion of the reactant, and hence decreased slope of the concentration profile as time progresses. Accordingly, the current (at a planar electrode) decays with time, as given by the Cottrell equation: i(t) = nFACD1/2/π1/2 t1/2 = kt-1/2 Such an it1/2 constancy is often termed "Cottrell behavior". Deviations from such behavior occur at long times (usually over l00 s) as a result of natural convection effects, or when using microelectrodes with high perimeter-to-area ratios. In the latter case, a time-independent current (proportional to the concentration) is obtained for t > 0.1 s due to a large radial diffusion contribution. Similar considerations apply to spherical electrodes whose current response

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NP. i. in which the enriched species on the working electrode is re-dissolved under voltammetric conditions. DP. without stirring. SW). For determinations at rather low concentrations (10 -11-10-6 M) mostly stripping methods are used.e. II. The electrolytic determination itself.e. 2. In this measurement mode higher frequencies can be used to enhance the sensitivity.g.6. i. Square-Wave Mode (SW) In this method developed by Barker and Jenkins [1]. which is of interest. As in the AC mode. All these methods give current peaks in function of the electrode voltage.following a potential step contains a time-dependent and time-independent terms. UPE = UP ± (200 to 400) mV. in which the species to be determined is electrochemically enriched on a suitable stationary working electrode with constant mixing of the solution during a precisely defined time (t PE: pre-electrolysis time) and a suitable voltage (UPE: pre-electrolysis voltage).. for example. Additional transient background contributions (associated with surface redox reactions) are common to solid-electrode chronoamperometric experiments. 20 sec) without stirring of the solution. The DC component is filtered out by the detector and the resulting i F is amplified. for constant instrumental parameters and a distinct species i P is directly proportional to the concentration of the species. decreases far more slowly during the same time. the greater the elimination of the iC due to the greater relaxation time for this current to decrease. a square-wave (SW) AC voltage of a modulation of small. In order to minimize the capacity current (i C arising from the superimposition of the AC voltage.. by altering the electrode DC voltage continuously. This is normally followed by a resting or "quiescent" period (e. For reversible 156 . i P = constant ⋅ n2 ν1/2 ∆U C. For each changing DC voltage step. Recall also that for small values of t (t < 50 ms) the chronoamperometric signal contains a background contribution of the charging current.5. rectified and recorded as a function of the DC ramp voltage. The Faraday current (iF).. The pre-electrolysis. metal ions in biological materials in concentrations down to the sub-ppb range.4. either one square-wave cycle or a burst of several cycles is applied. constant amplitude (∆U = 5-30 mV) and constant frequency (10-500 Hz) is superimposed on a linearly changing DC voltage ramp. Stripping Voltammetry Voltammetric methods are suitable for the determination of. the current measurement is made toward the end of each half cycle of the wave. This voltage-current curve can be measured with any of the normal voltammetric methods (DC. The DC voltage (Up) at the current maximum (i P) is indicative for the kind of species. AC. Square-wave voltammetry is especially well suited for reversible systems. The lower the frequency. Peak-shaped voltammograms are obtained. Stripping methods consist of two basic steps: 1.

g. Cu. Mn+ + ne. Mn+ + ne. re-oxidation). Several species can be determined in the same aliquot of the analyte: IP = constant . ν = sweep rate of the continuously changed DC voltage (∆U/∆t). Anodic Stripping Voltammetry (ASV) For anodic stripping voltammetry (ASV) mostly HMDE and MFE are used as working electrodes. Sn.e. The enrichment step can occur by three different kinds of reactions: 1. D = diffusion coefficient. TI. A = surface area of the electrode. and Hg. The cation is reduced to a lower oxidation state. Cathodic Stripping Voltammetry (CSV) The enrichment in cathodic stripping voltammetry (CSV) occurs by the formation of sparingly soluble compounds of the species to be determined with ions from the material of the electrode forming a film on the surface of the WE.+ Hg  M(Hg) 2. M(m-n)+/electrode The metal is determined by measuring the current-voltage curve with the applying of an anodic (positive) voltage sweep and recording the anodic dissolution current (i. The enrichment voltage (U PE) is such that the material of the WE is oxidized in presence of the substance being analyzed.electrode reactions the iP is directly proportional to the concentration of the species in the original solution [6]. This method is above all suitable for the determination of Bi. Zn. Cu and Hg [6]. The cation is reduced to a metal film on the electrode surface. e. Pb. The cation is reduced to the metal that forms an amalgam soluble in the mercury of the working electrode. Mm+ + ne. Pb.. Tl. the latter by the use of a gold electrode. The commonly used WE is the HMDE. Cd. C = concentration of the species. This method has been applied to the determination of different metal ions in body fluids. which forms a sparingly soluble deposit on the electrode surface. M/electrode 3. The more 157 .. n3/2 A D1/2 ν1/2 C where n = number of electrons involved in re-dissolution.

g. Au. HgO + Se2The concentration range that can be determined depends on the type and size of the WE. This relatively new method has considerably enlarged the number of trace elements that can be determined by stripping voltammetry. The measured cathodic current arises from the reduction of the cations (mostly Hg 2+ or Hg22+) present in the film of the sparingly soluble substances. there is no direct interaction between the species and the material of the WE. During the accumulation.. e.g. Hg0  Hg22+ + 2eHg22+ + 2Cl. this element must be in the oxidation state +IV.g. In some cases the integral of the cathodic current and not the iP is proportional to the concentration in the analyte. for the determination of Se [6]. glassy carbon) can be used. At higher concentrations the film of the sparingly soluble substances adsorbed to the electrode surface becomes too thick and the observed current peaks are broad and may even be split. Hg2Cl2 The species to be enriched may also be formed by an electrochemical reaction on the surface of the working electrode. HgO + 2CIHgSe + 2e. e.+ 6H+  Se2.insoluble is the compound formed. Thus. the more negative will be the voltage at which the oxidation of the electrode material will occur (Nernst equation).+ 6e. sparingly soluble. which do not form amalgams soluble in Hg as needed in ASV. Therefore. This method has also been applied to biological material.+ 3H2O Hg0  Hg2+ + 2eHg2+ + Se2.. Pt. metal ions. SeO32.. e. and maximally hydrophobic in the applied milieu.g. WEs of different materials (e.. Hg2Cl2 + 2e. Hg. HgSe The determination occurs by measuring the current-voltage curve by applying a cathodic voltage sweep (negative) to the WE. The complexes must be very stable. can also be determined with very high sensitivity 158 . the accumulation of the species of interest occurs by adsorption of a suitable complex of the species to the surface of the WE. For the determination of Se. Their structure should facilitate the electron transfer between the WE and the complex or the central ion. Adsorptive Stripping Voltammetry (ADSV) In adsorptive stripping voltammetry (ADSV).

We are continuously witnessing the introduction of new electrochemical sensing devices. Yet. Zn. Automation of all steps of a determination is to be preferred.g. 159 . above all surface-active ones. Such devices are being coupled with light and user-friendly microprocessor-based instrumentation. the complex formation with a suitable ligand (L) and its adsorption to the surface of the WE at a voltage (U PE) during a defined time (tPE) without any Faraday electrode reaction: M + L  mL (complex formation) ML + WE  MLads/WE As adsorption can take place in some cases without an applied voltage to the WE. and hence cannot solve all environmental monitoring needs. based on a wide range of chemical or biological recognition materials. In some cases neither the metal ion nor the ligand undergoes an electrode reaction but. and yet inexpensive (disposable). M + L + WE -U 2.down to the ppt range. ADSV is in most cases more sensitive and much faster than ASV and CSV. a vast array of electrochemical sensors has been applied in recent years for monitoring a wide range of inorganic and organic pollutants. Co. Mn+ + Lred + WE -U 3. MLads/WE + ne. The magnitude of the corresponding current is in a limited range directly proportional to the concentration of the adsorbed species. Cu. Pt. The determination may take place by different electrode reactions: 1. Mo [6]. Ni. MLads/WE + ne. the time elapsing between exposing the electrode surface to the solution and the start of the adsorption step should be well defined. i. In general. But this method demands a scrupulous sample preparation because many substances. the accumulation occurs in two steps. sensing devices.e. iP = constant · C and UP is indicative of the kind of metal. by the adsorbed complex hydrogen is catalytically evolved. The voltage at the WE is continuously changed to more negative values (CSV) and the current resulting from the reduction of the complexed metal ion is measured.. In addition. Metal ions that cannot be reduced in the applicable voltage range may be complexed with a ligand that can be reduced (oxidized). Electrochemical sensor technology is still limited in scope. interfere.. mass production techniques (adapted from the microelectronic industry) enable the fabrication of extremely small and reproducible. e.

Wiley-VCH. Angerer and K. 2. Marcel Dekker. On-going commercialization efforts. are certain to have a major impact on pollution control. “smart” sensors and molecular devices. Other advances of selective and stable recognition elements. T. M. 5. Marcel Dekker. VCH. H. José MARTÍNEZ CALATAYUD II. Bond. 1988. VoI.Fast-responding electrochemical sensors are also being adapted for detection in on-line monitoring or flow-injection systems (as needed for continuous monitoring or field screening applications). and introduction of multi-sensor systems for simultaneous monitoring of several priority contaminants. A. Laboratory Techniques in Electroanalytical Chemistry. P. Heidelberg. 1994. Marcel Dekker Inc. 1984. 2000. H. ISBN 0471-28272-3. eds. Seiler. remote electrodes. P. 1980. II. in Analyses oJ Hazardous Substances in Biological Materials. Weinheim.). CHROMATOGRAPHY Monica CATALÁ ICARDO.4.1. Heineman (eds. to the development of immunoassay-based electrochemical sensors and of remote electrodes for unattended operations. multi-parameter sensor arrays or micromachining and nanotechnology. to the design of new electrocatalysts (that facilitate the detection of additional priority pollutants). 4.6. to address the fouling and degradation of electrochemical sensors during use. Introduction 160 . Hiithig. New York. J. should lead to the translation of these and future research efforts into large-scale environmental applications. Kissinger and W. 3. Schaller. A.6. REFERENCES 1. H. Rach and H. Analytical Electrochemistry. 1987. Wang. New York.Sigel.). Handbook on METALS IN CLINICAL AND ANALYTICAL CHEMISTRY. Additional efforts should be given to the development of new immobilization procedures (that increase the stability of the biocomponent). to the replacement of classical mercury electrodes with well-defined solid surfaces. ISBN 0-8247-9094-4. 6. coupled with regulatory acceptance. Sigel. 2 (I.4. Polarography and Voltammetry in Trace Analysis. Modern Polarographic Methods in Analytical Chemistry. Seiler.

hence the name of the new technique [from the Greek khroma (colour) and grafein (to write)] according to the much-repeated “historical” version. Separation was done in space —temporal separation was accomplished at a later time.4. Sthal and Kirchner developed thin layer chromatography by transposing the original operating principle to thin layers of sorbent. in 1931. the Greek god of time. Kuhn and his coworkers. Goppelsröder in 1861 and Day in 1900. the Russian botanist Mikhail Tswett (1872–1920). Albrecht and Engler in the same period. in 1906 he succeeded in isolating chlorophylls and xanthophylls in a plant extract in petroleum ether by passing it through a glass column packed with finely divided calcium carbonate.16. To the author. Figure II. the name comes from Chronos. who is currently held as the father of chromatography. chromatographic techniques have evolved dramatically. Thus. Schönbein in 1861.4. by Runge in 1850. It was not until about thirty years after Tswett's discovery that the usefulness of adsorption chromatography for separation purposes was envisaged by R. and also by Kvita. 161 . Tswett refined his invention. A few years later. hand-in-hand with technological breakthroughs. however. The isolated species formed colored bands in the column. In 1903. Since then.The concept of chromatographic separation has a long history.16). Aristotle's contemporaries used various types of sorbents (earths) to process seawater. In subsequent years. Drawing of the original “chromatograph” from the first Tsweet article. conducted his pioneering experiment involving the passage of a plant extract through a column filled with a sorbent material (Figure II. More “scientific” applications were developed much later. Thus.

Those components that are strongly retained by the stationary phase will move slowly in the mobile phase flow. thus. the former are filled with particles containing the stationary phase (SP) and the latter consist of hollow capillaries the walls of which are coated with the SP. Types of Chromatography Chromatographic methods can be classified according to various criteria. Both types of chromatography rely on identical equilibria.” Chromatographic separation involves an interaction and the partitioning of the analyte between two immiscible phases for which it exhibits some affinity. Once each component has been isolated. gas and supercritical fluid chromatography. If the chromatographic process is allowed to proceed for a long enough time. There will thus be a spatial separation: each sample component will move separately from the others. on the other hand. can be a gas.4. namely: the mobile phase and the stationary phase.e. Thus. One is based on the way the stationary and mobile phases are brought into contact. the type of 162 . there is liquid. In planar chromatography. as the mobile phase. As a result. liquid or supercritical fluid that sweeps the sample components as it goes through the stationary phase. a gas and a supercritical fluid. i. Liquid chromatography can be implemented in a column or on a planar surface. the stationary phase is held in a narrow tube (a column) through which the mobile phase is passed by gravity or under pressure. however. there is column (three-dimensional) chromatography and planar (two-dimensional) chromatography. each sample component will reach a preset point (the detector) after a different time. the stationary phase is placed on a flat plate and the mobile phase travels across it by gravity or capillarity. which dictates the velocity at which it will travel or migrate across the stationary phase. also called the “eluent”. so there will be both spatial and temporal separation between all. II. whereas those that are weakly bound to such a phase will move faster. The mobile phase. The stationary phase in a chromatographic column can be solid (packing the column) or liquid (anchored to a solid surface). which use a liquid.2. A third criterion is the underlying retention mechanism. Columns can be of the packed and open-end types. respectively. gas and supercritical fluid chromatography can only be performed in a column.This inspired the following “historical definition” of chromatography: “a separation method based on the different velocity at which the components of a sample go through a stationary phase pushed or swept by a mobile phase. it can be identified and/or quantified individually. differences in migration velocity between the sample components eventually result in their separation. The way an analyte distributes itself between the two phases depends on its partition coefficient. In column chromatography.6. One other classifying criterion is the type of stationary and mobile phases used.

Selectivity here can be modulated by choosing an appropriate group to interact with the analyte and exploiting both chemical bonding and steric effects.3. the retention–elution cycle is successively completed. whereas the smaller ones can also penetrate the gel via tortuous paths. As a result.17. 163 . (e) Affinity. The larger molecules in the sample can only pass between the gel particles. The column is packed with a solid support coated with a liquid immiscible with the mobile phase or eluent. This is a liquid–liquid extraction process by which the sample components are separated as a function of differences in polarity. The mobile phase is usually non-polar (an organic solvent) and the stationary phase polar. By the composition of the mobile phase (liquid chromatography only).4.6. By the interaction between the analyte and stationary phase. For example. the overall phenomenon behind the separation usually involves more than one. 3. Classification of Chromatographic Processes The classification can be made according to different criteria. the stationary phase is a resin with negatively or positively charged covalently bound groups that attract ions of the opposite sign via electrostatic forces. In affinity chromatography. like 1. This type of chromatography is dealt with in detail later on. By the nature of the phases. (c) Ion exchange. In addition. Such a mechanism can be essentially of five different types – however. This occurs when the stationary phase consists of particles of a solid sorbent the surface of which retains the sample components that will compete for the mobile phase molecules. These are the ingredients of so-called “normal chromatography” as opposed to “reversed phase chromatography”. In this way. By the configuration of the separation system. (d) Exclusion. relies on a screening effect dependent on the size of ions and molecules. the bulkier particles will leave the column before the smaller ones. adsorption of the analytes onto the gel surface can give rise to partition coefficients greater or less than unity. namely: (a) Adsorption. In ion-exchange chromatography. A more complete and general classification scheme is depicted in Figure II. The column is filled with a porous stationary phase or a gel. and 4. the latter uses a polar (aqueous) mobile phase and a non-polar stationary phase. Exclusion chromatography. II.4. the solute interacts in a specific manner with a functional group in a molecule covalently bound to the stationary phase (immobilized on it). also known as “gel chromatography”. only the protein reacting with an antibody will bind to a column if the antibody is immobilized on the stationary phase. (b) Partitioning. 2. so the packing particles possess inner channels.physical–chemical interaction between the stationary phase and the sample components.

(e) a detector. processing and delivery system. 164 . and (f) a recorder or a data acquisition.18) essentially includes the following elements: (a) containers for the mobile phase and solvent treatment. (b) a pumping system.4.Components of Column Chromatography A column chromatography assembly (Figure II. (c) a sample insertion system. (d) a column.

165 . HPTLC Thin-layer LSC adsorption LLC partition SEC size exclusion /gel permeation IEC ion exchange BPC Bonded phase GPC gel permeation GGC gel filtration Figure II.Column Chromatography Character mobil phase Gas Liquid GLS partition Liquid Character Solid stationary GSC phase adsorption Planar Columna CL or HPLC PC paper TLC.17.4. General classification scheme.

Solvent/s container Data collection and presentation Pumping Injector Column Detector Figure II. As noted earlier. A comprehensive description of the influence of each experimental variable is obviously beyond the scope of this chapter and can be found. Rather. the stationary phase is packed in the column and the sample is inserted at one end and swept by a continuous (or. intermittent) flow of the mobile phase. The optimization process should be aimed at improving such efficiency. 165 . however.and also the thickness of the liquid film when the stationary phase is liquid. this entails minimizing band broadening and altering the relative migration rates in order to achieve complete separation of species at the column offset. An appropriate detector can be placed at the end of the column in analytical chromatography to identify and/or quantify each species ( viz. the length and diameter of the column. The most influential experimental variables in addition to the nature of the stationary and mobile phases . an increased dispersion of the sample components in the bulk mobile phase reduces the separation efficiency of the column.and the temperature in gas chromatography . each species can be collected in a separate vessel for subsequent measurement with an appropriate detector.4. the analytes will be separated into more or less sharp bands. If velocity differences between analytes are sufficiently large and the column sufficiently long. alternatively.18. to record a chromatogram. and the degree of uniformity of the packing . The longer the column is. the more efficiently can two species be separated. in any book about chromatography. it will travel at a different velocity through the column. this chapter summarizes the general theory of chromatographic separation. This requires the prior identification of the variables influencing the analyte migration rates and the factors resulting in band broadening. less often.are the flow-rate of the mobile phase and various characteristics of the column including the size of the packing particles. because each analyte interacts differently with the two phases. together with a definition of the most commonplace chromatographic terms. Whichever the type of analyte interaction is involved. Block diagram of a liquid chromatograph. where each band will show as a peak at a position dependent on the retention time of the analyte concerned).

In 1941. Martin and Synge developed the earliest theoretical description of chromatography. The central element in their theoretical development is the upstream partitioning concept and the definition of “theoretical plate” . which is the column section where the average concentrations of the analyte in the stationary and mobile phase are consistent with the above-described partition coefficient. the size and uniformity of the packing particles. Chromatographic Theory. HETP Figure II.6. The ratio of the column length to the number of theoretical plates it contains is called the “plate height”. An Overview The analyte (i.4.e. The size of a theoretical plate is defined as the height equivalent to a theoretical plate (HETP).4. and (d) miscellaneous effects including interactions between solutes.19. which is a function of various column-related factors including the following: (a) the effect of mass transfer. and 166 .by analogy with fractional distillation -. which earned them the Nobel Prize in 1952. XM ↔XS K = [X S ] [X M ] The number of theoretical plates of a column dictates its separation efficiency. the efficiency of a separation relies on the number of theoretical plates of the column. Therefore. (c) the diffusive effect. (b) the eddy effect.II. A straightforward description of the procedure used to determine the size (length) of the theoretical plate (and hence the number of plates in a column) based on the dynamics of chromatographic separation is provided below. the sample component to be isolated) partitions itself between the stationary and mobile phase depending on its specific affinity for each. the thickness of the mobile phase. Theoretical plate in a chromatographic column.4.

5 → complete separation W +W A B N (α − 1)  k´B    RS =  4 α  1 + k ´  B  CS: solute concentration in the stationary phase.4.variations in the flow-rate of the mobile phase across the column (as a result of gaseous phases being compressible). Capacity factor Describes the migration velocity of a solute into the column. Theoretically K V t −t k´A = A S = R M 1<k´A<5. (k´A<15). RS = RS ≥ 1. theoretical >N and <H → >efficiency 2 plates  tR  N = 16  W  Resolution Indicates the column ability to 2[ (t R ) B − (t R ) A ] separate two different solutes. Table II. mobile phase composition and filling in the column.4. H: plate height H and N indicate the efficiency of the N: number of column. Depending from mobile and stationary phases composition. Parameter Parameter name Distribution constant (coefficient) Retention time Definition Equilibrium constant Amobile↔Astationary K= tR cS cM Time interval from sample insertion to maximum peak appearance tM Dead time Time interval required for a nonretained solute to be transported through the column. Auxiliary parameters and equations in chromatography.4. 167 . Depending on the VM tM T. In Table II. Selectivity Indicating the relative situation of factor two peaks: being B the most retained K k´ (t ) − t α= B = B = R B M and A the most quickly eluted → K A k´A (t R ) A − tM α>1.4 are presented auxiliary parameters and equations in cromatography.

The transfer is not instantaneous.CM: solute concentration in the mobile phase. This effect is especially prominent in gaseous phases as a result of the increased transfer coefficient within a gas. short diffusion distances and large interface areas between the two phases. dg the thickness of the stationary phase. v the velocity of the mobile phase. the solute will have to travel long distances to reach the stationary phase and vice versa. In between these two extremes. the analyte passes from the stationary phase to the mobile phase and back many times. The diffusion rate in turn depends on the prevailing concentration gradient. rather. These variables (marked with Cv) contribute to the HETP as follows: Cv = k dg2 v / π2 (1+k)2 Ds where k is the effective partition coefficient. W = 4σ. if the mobile phase is thick. In summary. the travel of the mobile phase through the stationary phase can be compared to that of a mountain stream flowing along an uneven bed splitting into several independent courses at different points. (c) a large interface area. it is lower near the stationary phase than in the middle of the stream. The transfer of an analyte from the mobile phase to the stationary phase and back is favored by the following: (a) a high diffusion coefficient. is favored by high diffusion coefficients. (b) a small distance between the two phases. W: base-wide peak. 168 . the actual chromatographic peak will be broader than expected. Thus. actual analytes are retained to a variable degree and require the use of specific conditions for separation. Equilibrium. which can never be fully reached in a dynamic system of this type. and Ds the diffusion coefficient of the analyte in the stationary phase. the analyte will travel farther down the column than one would expect from its partition coefficient alone. As a result. one not retained at all by the stationary phase would migrate with the eluent front. Mass Transfer Effect An analyte that was very strongly retained by the stationary phase would stop at the first plate and require a large volume of mobile phase for elution. On the other hand. In fact. Eddy Effect The velocity of the mobile phase is not the same at every point of a theoretical plate. and (d) a low velocity of the mobile phase. it depends on the rate of diffusion of the analyte in the two phases. During the separation process.

However.4. some analyte molecules (or ions) will travel very rapidly. This phenomenon (marked with B/v) has the opposite effects of mass transfer.As a result. Its contribution to the HETP is: B/v = 2 γ Dm / v where γ is the tortuosity coefficient. Dm the diffusion coefficient of the solute in the mobile phase and v the velocity of the mobile phase (in cm s–1).20. Thus. b) Types of particle packing and resulting peaks. while others will be delayed and give rise to a new dispersion factor and hence to broadened chromatographic peaks. The mass transfer.e. In some cases. the velocity at which the analyte travels varies between two extreme values. The resulting dispersion will be higher for solutes with high diffusion coefficients and mobile phases flowing at a high rate. column efficiency can be improved by using a stationary phase of low viscosity with an increased diffusion coefficient for the solutes. the maximum of the chromatographic peak). As can be seen in Figure II. Diffusive Effect A solute in a mobile phase can travel (diffuse) freely in any direction. they have a 169 . so they do not alter the mean velocity of the solute (i. a) b) Figure II. a) Illustration of the eddy effect.20 minimizing the adverse effects of this phenomenon entails using the thinnest possible a stationary phase. not only towards the stationary phase or away from it. The contribution of the eddy effect (A) to the HETP is: A = 2 λ dp where λ is a measure of unevenness in the stationary phase packing and dp the average diameter of the support particles. eddy and diffusive effects in combination do not delay retention of the solute by the stationary phase.4. Diffusion causes displacements from the regions of increased concentration by effect of a concentration gradient.

With provision for these three effects. In order to expedite separation. known as the Van Deemter equation (see Figure II.21) clearly reflects the presence of a minimum HETP value that coincides with the optimum value —that to be pursued for optimal separation. it may be preferable to use a v value slightly higher than that resulting in the lowest possible HETP. HETP B C A voptimum Flow-rate mobile phase (v) Figure II. Graphic aspect of the Van Deemter equation. in non-linear chromatography). Such interactions are absent from linear 170 . a few others exist that contribute to peak broadening and thus influence HETP. Miscellaneous Effects The three above-described effects are not the only ones influencing chromatographic development. solutes may be eluted sooner than expected (e. the HETP for a column can be calculated from: HETP = 2 λdp + 2 γ Dm / v + 8 k dg2 v / π2 (1+k)2 Ds Because v is the sole variable that can be altered by the operator once the chromatographic system has been set up. however. (a) Each solute molecule or ion can act independently of the others.they result in broader peaks and hence is poorer analyte separation. In fact.4. the previous equation can be simplified to: HETP = A + B / v + Cv A plot of this expression. In the presence of interactions between one another. Thus. The operator should thus choose the v value leading to the minimum HETP.decisive influence on the solute dispersion within the chromatographic system .4.g.21.

it continues to be widely used in routine applications. and ( b) the neutralization or suppression of the large amount of salt species produced in the previous step by formation of non-conducting species (water or carbonic acid) and the enhancement of the intrinsic conductivity of the analytes by conversion into strong acids or bases.22 is presented the general scheme of an ion chromatograph. EC MP P I SC CD Figure II. the previous equation is incomplete.5. MP vessel holding the mobile phase.22. Originally. (b) The affinity of the stationary phase for the solutes varies across its surface.4. (c) The thickness of a liquid stationary phase is not uniform throughout the chromatographic system. In Figure II. CD conductimetric detector. II.4.chromatography. so partition coefficients are identical at any point in the chromatographic system. Although the subsequent inception of a new generation of chromatographic methods has lessened its significance. Ion Chromatography Monica CATALÁ ICARDO. 171 . SC suppressor column. so it should be replaced with (C + C′)v. their flow-rate changes with the distance traveled through the column. The most important parameters and equations in chromatography are presented in Table II. I sample injection system. ion chromatograph relied on two successive processes. Jose MARTÍNEZ CALATAYUD Ion chromatography peaked in popularity in the 1960s and 1970s.4.6. The latter step was dictated by the type of detector used (a conductimeter). which includes diffusion in both.4. (d) Because gaseous mobile phases are compressible. Thus. General scheme of an ion chromatograph. EC separation column packed with ion-exchange resin. Some authors have proposed alternative expressions descriptive of a similar variation of HETP with v.4. sorbent surfaces can be partially blocked by other polar substances such as water. For these reasons. P mobile phase propulsion system. (e) The term Cv encompasses diffusion in one phase only. namely: (a) the chromatographic separation of ionic compounds by interaction and/or exchange with charged sites on the stationary phase.

Ion Exchange A. Stationary Phase The mobile phase consists of a resin with a backbone bearing of a specific functional group capable of exchanging ions. This forms a three-dimensional hydrocarbon structure containing many instances of the following chemical sequence: This backbone can be easily obtained and is physically and chemically stable under certain conditions. (I) (II) The backbone of a typical ion exchanger consists of a styrene-divinylbenzene copolymer. For use as an ion exchanger. it is supplied with ionic groups as follows: 172 .

they can be eluted by using a solution containing an anion with a higher affinity for the resin (a strong acid) in order to displace the equilibrium back to the left. which bears charge of the opposite sign. cationic and basic (anionic). Ion-exchange Processes A resin bearing sulfonic groups (R–SO3–H+).size here means the actual size of the ion in solution. This latter portion can be exchanged with other ions present in the mobile phase. C. the other (counterion). However. Some lists rank ions for affinity to specific resins. 173 . Any conventional buffer is theoretically useful for this purpose. As a rule. -CH2CH2N(CH2CH3)3+OHAmine: -NH3+OH-. -CH2COOH Quaternary amine: -CH2N(CH3)3+OH-. Table II. the rankings. Types of exchangers. hydration sphere included. One end of the functional group is covalently bound to the hydrocarbon backbone. binds to it via electrostatic forces. the mobile phase is usually an aqueous solution occasionally containing some miscible organic solvent and an ionic species with buffering properties. can change slightly depending on the particular experimental conditions. all ionic sites in the resin will be occupied by hydrogen ions.Depending on the character of the ionic group introduced. Ionic exchangers Cation exchange Anion exchange Type Strong acidic Weakly acidic Strongly basic Weakly basic Chemical group Sulfonic acid: -SO3H. Once the target ions have been bound to and retained by the resin. -CH2CH2NH(CH2CH3)2+OH- B. The lists for the most commonplace resins can be used as guidance. such H+ ions can be displaced by other ions.4. and a small size . will establish the following ionic equilibrium: n R-SO3-H+ + Mn+ ↔ (R-SO3-)nMn+ + n H+ solid solution solid solution In an acid medium. -CH2CH2SO3H Carboxylic acid: -COOH. the exchanger can be of various types the most prominent of which are acid. the affinity of the resin will be maximal for ions possessing a high charge and charge density. for example. however.4. Table II. Mobile Phase In ion chromatography.5 shows selected examples of each type of exchanger.5.

E. Detectors The ideal detectors for ion chromatography are those based on conductivity. D. new types of suppressors such as the following have been developed: (a) Hollow fibers of polymeric ion-exchange material that can be regenerated by passing an appropriate solution over their outer surfaces. As stated above. the suppressor was the acid form of a cation-exchange resin and the eluent a carbonate or bicarbonate solution. Ion Suppression The earliest suppressors used in ion chromatography were ion-exchange resins that converted the solvent ions into scarcely ionized molecular species without altering the analyte ions. the eluate is passed through one of the cartridges only. Eluent power and elution selectivity depend on the particular type and concentration of the species added to the mobile phase. easily miniaturized. (b) Micro-membrane suppressors based on ion-exchange membranes that are described later on. so the exchange process was as follows: H+(aq) + Cl–(aq) + Resin+OH–(s) → Resin+Cl–(s) + H2O For anions. the composition of the mobile phase is changed during the chromatographic process. In its starting position. inexpensive. with HCl as eluent. which use two suppressor cartridges connected to a 10-port valve. (c) Electrolytic membrane-based suppressors. when the pH indicator it contains exhibits 174 . this problem can be overcome by using appropriate suppressors behind the ion-exchange column. the suppressor column contained an anionic exchanger in hydroxide form. ensuring that all analytes will be eluted from the column within a reasonable time entails using a high electrolyte concentration.phosphate buffer can usually be used with both types of exchanger.Anion exchangers are used with buffers consisting of positively charged species and the opposite is true for cation exchangers . Their lack of selectivity poses no problem here as the analytes are previously isolated. this decreases the sensitivity of the determination because of the conductivity of the eluent concealing that of the sample components. this is known as “gradient elution”. More recently. Thus. Suppressor columns must be regenerated on a regular basis (every 8–10 h) by converting the packing back to the original acid or basic form. In some applications. When the regeneration capacity of such a cartridge. which is transparent. where ion transfer across the membrane is favored by applying an electrical field. is exhausted (viz. (d) Packed-column mini-suppressors. robust and long-lasting. which are universal for charged species and can be highly sensitive in addition to simple. However.

a color change that signals the need for regeneration), the valve is switched to have the eluent pass through the second cartridge and the first is replaced. (e) Continuously regenerated packed-column suppressors, which rely on the ion reflux principle. This is an ion-exchange technique involving the passage of water over an electrically polarized resin bed and using an electrolytic reaction to produce the eluent and suppressor medium. Applications The quality of environmental water (rain, lake, underground, river) is usually assessed from analyses for inorganic ions such as sulfate, chloride, nitrate, sodium, potassium, ammonium, magnesium and calcium. Monitoring ion contents in water involves the simultaneous separation and determination of anions and cations by ion chromatography. A number of approaches have been explored for this purpose including the use of mixed beds of cation and anion exchangers or two individual columns and as many detectors. A. Determination of anions (CrO42–, MoO42–, BrO3–, SeO32–, SeO42–, HAsO42– and WO42–) and cations (Cu2+, Ni2+, Pb2+ and Cd2+) This is a joint determination of several ions in river water samples. The analytes are all known to be toxic to humans, animals and plants. Toxicological analyses must not only be highly selective, but also allow the speciation of ions as their deleterious effects depend on their specific valence states. This determination involves the chromatographic separation of both metal ions (following chelation with Na2EDTA) and non-metal ions (by suppressed anion-exchange chromatography). The novelty here is that the analysis time is reduced by performing gradient elution under optimal conditions; in this way, each analysis takes less than 20 min. The use of a gradient introduces a gradually increasing competitive advantage in the elution process (i.e. the sweeping of retained ions); however, the use of a conductimetric detector can lead to baseline drift and substantially increased salt concentrations in the eluent as a result. In this particular determination, elution gradients were programmed by using a baseline balancing method. The operational procedure was follows: a volume of 50 μL of sample was inserted into the chromatographic system following in-line removal of organic matter by using a Dionex OnGuard-RP cartridge and metal cations were pre-chelated with 0.25 mmol L–1 EDTA. The experimental set-up consisted of a Dionex AG9 4 × 50 mm i.d. column followed by a Dionex AS9 4 × 250 mm i.d. column packed with a 15 μm thick bed of polystyrene–divinylbenzene substrate with a completely aminated anion-exchange latex —the backbone of which was polyacrylate-based— as binder. The suppressor was of the membrane, sandwiched layer type [viz. an Anion Micro-Membrane Suppressor (AMMS-II)] and the regenerating solution 25 mmol L–1 H2SO4. The mobile phase was 3.5 mmol L–1 NaHCO3 at pH 9.75.

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The eluent was passed between two ion-exchange resins over the outer surface of which the regenerating solution was circulated upstream. The limits of detection thus achieved ranged from 2 μg L –1 for Ni, Cd, Cu and Cr to 6 for SeO32–. B. Determination of Na2+, NH4+, K+, Mg2+, Ca2+, IO3–, BrO3–, Br–, NO3– and Cl in various types of water (river, pond, tap) The process here involved the use of two columns packed with and anion exchanger and a cation exchanger, respectively. The columns were placed one after the other or accommodated in the loops of two injection valves. Figure II.4.23A shows the assembly with the two columns arranged in a serial manner. The sequence in which the two were placed was found to influence the elution profile. A high cation concentration can result in the formation of ion-pairs with the anions; therefore, the cation-exchange column should precede the anionexchange column when the eluent contains many cations. However, the peaks for the cations deteriorate when these are passed through the anion-exchange column, which is not the case with those for the anions. Also, ions such as calcium, iodate, bromate and nitrite cannot be determined under these conditions owing to the resulting peak overlap. These led to the development of the alternative depicted in figure II.4.23B, in which two 6-port valves were used in such a way that the cations separated in the cation-exchange column accommodated in the loop of the first valve were not passed through the anion-exchange column located in the loop of the second valve. When the sample volume (20 μL) was injected, the two valves were in their injection positions, so the sample was allowed to reach the loops accommodating the two columns. Cations were retained by the cation-exchange column while anions were passed through it and reach the anion-exchange column. Within 1.45 min, all anions were retained by the latter column, the second valve then being switched to the next position.

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(A)
FM

S

P

Columns cationic and anionic

UV

C

W

(B)
FM

S

P

V1

V2

UV
Cation exchange Anion exchange column column

C

W

Figure II.4.23. Schematic diagrams of the chromatograph configuration. S: sample (Injected volume, 20 μL); P: Pump (1 mL min -1); FM: Mobile Phase (H2SO4 1.0 mM + L-hystidine 0.1 mM); UV: UV detector (210 nm); C: conductimetric detector). The anion exchange column was a 50x4.6 mm TSKgel IC-Anion-SW; the cation exchange column was a 150x4.6 mm TSK gel Super IC-Cation. Separating the cations took 10 min, after which the valves were switched and all analytes allowed to reach the detector. For comparison purposes, the detection was done with a spectrophotometer and a conductimeter. Cations were detected by UV-VIS absorption spectrophotometry in an indirect manner and the mobile phase was supplied with an absorbing additive (viz. the aminoacid L-hystidine). The passage of each cation through the detector produced a negative peak. Absorbance measurements were made at 210 nm. The limits of detection thus achieved, in mg L –1, with UV and conductimetric detection, were as follows: Na + (0.092, 0.1), NH4+ (0.059, 0.09), K+ (0.14, 0.2), Mg2+ (0.14, 0.1), Ca2+ (0.4, 0.3), IO3– (0.2, –), BrO3– (0.2, –), Br– (0.042, –), NO3– (0.042, –) and Cl– (–, 7.1). The process took roughly 20 min. The presence of L- hystidine slightly increased the retention times for the cations as it competed with them for active sites. C. Simultaneous determination of inorganic nitrogen, nitrate, nitrite and ammonium in a micro-column (Figure II.4.24) Nitrogen can occur as various chemical species with valences ranging from +5 to –3. Also, the nitrogen cycle encompasses a wide variety of chemical and

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biological processes in the environment. In this example, the analytical method was applied to river water as the continuous release of nitrogen species into rivers increases eutrophication and environmental pollution. A micro-ion chromatographic column was used to speciate inorganic nitrogen and the separation system was coupled to two serially arranged detectors (a spectrophotometer and a spectrofluorimeter). Nitrate and nitrite were detected at 206 nm with the former. On the other hand, ammonium was post-column derivatized with o-phthalaldehyde in the presence of 2-mercaptoethanol to measure the light emitted by the resulting derivative at 470 nm upon excitation at 410 nm.

+ HOCH2CH2SH + NH2R →
MEC

OPA

amine

isoindole

The stationary phase was an IC-Anion SW anion-exchange resin packed into a fused-silica tube 100 mm long × 0.32 mm i.d. The mobile phase was a 20 mmol L –1 solution of sodium sulfate at pH 5.7. The analytical response was linear over the range 0.02–0.1 mmoL –1 for the three species and the limit of detection was 1.6, 2.3 and 17 μmol L –1 for nitrate, nitrite and ammonium, respectively. The repeatability in retention time and peak height and area at an analyte concentration of 0.1 mmol L –1 was always better than ±2%. Each analysis took less than 10 min. Samples were previously passed through a Develosil C30 column to remove hydrophobic substances.

S FM R

P

C V

UV

T-L

F

W

Figure II.4.24. Flow assembly for simultaneous determination of inorganic nitrogen. P: Syringe pump; V: sample injector; C: separation column; T-L: water bath (65 ºC) and reaction coil (fused-silica capillary tube of 50 μm i.d. x 2 m); UV: UV detector; F: fluorescence detector; S: sample; FM: eluent; R: reagent solution; W: waste

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25. P: gradient pump (1 mL min -1). The elution was done in the gradient mode.5.3. 3.4. AS: auto-sampler.25) included on-line pre-concentration. a total of 14 phenols and chlorophenols were determined as products of the decomposition of the fungicide tecnazene upon irradiation with UV–Visible light. The pre-concentration was done in an NG1 5 cm × 4 mm guard column accommodated in the loop of a 6-port injection valve.6. and column pre-concentration and elution 12 min in all.1 μg L–1.D.4. ion-exchange chromatographic separation and amperometric detection with a glassy carbon working electrode and an Ag/AgCl reference electrode. D: amperometric detector. using a constant concentration (10 mmol L–1) of sulfuric acid and a variable concentration (36–65% v/v) of acetonitrile. AS FM P GC AC D UV W Figure II. Then. The experimental set-up (Figure II. UV: UV reference detector. The real samples used were drinking water collected from the public supply network in various places.2% for concentrations over the range 50–250 nmol L–1. The repeatability (n = 5) in both retention times and peak areas was better than ±3. persistency and —in some cases— carcinogenicity. AC: analytical column. the valve was switched to the insertion position and eluted substances were passed through the separation column (an NS1 25 cm × 4 mm ion-exchange column). Chlorophenols have been widely used for more than fifty years despite their toxicity. using a potential difference of +1.2 V between the two. Separation took less than 30 min.6-dichlorophenol (1 nmol L–1). A sample volume of 5 mL was passed through the column to retain the target analytes. II.4. Gas Chromatography (GC) 179 .5-dichlorophenol (5 nmol L–1) and 2.6-tetrachlorophenol (1 nmol L–1). W: waste. In the following example. European legislation has set the maximum tolerated total phenol concentration in water for human consumption at 0. Flow assembly for phenols and polyphenols determination. FM: mobile phase.5 μg L –1 and that for individual phenols at 0. The limits of detection achieved ranged from 200 to 500 pmol L –1 for all phenols except 2. Determination of phenols and chlorophenols in water for human consumption The separation of inorganic ions is not the sole application of ion chromatography. GC: pre-column.6.

The elution is done using an inert gas as the mobile phase. namely: gas-solid chromatography (GSC) and gas-liquid chromatography (GLC). Its scope of application is highly restricted as a result of it usually giving tailed peaks . The sample should be volatilized immediately upon injection into the chromatographic system. it is more commonly used with liquid samples the components of which can be readily volatilized at the working temperature by derivatization.a consequence of non-linear adsorption . Gas chromatography can be of two different types depending on the nature of the stationary phase. it is usually applied to species of low molecular weight only. 180 . José MARTÍNEZ CALATAYUD Gas chromatography can be directly applied to volatile compounds.and of strongly polar molecules being retained almost permanently. henceforward called simply “gas chromatography”.4. as this is intended to carry the analyte through the column rather than interact with it unlike most liquid chromatography applications.26. Gas-liquid chromatography. Instrumentation for Gas Chromatography The basic components of a gas chromatograph are depicted in Figure II. For these reasons. relies on the partitioning of the analyte between a gaseous mobile phase and a stationary phase consisting of a liquid immobilized onto the surface of an inert support. however. Gas–solid chromatography uses a stationary phase that retains the analytes by adsorption.Monica CATALÁ ICARDO.

only a small fraction of sample reaches the column while the rest is sent to waste. requires the use of rotary valves similar to those employed in HPLC and FIA. waste. in this way. The mobile phase then sweeps the sample to the first plate in the chromatographic column. a stream splitter is used to circulate the sample through several channels only one of which leads to the column. the gas container must be equipped with pressure control and measurement facilities. The most common insertion method involves using a micro-volumetric syringe to inject the liquid or gaseous sample via a silicone rubber septum into a vaporization chamber located at the column top. Injected sample volumes usually range from 10 –3 to 20 μL. is usually helium. W. detector. Solid samples can also be introduced into the chromatographic system. The sample introduction system should allow the insertion of a sample plug of appropriate size into the system. nitrogen. separation column. reference column (nor present in any commercially available model).Figure II. C. computer. Schematic figure of a gas chromatograph. sc. The carrier gas. control of the gas flow-rate. and. Quantitative work.26. rc. carbon dioxide or hydrogen. The choice is frequently dictated by the type of detector used. argon. The chamber is about 50 ºC below the boiling point for the least volatile component of the sample. Also. 181 .4. gc. which minimizes dispersion. a flow meter and a molecular sieve (to remove water or other impurities). injection sample. For sub-microliter volumes. using glass vials of very thin walls that are broken from the outside once the vials have entered the injection chamber at the top of the column. D. which must be chemically inert. is. where reproducible insertion of the sample is crucial.

4. 2.0 0. Table II.27. Characteristics of the different types of columns.1-0.5-3.0 0.27). Gas chromatographic columns can essentially be of the packed or open-end types (see Table II. Stationary phase film.4. b) Porous layer capillary column. c) Open tubular capillary.5 mm 5-100 m/ 0. 4.Throughput rate /sample gas. 3.Portable chromatographs for the determination of the typically low concentrations encountered in fieldwork can use sample volumes from 10 L to 1 mL.6.0 ← Diminution ← ←Increasing ← a) 2 3 b) 2 1 c) 4 Figure II. Front view of different columns: a) Filled classical column. Supporting particle. and. 1.1-0.5-3. 182 . Tubing. Type of column “Classical type” Filled capillary Porous layer capillary Open capillary tubular Length/ Diameter 1-10 m/ 2-4 mm 10-50 m/ 1 mm 25-200 m/ 0.4. Stationary phase. dispersion mL/min 30-100 0.5-3.4.5 mm d.load S ← Increasing ← ← Increasing ← ← Diminution← ∼ 10 3-5 Flow.6 and Figure II.column Perme V M Loading ation V capacity d.

The earliest GC systems used packed columns where the stationary phase was a thin film of liquid coating the surface of an inert, finely divided solid support. A packed column consists of a glass, metal or Teflon tube 2–50 cm long and 1–4 mm in inner diameter. The tube is packed with a finely divided, homogeneous solid coated with a thin film (0.05–1 μm) of the stationary phase (a liquid). For easier accommodation into the chromatographic oven, the column is coiled to a diameter of about 15 cm. The efficiency of the column increases markedly with decreasing size of the packing particles; however, this also increases pressure within the system, so particles are rarely smaller than 150 μm in diameter. Packed columns afford larger injected volumes than do capillary columns; however, throughput and efficiency are better with unpacked columns of a very small inner diameter (a few tenths of a millimeter). These capillary columns are used with a stationary phase consisting of a uniform film of liquid a few tenths of a micrometer thick that is used to coat the inner walls of the capillary tube. This type of column is also known as open-end column. The materials from which these columns are made, and their coiled configuration, coincide with those of packed columns. Their inner diameters typically range from 250 to 320 μm, but can be smaller (200 or even 150 μm). The sample volume is usually very small, so the detector must be highly sensitive. The thickness of the stationary phase usually ranges from 5 μm for highly volatile species to 0.1 μm for less volatile ones. Although packed columns are more inexpensive and easy to use, capillary columns provide higher resolution. One problem with GC in both packed and capillary columns arises from the physical adsorption of polar compounds onto the surface of the stationary phase, which usually contains silicates. Overcoming it entails pre-treating the columns to remove SiOH groups that form on the support surface through hydrolysis by existing moisture. The liquid stationary phase used should be scarcely volatile, thermally stable and chemically inert; also, it should provide capacity and selectivity factor ( k′ and α) values within appropriate ranges for the analytes. The most suitable stationary phase must be determined on a case-by-case basis; in any case, the material should be similarly polar to the sample components. The column temperature is very important here and should be strictly controlled by using a thermostated oven. The optimum temperature in each case depends on the boiling point of the particular sample and the degree of separation required. Usually, the working temperature is slightly above the mean boiling point (bp) for the sample components. If the bp range spanned by such components is too wide, a temperature program is used instead to change the column temperature during the separation process. Usually, resolution improves with decreasing temperature, at

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the expense of longer elution times; a compromise must therefore usually be made in this respect. As a rule, the retention time doubles with each rise in temperature of 30 ºC. The chromatographic column is followed by the detector. The types of detectors used are rather different from those employed in liquid chromatography and can be classified as follows in terms of sensitivity: (a) Medium Sensitivity: Thermal Conductivity Detector, TCD or catharometer Gas density balance (to check other detectors, not commercial (b) High sensitivity: (b.1)- non-radioactive ionization: flame Ionization Detector, FID thermo-ionic (b.2)- radioactive ionization: electron capture detector, ECD ionization of argon The most commonplace - even in portable equipment - are the thermal conductivity detector (TCD), the flame ionization detector (FID) and the electron capture detector (ECD). In addition, a mass, IR or NMR detector is frequently used to facilitate the identification of individual components in mixtures. The thermal conductivity detector (TCD, see Figure II.4.28), also called “catharometer”, was used in the earliest GC applications. It relies on a combination of the thermal and electrical properties of the sample components. This type of detector is very simple, affords wide linear ranges, responds to both inorganic and organic compounds, and is non-destructive. On the other hand, it is scarcely sensitive, so it cannot be used with capillary columns, where sample size is usually very small.

Electric wire Gas outlet Detector hot block Gas inlet

184

Figure II.4.28. Detector of thermal conductivity. The flame ionization detector (FID, see Figure II.4.29) is the best for organic compounds and one of the most commonplaces in current commercial instruments.

Electrostatic Field

Ion current

+
High Voltage Electrode

-

Flame Collector Electrode

Air Sample Fuel

Figure II.4.29. Flame ionization detector. The effluent from the column reaches a burner where it is mixed with hydrogen and air that is electrically ignited. Burning of most organic compounds under these conditions produces ions and electrons that make the flame space conductive. By applying a potential difference between an electrode that can be the burner end itself and the collector electrode, placed above the flame, an electrical current is produced that represents the detector response to a sample component concentration. This type of detector is scarcely sensitive to flow-rate changes in the mobile phase and not responsive to water, CO 2, SO2 or nitrogen oxides, so it is unaffected by the presence of moisture or oxide impurities in the sample; however, it is highly sensitive to the target species. Also, it exhibits a very broad linear response range and low background noise, and is robust and easy to use, but has the disadvantage of its destructive character. The electron capture detector (ECD, see Figure II.4.30) irradiates the effluent column with γ radiation to alter the electrical conductivity of the gas. The presence of electron-withdrawing organic molecules decreases the current. This type of detector is selective for organic molecules bearing electronegative functional groups (halides, peroxides, quinones and nitro compounds), but is insensitive to amines, alcohols and

185

hydrocarbons. It exhibits a high analytical sensitivity and scarcely alters the sample. Its linear response range, however, is somewhat narrow.

Figure II.4.30. Electron capture detector. Automated systems for continuous on-line monitoring collect samples online, condition them and perform their analysis to provide relevant information in real time; this avoids potential contamination of samples during storage and transport. Also, the analytical results can be used as feedback for process control purposes. Conventional gas chromatographs are bulky and heavy. This entails transferring samples from the collection site to the laboratory for analysis, which, as noted earlier, makes it difficult to preserve their integrity. This has prompted the development of portable equipment exploiting recent micro-technological advances. Portable GC instruments are currently available from manufacturers such as Varian, Agilent and Perkin–Elmer. These instruments, however, only afford the analysis of volatile components (gases), as they cannot provide the temperatures required to convert semi-volatile substances into volatile ones. One instrument that overcomes this restriction is the microFAST GC, which is ten times faster than conventional instruments and affords the determination of environmental pollutants in real time. The instrument uses dual separation columns to ensure that each sample is analyzed individually in a simultaneous manner in each column. Columns in conventional GC instruments are heated by conduction; however, the microFAST GC uses a proprietary system where the heater is inserted between the two columns to heat them not only by conduction, but also according to a temperature program. This heating system is more precise and efficient, and uses less energy than conventional ones. Each column is 1–3 m long and 100–320 μm in inner diameter. It consumes little gas (less than 5 mL min–1) and also little hydrogen (less than 50 mL min –1), which allows the use of small, light gas containers.

186

187 . The analyte was concentrated at room temperature in the trap and then desorbed at fixed intervals by pulse flash electrical heating as a concentrated sample plug for injection into the column (an MXT1 3 m × 0. detract from resolution and can deteriorate the column. 0.constitute a major source of urban pollution. analyte concentrations and trapping temperatures. Very large injected sample volumes give rather broad bands. This allows the sample to be injected in a single volume or in several small ones. organic compounds were determined with a flame ionization detector. The trap consisted of a helical sorbent accommodated in a silicosteel tube. a helical sorbent trap and a gas chromatograph was used for this purpose.32 mm i. The limits of detection achieved were in the picogram-permilliliter region and depended on the trap pre-concentration time and on the variables influencing permeation through the membrane. Following separation.025 mm thick and 163 mm 2 in surface area. Safe sampling of the helical sorbent microtrap was found to be improved by the helical configuration of the sorbent. For continuous on-line sampling. Applications A. A portable set-up consisting of a membrane module.d. Automobile exhaust gases particularly those from diesel engines . The sample can be circulated in a continuous manner (through an on-line microtrap). The method was tested on diesel engine exhaust. the analytes were continuously collected and enriched by the membrane-and-trap interface and directly transferred to the chromatographic column by thermal desorption.One of the most critical components is the sample collection unit. was made of poly(dimethylsiloxane) (PDMS) or bisphenol A polycarbonatepoly(dimethylsiloxane) and used to adsorb the sample. alternatively. which are rapidly released by electrical heating for passage through the separation column. Under pressure from the carrier gas. concentrate and inject samples into the GC column -conventional injection systems are useless with trace level concentrations. the microtrap can be serially connected to an injection valve to construct a “sequential valve microtrap” (SVM). which was placed in an oven at 60 ºC. hence the need to pre-concentrate samples. Hydrogen at a flow-rate of 5 mL min –1 was used as the carrier gas. which generated a turbulent rotation flow on the surface of the sorbent. Automatic on-line pre-concentration and gas chromatographic monitoring of four volatile organic compounds by use of a helical sorbent microtrap. and by the use of low carrier gas flow-rates. It acts by trapping organic compounds. The microtrap consists of a sorbent material packed in a capillary tube that is placed in front of the chromatographic column. the sample was released from the membrane and transferred to the collector trap. silicosteel capillary column coated with a 3 μm thick PDMS film). which must condition. One alternative here is the use of a micro-sorbent trap (a microtrap) for both injection and pre-concentration. The membrane.

25 mm i.25 μm thick film and the mobile phase H2 at a flow-rate of 1 mL min –1. which is straightforward. The sole sample treatment required was the addition of 500 μl of 2. a field-portable GC–MS system was used for the determination of a chemical warfare agent.B. The oven was heated to 40ºC at 250 ºC min–1 and the temperature was then held for 2 min. the contact surface between the mobile phase and stationary phase should be as large as possible. Field analyses were performed on an electron impact ionization GC–MS–EI system mounted on a van. Sampling was done by solid-phase micro-extraction (SPME).5 M NaOH and methanol (in a 1:1 ratio) to effect the catalytic degradation of VX to bis(di-isopropylaminoethyl)disulphide (DES)2.6. reliable identification of analytes. Desorption from the SPME fiber was done in the splitless injection mode for 2 min. Mass spectra were recorded over the m/z range 35–350. using a 100 mm thick polydimethylsiloxane fiber coating. The GC–MS tandem was originally conceived more than thirty years ago for use on planetary aircraft and space tests. In this particular determination. The column was an HP-5MS 30 × 0. field-portable GC–MS systems are highly useful for the rapid. The injection temperature was 270 ºC. High Performance Liquid Chromatography (HPLC) Monica CATALÁ ICARDO. Thus. the greater will be the number of theoretical plates of the column and 188 .4. The most widely used type of mass analyzer is the linear quadrupole. II. However.7. viz. The analysis time was less than one hour and the method allowed (DES) 2 concentrations as low as 1 μg/g to be detected.d. a degradation product of the nerve agent VX. during which SPME headspace sampling took place. model coated with a 0. followed by injector purge at 50 mL min–1. O-ethyl S-(2-diisopropylaminoethyl)methylphosphonothiolate (VX). VX cannot be determined as such. compact and durable. Detection of bis(di-isopropylaminoethyl)disulphide. Both vehicle-mounted and hand-carried equipment of this type has been developed in recent years. however. the smaller such particles are. A vial containing a contaminated soil sample and the reagent was heated at 30 ºC for 30 min. in fact. scientists soon envisaged other possibilities in this hyphenated technique. so it must be previously converted into a detectable degradation product. This makes the size of the particles constituting the stationary phase (or its support) especially influential. José MARTÍNEZ CALATAYUD Based on the general theoretical principles exposed in introducing chromatography.

The use of very small particles in order not to allow the mobile phase to travel by gravity was started in 1964 by J. this signaled the inception of liquid chromatography as we know it today. Calvin Giddings. Pump 189 . This in turn calls for a stronger chromatographic system consisting of materials capable of withstanding the high pressures to be used. which can provide high pressures but causes the flow to pulsate every time the piston is loaded or emptied. and (d) use carefully programmed gradients of the mobile phase.31).4. S.4. This shortcoming. however.R. Solvent proportioning valve Pulse Injection valve damper Drain valve Precolumn Waste Solid reservoirs Detector Column Figure II. (b) deal with complex samples by integrating some sample pretreatment operations on-line. poorly resolved chromatographic peaks.the higher its separation efficiency. Schematic diagram of a HPLC system. (c) improve sensitivity and detection limits by —usually post-column— derivatization of the resolved components in order to enhance their detection characteristics. Lipsky constructed the first assembly for what is currently known as “high performance liquid chromatography” (HPLC. can be greatly circumvented by using two pumps in opposite phases. in the absence of a pulsating flow). Two years later.e. The system is made even more complicated in relation to traditional liquid chromatography by the need to: (a) separate chemically similar components. Propulsion Unit and Elution Modes (isocratic and gradient-based) The propulsion system is intended to provide the pressure required to force the mobile phase to pass through the column at as controlled and uniform flow-rate as possible (i. The small particle sizes used now do not allow the mobile phase to progress through the column solely by gravity.31. see Figure II. The most widely used propulsion system in this context is the piston pump. Packing uniformity is also very important to avoid distorted. This entails using an external force such as that provided by pumping at a high pressure.

4. Some commercial equipment includes an on-line vacuum de-bubbler and passes solvents through a porous PTFE membrane prior to use. Helium can remove up to 80% and evacuation up to 60% of the air initially present in the system.9 or 4. diameter and content. however. Scheme of a six-port valve and its operation.6 mm in diameter are quite suitable for HPLC work. sample dispersion during this step should be virtually zero. Stainless steel tubes 3. In this way.32. Columns HPLC columns vary in length. The problem can be worsened by the use of mixed solvents (e. Many operators. They should be as chemically inert as possible and capable of withstanding high pressures. This entails the use of a de-bubbling system to reduce the air concentration below saturation levels.Gradient elution requires accurate. Mobile phase To column Mobile phase To column Sample loop Sample Waste Sample Waste Figure II.32.4. The outlets of several flasks containing the different solvents (or solutions) are connected to a valve allowing their flow-rates to be continuously controlled. ionic strength and pH to be altered during elution.g. mixing proportions can be changed as required at any time. Sample Insertion The sample volume to be inserted should be accurately known (the operational scale is in the microliter region) and reproducible. The most universal sample insertion system is a six-port valve similar to those used in FIA but constructed in stainless steel in order to withstand the high working pressures required. 190 . choose to use a helium pretreatment followed by evacuation. programmed control of the mixing of the solvents making up the eluent. Its operation is depicted in the corresponding sections on flow-analysis and Figure II. acetonitrile or methanol in water) as air is less readily soluble in solvent mixtures than it is in individual solvents. Also. This allows such variables as the solvent polarity. The solvents used must be highly pure and degassed as bubbling results in distorted or even spurious peaks.

Most HPLC work involves partitioning with liquid stationary phases chemically bonded to a support surface. The original irregular particles of silica gel and alumina have been subsequently replaced with regular spherical particles that provide more uniform packing and reduce the distortion in the separation zones (see the general theory of chromatography in its introduction). Thus. Especially commonplace among the ligands bonded to silica gel are hydrocarbon chains of 8 or 18 carbon atoms (C8 and C18.6 mm i.d. However. B. Mobile Phase The composition of the mobile phase in HPLC varies. As the eluent polarity is increased.5) and working temperature. the eluent composition need not be maintained throughout the chromatographic process. which is variable in ODS1 (it contains a specific number of OH groups) and zero in ODS. Also. and ( b) its absence shortens the column lifetime. by using mixed eluents consisting of two or more solvents). the previously retained solutes are gradually swept. is known as gradient elution. Stationary Phase. Packing Type (modified surfaces) In its original and still widely prevailing meaning. In this way. easily provides 60 000–90 000 theoretical plates per linear meter. manufacturers tend to give their columns a trade name not specifying the treatment they have received. 191 . programmed manner. Columns modified with octadecyl ligands are often referred to as “ODS columns”. In most cases. respectively). The pre-column should be regenerated or replaced on a regular basis.g. its properties can be altered by deactivating residual silanol groups using the “end cropping” method. Its importance lies in the facts that ( a) it can act as a filter by retaining solid particles (impurities) in the sample or solvents that might partially block the column and alter its efficiency and selectivity. “competition” with the solutes for binding sites in the stationary phase increases. A. chromatographic separation is synonymous with adsorption and/or partitioning. pure solvents do not allow all sample components to be separated. 5 μm in diameter tightly packed in a stainless steel tube of 4. There are ODS1 and ODS columns. where the eluent composition is changed in a continuous. it can be changed in a continuous manner (the eluent will initially sweep the less strongly retained components and leave the others anchored to the column). the two types differing in the proportion active residual OH groups. Particles are now typically 5–10 μm in diameter and have pores 60–100 Å in size. In fact. its eluting power can be altered by changing its polarity ( e. This operational mode. The problem with silica gel as a chromatographic support is its narrow operating pH range (3–7. The use of a 3–10 cm long pre-column packed containing the same packing as the column is usually advisable.A column consisting of particles ca. However.

a concentric column can be used to circulate water from a thermostated bath to adjust the temperature of the analytical column. with the separation of basic compounds).g. use two channels to carry the pure solvent and the column eluent. (b) Selective detectors. which respond to the presence of functional groups or specific structures in the analyte (e. fluorescence and IR detectors). measures changes in refractive index in the eluent after sweeping some solute from the sample. It provides detection limits over the range 1–100 ppm. There are some exceptions such as the refractive index detector. This has substantially expanded the scope of reversedphase chromatography (e. C. especially when the refractive indices of the solute and solvent are similar.g. 192 . There are various refractometer models. and measure the differences in refractive index by comparison. Baseline changes in gradient elution work do not allow chromatographic changes to be accurately detected. Temperature Control Unlike gas chromatography.Some polymer-based packing can work throughout the pH scale and at temperatures up to 150 ºC. If needed. The relative importance of each type of detector is to a great extent consistent with its use in other batch and continuous analytical modes. The differential refractometer. However. Detectors All types of available analytical detectors can be integrated with a chromatographic separation process provided they can be furnished with a flowthrough cell. which respond to mass. most. few types of detectors for HPLC are commercially available.or volume-related properties of the eluent (e. however.4. the universal character of which made it one of the most useful until the gradient elution mode gained widespread acceptance. liquid chromatography seldom requires temperature control. namely: (a) General detectors. A typical model is depicted in the Figure II. so it requires careful thermostating. There are two broad categories of HPLC detectors. also known as the “universal detector”. absorption in the UV–VIS region). the refractive index.33. The differential refractometer is extremely sensitive to temperature changes.g.

thus. so it can only measure concentrations at a few different wavelengths. 6. Fluorescence detectors can be more sensitive and slightly more selective than UV detectors. Two different models of the flow-refractometer detector: Left. Diode array detectors constitute powerful tools for qualitative analysis as they allow spectra to be directly recorded without the need to stop the mobile phase flow.Fresnel type. Steel plate. 5. The presence of a solute into the reference part changes the refraction of light beam.Lens Mask Mirror Detector Reference cell Optical zero Figure II. Light source 193 Sample cell . 2. The ability to resolve overlapped spectra by using spectral derivatives or an alternative chemometric technique results in further increased separation power. 3. this enables qualitative identification by stopping the mobile phase flow.4. 7. detector characteristics are illustrated for the UV detector. The UV detector often uses a single interference filter. respectively. Prism. Finally. a cell is divided in two asymmetric parts for sample and reference (pure eluent). Right . Lamp. Teflon mask.Deflection type.01 ppm or even as low as a few nanograms if an appropriate chromophore is used to derivatize the analytes post-column. amperometric detectors are widely used for the detection of electroactive biochemical substances. The more sophisticated models use a monochromator to select the most suitable wavelength in each situation.33. Incident light). 4. Liquid. reference and eluent are flowing through two channels carved into a prism (1. the former can detect concentrations down to 0. The UV detector is normally more sensitive than the refractive index detector. Operational Characteristics of HPLC Detectors In this section.

L D L D Figure II.0008/104 = 8 × 10–8 mol L–1 = 80 nmol L-1 This is the concentration the detector will be able to detect after the solute spreads during the separation process. (L from lamp.4. The latter is usually 10 mm. the absorbance of a solution depends on the concentration of the absorbing substance and the length of the optical path.0004 absorbance units.0008 mol L–1 Therefore. in the latter.34. Scheme of different flow-cells Z-shape (left) and U-shape (right).The flow-cell is usually U. will lead to: DL = 1600 nmol L–1 × 300 g mol–1 = 480 000 ng L–1 194 . With a dilution factor of 20. for a substance of molecular weight 300. with a molar absorptivity ε = 104 L mol–1 cm–1.4. the lowest detectable concentration referred to the sample inserted into column will be: DL = 80 nmol L–1 × 20 = 1600 nmol L–1 which. Based on the Beer–Lambert law. especially. the flow circulates upstream. D to detector). but can range from 1 to 10 mm. which allows small solid particles (impurities) and gas bubbles to be removed. the detection limit will be: DL = (2 × background noise) mol L–1 = 0. Z-shaped (see Figure II. The detection limit or lowest detectable concentration is usually taken to be twice background noise (the baseline).or.34). With a path length of 10 mm and a background noise (minimum reading) of 0. DL = 0.

in terms of variance. its width is also important as it dictates how efficiently neighboring peaks can be resolved. The wavelength of choice will be that best suiting the solutes as a whole. 195 . the height of the chromatographic peak).40 ng The lower limit of the linear range of this detector coincides with the detection limit and its upper limit with the deviations from the Beer–Lambert law that “bend” the calibration graph. Peak height is defined mathematically. In principle. Except for diode array detectors.If the volume of the injected sample is 5 μL. the void volume of both the detector and the column–detector connection should be as small as possible. slight temperature changes rarely alter the analytical response.4.e. all solutes are measured over the same wavelength range. The 254 nm line from an Hg lamp provides 90% of the total amount of light it irradiates. this line is suitable for most organic compounds. but may be far from the optimum values (maxima) for some solutes. even though they can modify the baseline. thus. the solute can spread throughout the chromatographic system. Figure II.35 reproduces the obtained peaks in a chromatographic system when the void volume of detector is changed. the effluent should not absorb at the chosen wavelength. as σ2total = σ2injector + σ2column+ σ2detector+ σ2connectors Based on this equation. Obviously. The choice of eluent is dictated to some extent by the type of detector used. The foregoing refers to the magnitude of the signal ( i. then the detection limit for the system will be: DL = 480 000 ng L–1 × 5 10–6 L = 2. With this type of detector.

e. (b) the derivatization reaction should be fast. which can also provide improved sensitivity and detection limits. and particularly in the pre-column mode .35. path length 1 mm. control. Off-line derivatization. is more difficult to automate. also. whether in the pre-column or post-column mode. Such selectivity can also be introduced via post-column derivatization. Obtained peaks comparing flow-cells: left. (c) based on kinetic and viscosity grounds. and stable in. the mobile phase. Post-column Derivatization The ideal detector would be the one exhibiting selectivity for various sample components.4. (f) the mobile phase should reach neither the reactants nor the support bed. medium. Post-column derivatization is done between the separation column and the detector. 0.Figure II. high void volumes) should be avoided as they result in increased peak width. 196 . the working temperature should be relatively high. using a mixing system and a reaction chamber with temperature regulation.subject to a higher risk of sample contamination and of a greater number of its components entering the separation column.8 µL cell (Chromatronix 210FC1X10T). path length 1 cm. Additional requirements to be met when the post-column derivatization reaction involves heterogeneous phases include the following: (e) the constituents of the reactant bed should be compatible with. The following requirements should be met for efficient operation: (a) the derivatizing reagent should be compatible with the mobile phase. it is more labor-intensive and time consuming. right 0.1 mL cell (common “micro” cell). and (d) increased reaction times and reactor lengths ( i.

but also to physical wear under the action of a continuous flow. As noted earlier. in some cases. with all reactants in solution) or a heterogeneous phase (by use of a solidphase reactor). 197 . a high pressure and. Scheme of a reaction detector based on packed-bed reactor.37 depicts a schematic diagram of the chromatograph designed by G. and (i) the reactor response should be reproducible.36. The Figure II.4. post-column derivatization can be done in a homogeneous phase (i. V: injection valve.36 depicts the scheme of a reaction detector based on packedbed reactor. D: Spectrophotometer Applications Some manufacturers such as SRI instruments produce equipment for fieldwork that affords both isocratic elution and gradient elution.(g) the solid-phase reactor should be resistant not only to chemical agents. an also high temperature. and can be furnished with a UV or conductimetric detector. PM Isooctane ethanol NaI Isopropanol Acetic acid isopropanol P PM P V Column Reactor D Figure II. P: pump. Baram for the fieldwork.4.e.I. Exemple 1 The Figure II.4. (h) any solid-phase derivatization reactions should be quantitative. PM: Pressure monitor.

(b) the concentrations of the sample components should fall within the range 1– 1000 mg L–1 (greater concentrations require pre-concentration of the sample). On the other hand. 198 . PT: Pressure transducer.According to this author. the sample and analytical procedure used should meet the following requirements: (a) the molecular mass of the substances should be less than 500 and their number not exceed 15 (1–5 is the usual number). and (f) it should be flexible enough for application to a variety of analytical problems. (c) it should operate over wide ranges of temperature and moisture. Schematic diagram of the chromatograph for the field work. (e) it should be compatible with the solutions typically used in field work. (b) it should use little energy and not be affected by small oscillations in electrical power. AS: auto-sampler. D: Detector. V: valve. (c) the sample volume should be 10 μL or greater. W: Waste. and be vibration resistant. Eluent 1 PT V P1 P2 injection AS Eluent 2 D W column Figure II.4. a liquid chromatography system for use in fieldwork should meet the following requirements: (a) it should be small and lightweight for easy carrying. (d) it should have as short as possible a warm-up time. (d) analysis times (sample preparation included) should not exceed 10–30 min. P: pump.37.

First pump drives the sample to the needle. The volume of sample (a methanol solution of an hexane extract of snow) was only 2 μL. a pressure transducer and a mixer. was used in this determination. and chlorinated aromatic acids and esters.15 mL min–1 and the working pressure 4 MPa. column. The presence of these compounds influences phytoplankton growth. It uses a UV detector that operates over the range 190–360 nm. working pressures up to 5 MPa and injected volumes of 5–20 μL.8 as eluent. The pump is connected to the column via a needle. The same column was also used. from Nauchpribor (Oriol. (c) The determination of 8 polynitro explosives with a Eurospher 80-5 C18. to separate triazine. The equipment dimensions are 530 mm (L) × 200 mm (W) × 300 mm (H) and its weight less than 15 kg. carbamate.1 methanol–water–trifluoroacetic acid and methanol. It uses a power supply of 100–120/200–240 V. The water surface layer (viz. under different conditions. (b) The determination of 16 polynuclear aromatic hydrocarbons by use of a Nucleosil 5-C18 PAH 75 mm × 2 mm i. one holding the sample and the other a reference solution. The column is accommodated in a solid-type heater.37 consists of two gradient piston pumps with an in–out switching valve. 50–60 Hz. The eluents used were 65:35 methanol-water and 85:15 acetonitrile-water. Russian Federation). under different conditions. the working pressure 1.12 mL min –1. The sample volume required was only 3 mL and the detection wavelength 230 nm. The sample volume was 5 μL and the detection wavelength 230 nm. column and gradient elution at 40 ºC. which is roughly 50–100 μm thick) provides the 199 . The equipment consists of a syringe pump loading up to 2600 μL and providing flow-rates of 2–600 μL min–1. The system was used to determine free fatty acids in natural water samples. constitutes the stopped-flow injector. column and gradient elution at 50 ºC. Example 2 A commercially available Milikhron-1 portable HPLC system. Elution was done at 45 ºC in the isocratic mode. urea pesticides. The flow-rate was 0.9:0. The flow-rate was 0. was used to determine 6 phthalate esters.The equipment of the Figure II. together with a tightening device. which. The flow-rate was 0. using a 50:40:10 mixture of methanol. The eluents used were 36:63. The same type of column. it is a single-beam detector furnished with a mirror that allows the light beam to be passed through two cells. 100 VA. 64 mm × 2 mm i.4. water and 0.1 M tetrabutylammonium phosphate at pH 6.d.5 MPa and the detection wavelength 250 and 260 nm.d. The set-up is completed by an auto-sampler capable of holding 46 vials and a double-beam UV spectrophotometric detector. 64 mm × 2 mm i. Its performance was tested in various determinations of toxic environmental substances including the following: (a) The determination of 11 phenols by use of a Eurospher 80-5 C18.d.28 mL min –1 and the working pressure 5 MPa. the topmost layer.

. 2003 (789) 405 . ISBN 007-237547-7.4. Cloths and K. Chrom. A. Chrom. II. D. Biomed. T.7. 2003 (786) 95 .7. Harvey D. 7. J. Santos. MASS SPECTROMETRY Andrei Valentin MEDVEDOVICI II. Rocha and Damiá Barceló. J. 5 μm particle size). 2. The time required to separate the 14 fatty acids. E. Wiley 2004. Biomed. P. Christian. J.. 4. Kenneth A. J.Y. Wittintgton. T. B. D. M. 6. Principles Mass spectrometry deals with both quantitative and structural information. J. 2003 (32). Prentice Hall Inc. J. Ptacek.d. Contemporary Instrumental Analysis. Calvin Giddings. using acetonitrile–water and ethanol– water mixtures. J. 3. Roskar. Anal. Inc. REFERENCES 1.2. D. Rubinson. 1061. J. column. by means of a mass spectrometer .most valuable information for this purpose. Kharash. reconditioning included. Mc Graw Hill Comp. Rubinson and Judith F.1. The method was applied to oil from sea buckthorn berries (Hippophae rhamnoides) and the results were compared with the fatty acid contents in laboratory culture media of Spirulina platensis algae. Gary D. Ducharme. M. ISBN 0-47145162-2 N. L. 9. 36 (1964) 1980 II.4. Macek and J. 2003 (31). was 45 min.12. 407. R. B. Tu and J. 200 . Chrom. Chem. 10.103. ISBN 0-13-790726-5. Modern Analytical Chemistry. B.7. Analytical chemistry (6th edition). 2000 (879) 3 . 8. 2003 (787) 243-253. Definition Mass spectrometry is the branch of science dealing with qualitative and quantitative interpretation of ions produced under controlled conditions by a sample submitted to analysis. Projean.1066. C. Sc. McErlane. Chrom.. Pharm. Sc. The determination required the derivatization of free fatty acids and their separation on a Separon C18 column (64 mm × 2 mm i. 200. the two being quite consistent in terms of both composition and concentration. 2000. Pharm. J.410.4. Klima. Elution was done in the gradient mode. Kmetec and R.412 5. C. V.

4.7. The basic schema of a mass spectrometer is given in Figure II. (3) the ion nominal mass represents the sum of the integer masses of the most abundant natural isotope corresponding to each of the forming atoms. (4) ion counting / detection. the sample is stroked to generate fragments. separation and characterization require an environment free of interferences. only positive (+) or negative (-) ions are further considered. In modern mass spectrometers.3. the mean atomic mass is calculated as a balanced average of exact masses of the existing isotopes and their natural occurrence. 201 . The ion mass can be expressed in the following manners: (1) the ion average mass representing the sum of the mean masses of the forming atoms. Structural information derives from a hypothetical reformation of the integer from the resulting ions of known masses. (3) ion analysis / processing (mainly separation according to their m/z). It is also worthwhile to note that ion formation. Ions are produced during the ionization stage of a sample representing a pure compound or a mixture. Selected ions are then characterized according to their mass / charge ratio (m/z). Quantitative information is produced by counting the total number of ions (positive or negative) that are produced or by counting the number of ions having a precise m/z being produced during ionization stage. (2) sample ionization. (5) records of the results and interpretation. The sequence between brackets in Figure II. Ion masses are expressed in Daltons (Da). Hence mass spectrometry deals with ions produced by the sample under controlled conditions and these ions are positively or negatively charged. Some applications require that stage (3) be expanded in order to improve the information amount to be taken from the ionization process or to increase detection selectivity.12. Against the fragments generated upon impact.4. according to the charge sign of the ions being analyzed.4. Principles and practice of mass spectrometry are detailed in general textbooks such as references 1 . vacuum levels ranges between 10 -5 and 10-12 torr). expressed in units of elementary charge (e).38 illustrates this alternative. The Mass Spectrum The mass spectrum is the plot of the individual ion abundance as function of their respective m/z.38. (2) the ion mono-isotopic mass represents the sum of the exact masses corresponding to the most abundant natural isotope existing for each of the forming atoms. II. meaning that a mass spectrometer is working in deep vacuum conditions (according to the constructive characteristics of the mass analyzer.Basically. but in no cases. Mass spectrometry is thus achieved by means of the following consecutive operations: (1) sample introduction. z represents the total number of charges existing on the ion. it clearly results that mass spectrometry should be addressed as (+) MS or (-) MS. simultaneous (+) and (-) ion analysis is affordable. analysis of positive ions can be alternatively switched to analysis of negative ions.

4. while the line view gives the intensities only for integer values of m/z.39. The molecular ion is the ion produced by the molecules of the sample by means of the removal or addition of one/more electrons.38.A. The ion with the highest abundance in the mass spectrum is referred as the major ion.09377 Nominal Mw = 215 100 90 80 70 60 50 Ion Relative Abundance ( R. 100 90 ATRAZINE Molecular formula: C8H14ClN5 Average Mw = 215. In Figure II. Normalization of the abundances of the other ions with respect to the major one leads to the relative abundance measurements (R. %) 80 70 60 50 40 30 20 10 0 Line Spectra 40 30 20 10 0 Profile Spectra 211 212 214 215 216 198 199 200 201 207 208 209 210 202 204 205 206 217 218 213 203 219 220 215 216 197 201 202 204 209 210 211 212 217 218 214 219 198 199 200 203 205 206 207 208 Mass to charge ratio (m/z) Figure II. The profile view assumes a definite resolution of the mass analyzer.4.39 a detail from the atrazine mass spectrum is presented (m/z interval ranging from 197 to 220 Da).69 Monoisotopic Mw = 215. Basic schema of a mass spectrometer. computing & interpretation Stage (5) Sample inlet Stage (1) Ion source Stage (2) Mass analyzer Stage (3) Detector Stage (4) Ion isolation (precursor ion) Ion isolation (collisionally induced) Product ions analysis n Vacuum System Figure II.4. 213 202 220 . Detail from the atrazine mass spectrum.Computer Hardware control Data acquisition.%) placed on the Oy axis.A.

p = primary.* →Ci+ + D • j M (3) M (4) • → Ai− +Bj → Ci− + D • j M = target molecule. Sample Introduction Samples can be brought in the ionization area either in a gas phase as well as in a condensed phase (liquid or solid). 203 . 7. An excess of energy is required to generate molecular fragmentation (reactions 3. For inorganic samples. B. If such structural information is required.)M). all separation techniques (thin layer chromatography. organic samples are submitted to MS analysis in order to obtain structural information or confirmation. simultaneous ionization of different molecular species should be avoided. Ionization arises according to the following patterns: (+) mode M +e − p →M + • +2e − s (-) mode M → M (1) + e − p − . s = . liquid chromatography. secondary: * = activated energy state. In such cases. II. Electron impact (EI) Achieving electron impact ionization means that focused and accelerated electrons collide the molecules of the analyte introduced in the ionization area in a gaseous state. A.5.* -• -• * (5) (6) (7) (8) + • * − M +e− → M M + e− + 2 es (2) p p →M M +. Ionization Modes in Mass Spectrometry Gas Phase Ionization A. 4. Samples subjected to MS analysis have either organic or inorganic nature. supercritical fluid chromatography. directly or by means of especially designed interfaces.II. More often.* − .P.* → Ai+• + B j M +. but sample ionization methods are more complex (ion bombardment. micellar electrokinetic chromatography – micelle staking technique. D = molecular fragments. inductively coupled plasma). capillary zone electrophoresis) have been successfully coupled to mass spectrometers.4. information deals with identification and assay of atoms and related isotopic occurrence. Nowadays.7. gas chromatography. C. generally from extremely low amounts.7. = impair electron The basic condition for ionization is that the primary electron posses an energy at least equal to the ionization potential of the target analyte ( Ee-p ≥ (I. no special features are imposed for sample introduction. 8). capillary electro chromatography.4.4. This means that multi component samples should be first separated in individual constituents and then subjected to MS analysis.

The major advantages of Electron Impact ionization mode are its stability. and generation of library searchable spectra.40. Within a mass spectrometer. 0. The reduction of the primary electron energy is not recommended on that purpose as long as ion formation yield and ionization reproducibility are strongly affected. The residence of the ions within the source ranges in the 10 -6 ÷ 5 x 10-6 seconds interval. Correlation between the molecular ion lifetime and characteristics of the mass spectrum. Less Practically no fragmentation. relative high sensitivity. while the time taken for ions to run through the mass analyzer region is higher (about 10-5 seconds). illustrates the relation between the lifetime of the molecular ion and the resulting mass spectrum (Figure II.As the ionization potential of organic molecules falls in the 8 – 12 eV range.1 1 10 Molecular ion lifetime (µs) 100 1000 Figure II. molecular ion in the mass spectrum. Loss of the However. The choice of a standardized value for Ee-p = 70eV is justified as long as lower energy values lead to a significant variance of the ionization yield.4. lack of contamination problems. it results that energies of the primary electron higher than 15 eV should provide proper ionization and fragmentation. Occurrence of Only the metastable ions molecular ion is probable. the fragment ions produced in the ionization area and metastable ions (produced by decomposition of molecular ions after leaving the ion source).40). spectrum. finding a way of reducing ionization energy is mandatory if their observation is required. signal of the observation of molecular ion the molecular in the mass ion is possible. fragmentation. is observable Intense signal in the mass of the spectrum. ease of operation and control of the beam intensity. For chemical species generating molecular ions with extremely short lifetimes. It is obvious that consistent structural information is obtained if the molecular ion and fragment ions are present with significant abundances within the mass spectrum. three types of ionic species exist: the molecular ions.4. Chemical Ionization (CI) 204 . is important. Very high Fragmentation fragmentation. B.

Ionization products in CI exhibit an enhanced stability due to their even electron state (compared to impair electron state for ions generated through EI). CHCl3 (Cl-). CH4 (CH5+). Specific interactions in the ion source are exemplified below on using methane as a reagent gas: CH4 + e-p →CH4+• + 2e-s CH5+ + M → [MH]+ + CH4 + RH 3 (14) anion abstraction 205 . N2O or mixed with N2 (O-. H2O (H3O+). For negative CI. CH2Cl2 (Cl-).→ H2O + [M – H]+ (16) - M + OH → [MOH] (12) electrophilic addition (17) (13) charge exchange + MH → M + RH = reagent gas. p = primary.+ H (15) [RH 2 ] + + M → [ MH] + + RH (11) proton transfer [RH 2 ] + + M → [ MRH2] + RH +• + M → M +• + RH [RH 2 ] + M + OH. H2 + He (OH-). H2NCH2CH2NH2 (H2NCH2CH2NH3+). CH3NH2 (CH3NH3+).). Existence of reagent gas molecules in large excess with respect to the target molecules (10000:1) imposes an increase of the energy of primary electrons (500 eV). ionization of the target molecule is made by interactions with ions produced in the source by means of the electron impact on reagent gas molecules. RH2+ = reagent ion. + RH +• + RH → [RH 2 ] + R (10) H2O + e-p → OH. CI is known as a “soft ionization” technique because energy transfer between reagent gas ions and sample molecules do not exceed 5 eV. CH2Br2 (Br-). s = secondary: • = impair electron The following reagents gases are commonly used in CI (reagent ions are placed between brackets): H2 (H3+). CHF3 (F-). the following reagent gases are commonly used (reagent ions are placed between brackets): NH 3 (NH2-). O2 (O2-. N2O mixed with CH4. CH3CN (CH3CNH+). C2H6 (C2H7+). CF2Cl2 (Cl-). Interactions in CI are summarized below: (+) mode +• − RH + e − + 2 es p → RH (-) mode (9) .In CI. NF3 (F-). NH3 (NH4+). M or MH = target molecule.). CH3OH (CH3OH2+). CH4 + He.

Basic construction of an ion source designed for EI or CI ionization modes. multiple checks of the molecular weight possible due to a large variety of ionization processes.CH4+• → CH3+ + H• CH4+• → CH2+• + H2 CH4+• + CH4 → C2H3+ + H2 + H• CH2+• + CH4 → CH5+ + CH3• CH3+ + CH4 → C2H5+ + H2 C2H3+ + CH4 → C3H5+ + H2 C2H5+ + M → [MH]+ + C2H4 C3H5+ + M → [MH]+ + C3H4 CH5+ + M → [MCH5]+ C2H5+ + M →[MC2H5]+ C3H5+ + M →[MC3H5]+ C2H5+ + M →[M .H]++ C2H6 The advantages of CI mode are: soft ionization. Both EI and CI ionization modes are realized in ion sources constructively similar to the schema depicted in Figure II.41.4.41. 206 . Reagent gas (CI) Magnet Renium Filament Valve Pressure gauge Accelerating and focusing plate Sample inlet (vapours) N Repeller Electrode Ionization area To Mass Analyzer Vacuum Anode Ion Source Magnet Primary electrons (70 eV) Screen system for acceleration and focusing product ions to mass analyzer S Figure II. allowing structural and thermochemical measurements. easy formation of negative ions. universal or selective action depending on the choice of the reagent gas.4.

ions occur. Ionization from Condensed Phases A. molecules of the analyte in gaseous state are brought close to a surface with a high curvature shape (generally tips. In the region above the surface of the frit. relatively nonvolatile liquid matrix on a porous surface and are subjected to a bombardment of atoms or ions having keV translation energies. The electrical discharge ionizes solvent molecules existing in the gas state. the ionization technique is also known as LSIMS (liquid secondary ions mass spectrometry). The screen system serves to focus and acceleration of product ions towards the mass analyzer (screens are charged to increased voltages of contrary sign with respect of the product ions to be analyzed). realized between a tip and a disk shaped counter electrode maintained at 1 – 4kV potential). Reagent gas line feeding the system includes also a control valve and a pressure gauge for controlling the reagent ions formed within the source. A liquid flow containing the target molecules is pumped through a heated vaporizer. The whole ion source is adequately vacuumated by means of turbomolecular or diffusion pumps. The most common viscous liquid is glycerol. whiskers or blades) subjected to intense electric fields (107 ÷ 108 V x cm-1). If high-energy ions are used instead of atoms. the anode. The jet of vapors containing also liquid droplets (continuously evaporating under heat) is oriented toward a discharge zone (typically of corona form. The electron beam collides with the molecules of the sample (or the molecules of the reagent gas) in the ionization area. The generation of protonated or deprotonated molecular ions ([M+H] + or 207 . having as a major drawback the lack in sensitivity. Liquid Phases Fast Atom Bombardment (FAB). These molecules are readily ionized by means of quantum tunneling of valence electrons from the molecule to the metal surface. The use of Xe atoms or Cs ions generates similar spectra. The method is suitable for polar molecules with Mw ≤ 20. The techniques should be considered as “soft” ionization ones. A combination of collisions and charge transfer reactions between solvent ions and target molecules leads to their ionization. The repeller electrode is used for the elimination of unwanted product ions (the electrode is positively charged for elimination of negative product ions and negatively charged for elimination of positive product ions). C. Field Ionization (FI) In FI mode.Thermal electrons produced by the Renium filament are focused and accelerated by means of the plate. Target molecules are dissolved in viscous. target molecules are interacting in a similar way as in CI. and/or the magnet poles. Atmospheric Pressure Chemical Ionization (APCI).000 Da. [M+H] + and [M-H].

The APCI source (orthogonal design). It is especially suited for polar compounds.4.42. A plume of charged liquid droplets is formed (Taylor cone).ions. Ions are ejected from the charged liquid droplets as [M+H]+ or [M-H].42.43 is illustrating the operating principle of ESI. The technique was never particularly routine and has been superseded by APCI. Ionization yield is highly influenced by the solution chemistry of the analyte in the carrier flow. It easily generates multiply charged ions. The ionization pattern is not greatly influenced by the solution chemistry of analytes in the carrying phase.4. It is however especially used for nonpolar compounds.[M-H]-) is thus possible. even with a non-volatile character. corona discharge + + Liquid flow N2 flow + + + Heated sheath (up to 600 oC) Gas phase Counter electrode Needle electrode Extracting capillary To mass analyzer Heated N2 curtain Corona discharge Vacuum Figure II. Figure II. An oriented counter current heated nitrogen curtain continuously determines the reduction of the volume of droplets with a corresponding increase of the electric field density on the surface until a critical volume is reached (Reyleigh limit). Electrospray ionization (ESI or AP-ESI). allowing determination of compounds characterized by a high molecular weight. ESI is intolerant to nonvolatile salts or buffers. 4. A liquid carrier containing target molecules is mixed with a nitrogen flow and forced through a stainless steel capillary maintained at 3-4 kV. The set-up is presented in Figure II. ESI is the softest ionization technique available. The forerunner of APCI was thermo spray (TSP). It is unsuitable for thermally labile compounds and rarely generates multiply charged ions. APCI is used for a wide range polarity of target molecules and is relatively tolerant to nonvolatile salts or buffers existing in the liquid effluent. The ions are extracted toward the mass analyzer via a capillary tube. 208 . Resulting ions are extracted from droplets and sampled through a capillary tube to the mass analyzer.

resulting in lower ionization yields. 2. concentration. and separation. intermediate electronic state (a resonance enhanced process). by means of proton or electron transfer interactions.4. The target molecules contained in a liquid carrier are nebulized by mixing with a nitrogen flow. leading to an extended range for detecting compounds and analysis throughput. sequential irradiation of this intermediate with m photons leading to final ionization. Schematic set-up of an AP-ESI ion source. The sample initially 209 .43. Introduction in the nitrogen flow through nebulization of a dopant can indirectly enhance on ionization. Dual APCI / AP-ESI ionization modes have been also experienced.Liquid flow N2 flow ∼ 4 kV Counter electrode Charged droplets dispersion (Taylor cone) Heated N2 curtain Extracting capillary to mass analyzer Original droplet (+ ions predominate) Vacuum Reyleigh limit + -+ + ++ -++ + + + -+ - Volume reduction -+ -+ +++ + + + -+-+ + -. Nanospray and Electrospray Emitter Arrays are increasing sample throughput. Two stages should be considered: 1. By increasing the power density of the laser source it is possible to change from soft molecular ion spectrum to ones dominated by fragmentation. Electrospray ionization is readily subjected to miniaturization. Multiphoton ionization (MPI). Direct photo ionization of the target molecules is statistically unfavorable. Solvent and analyte are then vaporized together in a quartz tube and are brought in the irradiation zone as a homogenous gaseous phase. C. sample preparation. The dopant is first ionized and the resulting ions are reacting with the analyte. Solid Phases Matrix Assisted Laser Desorption Ionization (MALDI). The use of pulsed tunable dye lasers are strongly enhancing on ionization yields. n photons coherently excite sample molecules to a real. realizing at on-chip scale.- -++ + -+ + + + + + + Explosion Solvention cluster + + Ion extraction Figure II.

PDI is nowadays superseded by MALDI. requires deposition of sample molecules in a condensed phase on the high curvature surface subjected to intense electric fields. although (+) MALDI is the most common. The pulsed laser fascicules generate a plume of ejected particles.000 Da). Both (+) and (-) modes are known. The laser pulse is repeated over a precise interval (commonly 1 Hz frequency). enzymically or chemically. the mixture is transferred to a sample probe.44. and H-). Once prepared. Time of Flight (TOF) and Fourier Transform – Ion Cyclotron Resonance (FTICR) mass analyzers are more often coupled to the MALDI source. MALDI is a soft ionization. Field Deposition (FD). with practically no fragmentation. These ions interact with nonvolatile sample molecules just after the thermal shock. sinapinic acid). expanding at supersonic speed from the surface of the probe. converting them to ions. PDI can be applied for large biomolecules (up to 50. Deposition on nitrocellulose yields simple and multiply charged ions as well. Similar to FI (see C. tending to produce singly charged ions (resulting in very simple spectra). The (-) mode is frequently used for nucleic acid analysis. A laser pulse takes from 1 to 10 ns and impacts an area of about 0. generally under right angles. The molar ratio analyte / matrix is an important operational parameter. Local cluster and size variations occur across the sample deposition site. Fission Fragment Ionization or Plasma Desorption Ionization (PDI).4. MALDI induces no fragmentation. readily varied by the analyst. Pulses of 252Cf fission fragments pass through the probe. allowing selection of the promising crystals for excitation with the laser beam. chosen for its ability to absorb and dissipate energy transferred from a laser beam. The thermal shock vaporizes mobile impurity ions (H +. Biochemical structural studies using MALDI will involve breakdown of the precursor molecule prior to ionization. The MALDI source is presented in Figure II. 210 .01 mm2. In the default operational mode. resulting in the co-crystallization of the sample with the matrix. Probe surface and geometry should also be considered as important for the results of the analysis. Sample molecules are deposited on a Nickel foil. belonging to matrix and analyte. After application. This parameter should be chosen in accordance with the type of mass analyzer that is used. including ions and neutral. UV lasers are usual (N 2 laser at 337 nm or Nd-YAG). IR lasers (CO 2 or Er-YAG) are used only for specialized applications. Applications of MALDI are covering identification of additives in food profiling and characterization of environmental macromolecules (correlation of the analytical data with structure and environmental impact).).mixed with a liquid matrix (eg. the sample and the matrix are converted to a condensed (solid) phase by gas evaporation or vacuum drying. The selection of “sweet spots” is sometimes assisted by a CCD (charge coupled device) camera. New instruments incorporate a microscopic movement sample handler. Na+.

The choice of the primary ions will determine the nature of secondary ones.Laser source Sample Matrix Probe Desolvation Desorption Proton transfer x Probe handler Matrix / Sample co crystallization mass CCD camera H+ + To mass analyzers y Figure II. Mass Analyzers The mass analyzer is the component of mass spectrometer acting on the specific ions generated within the source. Ionization efficiency approaches 100 % for most elements of interest. which are transferred to the mass analyzer. protonated molecular ions and sodium adducts are readily formed. In the latter case. Inductively Coupled Plasma (ICP). to ordinate them according to the m/z values. Kinetic and chemisorption theories are explaining ejection of the different types of secondary ions. Surface imaging (static SIMS) as well as 3D mapping (Dynamic SIMS) is possible. obtained from inert gases or from electropositive / electronegative elements) collides the sample deposited on a metal surface. Liquid or solid samples of organic or inorganic nature are dispersed in an Argon flow and introduced in an inductively coupled plasma torch. II. Temperatures around 8000 oK are atomizing sample and ionizing the resulting atoms. 211 .7. The basic functioning principle of a mass analyzer is the interaction of the target ions with external electrostatic and magnetic fields of precise geometry.6. A beam of ions (either monoatomic or polyatomic.4. positive or negative. Secondary Ion Mass Spectrometry (SIMS). Ions are sampled through a skimmer directly to the mass analyzer after formation of a supersonic jet in the differentially pumped region behind the plasma extraction cone.4. Secondary ions ejected from the surface are trapped and analyzed. The technique is suitable also for inorganic samples as well as for organic molecules. The MALDI source.44.

The electrostatic sector performs only an isokinetic arrangement of ions. m its mass. z its charge and e is the elementary unit of charge. the centrifugal force should equalize the magnetic force. d is the distance between curved electrodes. rE is the radius of the path followed by the ion in the electric field. (2) m e ×d 2 = × B 2 × rB z E × rE (5) The tandem between electrostatic and magnetic sectors acts as a mass analyzer either by scanning the magnetic field B or the electrostatic field E. v is the velocity of the ion. When the ions are introduced in a radial electric field. their pathways became stable only if the centrifugal force equalizes the electric force: m ×v2 z ×e × E = (1) rE d where E is the potential applied between the curved electrodes.Sector Mass Analyzers Two types of sectors are used for construction of the mass analyzers: magnetic (B) and electrostatic (E) sectors. if the velocity during the transfer is equal to the velocity in the electrostatic sector. However. An ion introduced in a magnetic sector moves on a circular path with the radius rB. As the velocity of the ion is the same in both the electrostatic and the magnetic sectors. m ×v2 = z ×e × B ×v rB (2) It is worthwhile to note that the magnetic field acts as a momentum ( m x v) separator. From relation (5) it seems that m/z is independent with respect to the initial acceleration potential V. the relations (1) and (2) should be combined as follows: v= z × e × E × rE m×d (3) din relaţia (1) z × e × B × rB = m It results that: z × e × E × rE m×d (4) introducând rel. again. an instrument defined correlation is found between V and E: 212 . for which. (3) în rel. given to the ion during the transfer from the source to the mass analyzer.

Pathways of a positive ion in electrostatic and magnetic sectors.A).45.4.g. Time of Flight Mass Analyzers (ToF) ToF mass analyzers are based on the measurement of the individual flight times of ions having identical kinetic energy through a specific path (field free drift tube) separating the ion source and the detection area (see Figure II. scan speeds are limited by the hysteresis and magnet heating.46.= E × rE 2 ×d (6) The former relationships are illustrated in Figure II.4. increased resolution of 100.45 . S + E d B + rB N rE V + Figure II. complex construction. The double focusing mass analyzer is used for high resolution measurements (e.000 Da. m ×v2 = z × e ×V 2 (7) The flight time is thus calculated according to the simple relationship: 213 . needs deep vacuum conditions (10 -10 ÷ 10-12 torr). the EPA method for dioxins imposes such a requirement) and fundamental MS studies. Characteristics: resolution of 10.000 over a mass range up to 15.000 over a mass range up to 100.000 Da can be obtained with a cost in terms of sensitivity.4. The kinetic energy of the ions produced within the source is established by means of the acceleration potential V.

214 .000) when using the reflector design. through generation of discrete packets of ions. the time measurement means m/z discrimination.46.4. high resolution (up to 20. high sensitivity. allowing a better control over the initial kinetic energy spread. The Hadamard multiplexing technique is usable together with the ToF principle. needs for high performance electronic controlling detection area. especially at high ion sampling frequencies. Characteristics: no instrumental parameters limit the upper margin of the m/z interval. The flight time of the ions typically falls in the 1 to 100 ns interval. The specific ToF resolution is enhanced by using the reflector design (see Figure II. The ion introduction in the flight zone should be pulsed.t= L = v L × m1 / 2 2 × z × e ×V (8) It is clearly resulting that: m  2 × e ×V  = t2 ×  2 z  L  (9) For given settings of V and L.B). and the spatial distribution. suitable for applications requiring both high resolution and sensitivity for high Mw compounds.

or +) (VDC + VRF cos(2πvt)) (10) The hyperbolic field generated within the electrodes should be written as following: Φ = (V DC + VRF cos(2πvt )) × x2 − y2 r02 (11) If VRF > VDC. Basic functioning of a ToF mass analyzer.V L Field free drift tube A Ion Source + + + + + Detection Area Acceleration plate V Field free drift tube Deflection Electrode + B Ion Source + + + Acceleration plate Detection Area + L + Figure II.47).4. interconnected two by two at positive and negative potentials consisting in both constant and radio frequency modulated components (see Figure II. low mass ions are lost on the Ox direction (collision with left / right electrodes due to the fluctuation of the RF potential) and heavy ions are lost on the Oy direction (collision with upper / lower electrodes due to their inertia toward the 215 . The Quadrupole Mass Analyzers (QMA) The functioning of the QMA is based on the interaction between ions and a hyperbolic field generated within 4 rod shaped electrodes arranged in a square array.4.46. The potential applied to the pairs of electrodes is: Φ0 = ( .

the upper electrode in a ring while the lower one is reduced to a mathematical point (see Figure II. The mass interval for ions having motions on the Ox and Oy directions smaller than ro (the ions stay within the rods) depends on the V DC/VRF ratio. υ is variable. VRF and VDC/VRF ratio.4. VDC. Ion Trap Mass Analyzers or Quistors (QIT) The fundamental working principles of an ion trap are the same as those described for the linear quadrupole for the single reason that a quistor results from the imaginary bending of a linear quadrupole to a closed loop. suitable for structural confirmation of low M w molecules.4.47. wide applications when coupled to GC and LC separation systems. To functioning modes are possible: a. Characteristics: upper mass limit falls in the 3. b.fast altering variable field).000 Da interval.10-5 torr). 216 . needs serial coupling of three devices for allowing MS/MS experiments. the VDC/VRF ratio is chosen such that 1 Da window is selected over the entire mass range. VDC. That imaginary operation transforms the left / right rod electrodes in hyperbolic calottes. accepts relatively high pressure regime (10-4 . Generally.4. low resolution.48). low costs. VDC and VRF are variable. y z o x +r o + - o + + z + x y o + z -Φo +Φo - Figure II. The hyperbolic surface calottes (named also end-caps) are perforated to allow ion extraction from the source and ion ejection to detection area. Ions can be readily formed in the trap or captured from an external source as well. Constant potential is applied to end-caps. resulting in simple vacuum generating systems. VRF and VDC/VRF are constant. This combination of both high and low pass filters yields to a stability window defined by υ.000 . The set-up of a quadrupole mass analyzer. υ and VDC/VRF are constant.

all ions covering a large mass interval are stored together within the ring electrode. because of the limitation of the spatial distribution (the field imperfection is minimal in the center of the trap). The filling / emptying frequency determines the sensitivity of the instrument.48.Ring electrode VRF x cos(ϖt) Trapping ions VRF low Ion ejection VRF high + Ion Source + Detection Area r0 Lissajou shaped ion orbits End-cap electrode VDC z End-cap electrode VDC Damping gas atoms (He) Figure II. a damping gas (usually He or H2) is feed at 10-3 torr pressure in the trap. Forcing an ion population on stable orbits within the ring produces the risk of an uncontrolled increase of the paths radii. the amplitude V RF of the radio frequency modulated potential starts to be scanned. on stable orbits. Within this three-dimensional arrangement ions travel on Lissajou shaped paths. This is also improving the resolution. An ion trap holds a fixed maximum number of ions and depending on the VRF scanning ramp. characterized by precise radii and z expansions. 217 . Increasing the V RF value forces the ions to be ejected from the trap to the detection area (through the holes of the end cap electrode). To avoid this. For low VRF amplitude. Collisions between ions and damping gas atoms reduce ion energies and force them to move closely around the center of the trap. The quadrupole ion trap (quistor). it can be filled and emptied sequentially.4. due to electrostatic repulsions (collisions of ions with electrodes become possible). starting with low masses. A radio frequency modulated potential (VRF x cos(ωt)) is applied to the inner ring electrode. When all ions are stabilized within the ring.

small dimensions. an inexpensive vacuum system is required.4. low resolution (typically 1 Da). The Ion Cyclotron Resonance mass analyzer. A strong magnetic field is used for curving the paths of the ions inside the ICR cavity. A small potential is applied to the trapping (front and back) plates (same sign as the ions) in order to prevent lateral ionic loss.4. As for the magnetic sector.49. The intensity of the magnetic field (B) is so high that ions will finally move on circular orbits within the cavity. suitable for structural confirmation.49. suitable for easy GC and LC interfacing.Characteristics: energy and spatial distributions of ions produced within the source are non critical for the quistor. inherent time controlled tandem MS capabilities. improved resolution is obtained for specific scanning profiles of VRF. The construction of an ion cyclotron resonance mass analyzer is presented in Figure II. low costs. Receiver plate (up) Trapping plate (front) Emitter plate (left) + + VDC B Trapping plate (back) Amplifier + ++ + + -V + + Ion beam (from source) + VDC Counter plate (right) Receiver plate (bottom) B + + + + B + B + + Figure II. the use of low potentials allows a relatively high pressure and therefore. with the proportional reduction of the mass interval or sensitivity. the following relation remains valid: 218 . Ion Cyclotron Resonance Mass Analyzer (ICR).

The receiver plates pick-up signals at different moments of time. electrons travel from the upper plate to the opposite one. When a radio frequency νi is applied to the emitter plate. Functions resulting for each individual cyclotron frequency are then converted to m/z values to produce the conventional mass spectrum. Once the trajectory of the resonant packets of ions comes closer to the receiver plates. high detection efficiency (1 molecule detection). high vacuum systems and demanding computer facilities. radio frequencies of kHz to MHz result for the trapped ions. as the mathematical function is periodic. Characteristics: unsurpassed resolution (around 50.m ×v = z ×e × B r The angular velocity of the ion (ϖ) is given by the relation: (12) ϖ= v = 2 ×π × v r (13) Transferring relation (16) in relation (15). inherent tandem MS capabilities. an “image current” is induced and consequently detected (if the ions approach the top plate. producing a detectable current). ions having the angular velocity ϖi = 2 x π x νi start to absorb the incoming energy (resonance phenomenon) and continuously increase their orbit radius (from circular. it clearly results that: m e×B = z ω (14) For an applied magnetic field of 5 T (Tesla) and a mass interval ranging from 15 to 1. electrons are attracted to this plate from the ground.500 Da. high costs due to the cooling systems of the superconducting magnets. 219 . suitable for extremely high resolution measurements.000). fundamental ion chemistry and stability. m/z scale higher than 100 Da (usually higher than 15. A Fourier transformation should be applied to the “image current” variation in time. A faster method (FT-ICR) involves the excitation of ions by means of a fast sweep of frequencies over a broad range. The signal at time t i corresponds to all ions reaching the resonant state by absorption of energy from the frequency sweep applied at this specific moment. when the packets of ions are situated in the proximity of the bottom plate.000). the paths became spiral shaped).000 for an m/z ∼ 10. the detection process is not destructive. Scanning frequencies νi over a given interval results in the observation of the signals for the corresponding m/z ions. difficult to couple to atmospheric pressure ion sources.

while the selection of precursor / product ions 220 . Ion detection is achieved mainly by means of electron (photon) multipliers.4. In such a post acceleration set-up (PAD) an electrode operated at high potential (up to 30 kV) is placed before the electron multiplier and strongly increases electrostatically the velocity of the incident ions. some manufacturers prefer to introduce a phosphorescent screen between the post acceleration electrode and the multiplier itself. The body of the channeltron is built up from a Lead doped glass with high secondary emissive properties and electrical resistivity. it may result a real need in isolation of a specific ion (precursor or parent) and its further fragmentation followed by the analysis of the resulting ionic fragments (product or daughter ions). Theoretically. A voltage applied between the ends of the conical shaped curved cavity generates a field gradient on the inner walls. A different approach is the continuous channel multiplier or channeltron.4. Secondary electrons are thus multiplied through successive acceleration / impact processes between electrodes (dynodes made from high emissive Beryllium – Copper alloys) having an increased positive potential (12 to 20 dynodes are usually coupled). for some specific purposes. tandem mass spectrometry represents an arrangement in which ions are subjected to two or more sequential stages of analysis (which may be separated spatially or temporally) according to the quotient mass/charge. MS hyphenation in time means the use of a single mass analyzer. Multiple Sequential MS (Tandem MS) As described already in figure II. The main objective of MS/MS hyphenation is to enhance the selectivity characterizing methods focused on quantitative aims or structural / stability studies for the target analytes. Ion Detection Systems Ion current intensities resulting from the mass analyzer are ranging in an extremely wide interval (10-9 ÷ 10-18 A).8. this loop may be repeated few times consecutively.4. MS hyphenation in space requires the use of two ore more mass analyzers coupled serially (one for each ion discrimination process).7.7. lower detection sensitivity may be obtained due to a poorer extraction capacity of the secondary electrons. Because photon multipliers are much more robust compared to electron multipliers. resulting in (MS)n experiments.7. with a corresponding gain in terms of secondary electron yield. II. The primary ions hit an electrode to generate secondary electrons.II. If the speed of the incoming ions is less than 1.38. A solution to this problem is to increase the velocity of the ions ejected from the mass analyzer just before detection.8 x 10 4 m/s. According to the IUPAC Compendium of Chemical Terminology.

7. Quadrupole / Time of Flight (Q/TOF). reproducible CID processes are obtained by means of the controlled acceleration of the precursor ions. The following modes for achieving dissociation of precursor ions are used in practice: 1. at different moments with respect to the beginning of the process. As the energy control upon neutral species (He atoms) is difficult. The process requires Helium atoms to collide precursor ions resulting in their further fragmentation. The typical illustration for “in time” MS hyphenation is the ion trap (quistor). As ions obtained from the source are all trapped within the ring electrode. 3. Sustained Off-Resonance Irradiation (SORI).4. ejection of all ions excepting the parent ones is realized without activating detection. The most common mode of dissociation of precursor ions is undoubtedly CID. Surface Induced Dissociation (SID). Once the precursor ions are trapped. 2. although more than MS 4 experiments are not commonly required. this time with an activated detection system. Collision induced dissociation arises. Collision Induced Dissociation (CID). Time of Flight / Time of Flight (TOF/TOF). Infrared Multiphoton Dissociation (IRMPD). it is necessary to initiate its dissociation to produce the corresponding fragment ions (products). Blackbody Infrared Dissociation (BIRD). 4. 221 . Electron Capture Dissociation (ECD). Post Source Decay (PSD). 2. 3. As example for a tandem MS instrumentation (spatially developed) the triple quadrupole (QQQ) set-up is further discussed (see Figure II. modes 3-6 are characteristic for trapping mass analyzers while the last mode is dedicated to MALDI sources. while VRF is set again at lower values to trap all product (daughter) ions. Once the precursor ion has been isolated. Scanning V RF amplitude results in ejection of the product (daughter) ions in the increased order of their m/z value. as the use of a single mass analyzer is required. 6.50). an increased constant potential is applied to the calotte electrodes to produce their acceleration. Multiple magnetic sectors. The second quadrupole is used only for acceleration of the precursor (parent) ion to achieve CID.is made successively. This procedure can be repeated many times (commercially available systems are allowing up to 13 repetitions). It seems clear that MS hyphenation in time is less expensive with respect to spatially developed tandem instruments. 5. Other arrangements designed for spatial tandem MS experiments are: 1. The ICR based instrumentation is also suitable to work under time delayed MS hyphenation. Modes 1-2 have wide spread applications.

f(m’i/z) R.n mi/z. i = 1. i ≠ j: blocked Detector Technique R. …. mn/z arranged and transmitted mj/z: transmitted mi/z.n Mass Analyzer 1 mj/z: transmitted mi/z. m’2/z. Triple quadrupole arrangement for tandem MS.Collision Induced Dissociation (CID) He atoms Ion Source + + + + + Acceleration electrode + + Precursor ions + + + + + + + + + Product ions Quadrupole 2 Quadrupole 3 Figure II.(mq)/RA(mk) mi/z.A.n R.n mj/z.A.(mj)/RA(mq) R. …. i = 1.. mn/z Mass Analyzer 2 m’1/z. R.4. i ≠ j: blocked Detector R.000) Multiple Ion Detection (MID) Quantitative analysis (with increased sensitivity) + structural confirmation mi/z.….9. Mass Spectrometry – Working Modes The possible ways of achieving MS experiments are illustrated in the Figure II.A. ….. m2/z.51: SINGLE MS STAGE Ion Source mi/z.50. i ≠ j: blocked m1/z. mq/z. II.(mj)/RA(mk) R.A.A. m2/z.7. i = 1. m’n/z transmitted and arranged m’j/z: transmitted m’i/z.…. mk/z: transmitted mi/z. i ≠ j. mk/z).…. m2/z. f(m’j/z) = Product Ion Scan Precursor Ion Scan = Electron Multiplier 222 . = f(mj/z) R.n Mass Analyzer m1/z.A. = f(m/z) Technique Full Scan Mode Qualitative and quantitative purposes (less sensitive) Selected Ion Monitoring (SIM) Quantitative analysis (sensitivity gain ∼1. i = 1.4. k: blocked MULTIPLE MS STAGES Ion Source mi/z.n m1/z.…. mn/z transmitted and arranged CID mj/z fragmente d to m’i/z.…. = f(mj/z.4. i = 1.…. i = 1.A. mq/z.A.. q.

Busch.. R. 5.…. K. i ≠ j: blocked R. q. 2nd 3. R. i = 1.n m1/z. i = 1. k: blocked / / / Figure II. m’n/z transmitted and arranged ∆ m = ct.(m’q) RA(m’k). Chichester (2002).51. i = 1. Mass Spectrometry Basics.. i = 1.…. Herbert. R.. Barker. Mass Spectrometry: Analytical Chemistry by Open Learning . i ≠ j.n mj/z: transmitted mi/z. New York (2001). R. Mass Spectrometry: Principles and Applications . Advances in Mass Spectrometry .A. Budde Editor. W. V. CRC Press. Constant neutral loss/gain scan mi/z. 2nd Edition.. 6. J. American Chemical Society. 26 (2002). REFERENCES..n mj/z: transmitted mi/z.A. J. E. f(m’j/z. J.. Boca Raton. C.4. Edition. m’2/z.….n Mass Analyzer 2 Detector Technique mi/z.A. Wiley & Sons.L. Chichester (2001). Mass spectrometric modes (for single and multiple MS stages). 4. i = 1.(m’j) RA(m’k). m’k/z). i = 1.A.n m’j/z.….…. = Single Reaction Monitoring (SRM) = Multiple Reaction Monitoring (MRM) mi/z. Wiley & Sons. Mass Spectrometry. de Hoffman.Ion Source Mass Analyzer 1 CID fragmente d to m’i/z. Gelpi Editor. J. Johnstone.A. m’q/z.n mj/z fragmente d to m’i/z. i ≠ j: blocked mj/z fragmente d to m’i/z. David Editors.A.. 17(6S). J. Oxford University Press Inc.n m1/z.L. Chichester (1999). mn/z transmitted and arranged m’1/z. Stroobant. 1. f(m’j/z) R. 2..(m’j) RA(m’q). m’k/z: transmitted mi/z. E. m2/z. i ≠ j: blocked m’j/z: transmitted m’i/z. m’q/z. Analytical Mass Spectrometry Strategies for Environmental and Related Applications.…... Wiley & Sons. 223 . i = 1. mn/z fragmente d to m’i/z. m2/z.….

Turecek. Lewis Publisher.uic.M. the selectivity in gas chromatography only depends on the analyte / stationary phase interaction. confirmation and quantitation of volatile or semi volatile analytes in complex mixtures. University Science Books. 8.Florida (2002).scripps. New York (1983). www. 12.8. Because interaction between analytes and the mobile phase does not exist practically.4. 11. II. 3.W. F. b.edu. Principles Gas chromatography is a separation technique suitable for volatile / semi volatile compounds based on their differentiate partition between a moving carrier gas (mobile phase) and a stationary phase consisting in a granular solid adsorbent or a viscous liquid phase uniformly distributed on the surface of an inert support. New York (1989). GAS CHROMATOGRAPHY / MASS SPECTROMETRY Andrei Valentin MEDVEDOVICI II. Marsh. 2nd Edition. 10.A. R. 7. McLafferty Ed. VCH. Definition Gas chromatography / mass spectrometry (GC/MS) is a coupled analytical technique used for the following purposes: a. 102.4. Tandem Mass Spectrometry. II. 1. Quadrupole storage Mass Spectrometry.chem.8.com. 2. John Wiley & Sons. c. http://masspec. 9. www.E. California (1996). Interpretation of Mass Spectra. Wiley Interscience (1998). http://chipo.asms. B. F. Useful Net sites.8.i-mass. Weiheim (1991). Handbook of Mass Spectra of Environmental Contaminants. R. John Wiley & Sons. 4th Edition. Florida (1992). Boca Raton.1. Sansalito... determination of molecular weights and / or elemental composition of volatile / semi volatile unknowns.4. 4. F. 224 . vol.org. Hites. Smith. structural determination of volatile / semi volatile unknowns in a mixture by means of spectral matching or spectral interpretation.edu. Understanding Mass Spectra: A Basic Approach .W. FT-ICR MS: Analytical Applications of Fourier Transform Ion Cyclotron Resonance Mass Spectrometry. www. R. Asamoto Ed.arizona. McLafferty.2.edu.chem. 5.

usually at a temperature at least 10 oC higher than the higher column operating temperature over the whole chromatographic run. 225 . Mass spectrometry generates both qualitative and quantitative information related to the target compound. To avoid analyte condensation through the bypass zone from the GC oven to the MS ion source. In such case the direct coupling between MS and GC is allowed.5 – 1.4.7. polymethylcyanopropylsiloxanes. special requirements are imposed in terms of selectivity to obtain acceptable resolution.5 mL/min. polyphenylmethylsiloxanes. When using open tubular capillary columns having internal diameters below 0.52. it is not surprisingly at all that the coupling of these two techniques was suggested shortly after the GC development period placed in the mid fifties. This results in a wide variety of stationary phases routinely used. 4.4. the mass spectrometer acts as a detector of the gas chromatographic system. Stationary phases for capillary gas chromatography (CGC) are basically belonging to five classes of polymeric materials. by data interpretation of ionic fragments characterized upon their mass to charge (m/z) ratio and specific formation yields.Gas chromatography is achieved on both packed (in practice is considered already obsolete) and open tubular capillary columns (present state of the art).8. Both GC and MS bring something unique to their union. Complementarities and compatibility features of the techniques are reviewed in the Table II. The bidimensional information routinely extracted from a “classic” chromatogram (detector response as function of the time period elapsed from sample injection) is expanded by the addition of a third dimension (detector response as function of the m/z value) – see Figure II.4. the operating pressure of GC and MS instruments is the main parameter introducing major coupling difficulties. Therefore. Interfacing MS to GC As resulting from Table II. The direct interface requires the introduction of the capillary column exit in the ion source of the MS instrument. Otherwise speaking. classified according to their increasing polarity (polydimethylsiloxanes. Gas chromatography on open tubular capillary columns is characterized by a tremendous increase of efficiency (extremely narrow peaks are produced) resulting in a moderate need for selectivity. polymethyltrifluoropropylsiloxanes and polyethyleneglycols). optimal carrier gas flow through the column (as resulting from the Van Deemter curves) falls in the 0. Because packed column gas chromatography exhibits less efficiency (generates broader peaks). column heating is mandatory.7.32 mm.3. as long as modern vacuum systems achieve 10 -5 – 10-8 torr pressure level under continuous gas flow feeding up to 1.5 mL/min interval.

2. an interface (separator) should be inserted between the chromatographic column and the inlet of the MS ion source.4. In such situation. 3D result of a GC / MS analysis. carrier gas flow is much higher (5 – 40 mL/min).Table II. Parallel between characteristics of GC and MS analytical techniques. to improve sensitivity. reduction of the carrier gas flow to an acceptable value allowing adequate vacuum level in the ion source. # 1 2 3 4 5 Characteristic Ability of handling mixtures Ability to provide structural information Ability to accept vapor state samples Ability to consider ng amount of samples Working pressure GC     MS     atmospheric (at the deep vacuum column exit) When packed columns (or megabore open tubular columns) are used. 226 .7. Ion Abundance A Mass/Charge m/z Typical Mass Spectrum Retention Time tR (min) Typical chromatogram Figure II. The separator has two main functions: 1.4. increasing concentration of the analyte in the carrier gas flow fraction transferred in the ion source.52.

35 1-5 1 .8. Teflon. silicone. porous membrane separator (membranes are made from glass.53 (A.4.18 7 .4.20 1-5 105 2 . accepted operating temperature (oC) 350 350 280 250 350 250 250 250 350 # 1 2 3 4 5 6 7 8 9 Interface Supersonic molecular jet Porous glass tube Porous Teflon tube Silicone membrane Silver membrane (type 1) Silver membrane (type 2) Porous stainless steel membrane Combined silver frit and silicone membrane Slit N 40 .50 1-5 4 .100 227 . Technical characteristics of some separators used for GC / MS interfacing.B and C).108 105 10 – 60 Flow interval (mL/min) 1 . The characteristics are summarized in Table II. The following devices are used as GC / MS interfaces: supersonic molecular jet separator. respectively. and CF GC and GFMS are carrier gas flow exiting column or reaching the MS ion source.100 4 .  % 50 50 50 50 50 50 50 40 30 Max. The separator behavior is characterized by means of separation yield ( η) and concentration factor (N) calculated according to the following relations: η= QMS × 100 . silver.4. The working principles are illustrated in Figure II.8.The introduction of a separator between GC and MS means already the insertion of a supplementary void volume between column and detection area. QGC N = η× CFGC CFMS where QMS and QGC are analyte amount reaching the ion source or exiting the chromatographic column.86 5 .24 10 . respectively. resulting in a significant loss in terms of separation efficiency (up to 60%). Table II. combined silver and silicone or stainless steel) and slit separator.60 1 .60 2 .30 1 .

note that for routine GC / MS analysis.25 mm. the voltage output from the preamplifier of the electron multiplier is converted from an analog signal to a digital value (using an analog to digital converter) at a rate of 10. m/z scales are much broader: up to 800 or even 1000).53. each channel should be sampled for 8 ms (accepting a spectral resolution of 1 Da and a m/z interval ranging from 0 to 250.4. However. for accurate reproduction of the Gauss profile. 10 points are needed.L1 d1 L2 d2 d3 From column To ion source Carrier gas Analyte V1 A d1=0.1 mm.4. it clearly results that a MS spectrum should be collected every 2 s. meaning that each MS signal has a Gauss shape. The line spectrum appearance more often used in practice is in fact a profile spectrum. C. d2=0.000 to 100. V1=10-3-10-4 torr.15 mm. Basic construction of some separators used for GC / MS interfacing: A. MS instruments are characterized by a defined resolution. V2= 10-4-10-5 torr V2 B d From column Membrane To ion source From column C V To ion source V Figure II. Assuming that a GC peak exhibits a width (6 σ) of about 20 s and that a correct peak profile as a Gauss shaped function could be obtained with at least 10 points (see Figure II. As long as a mass spectrum is obtained by sequential scanning on all m/z channels. membrane separator. supersonic molecular jet separator. Data System for GC / MS Instrumentation A GC / MS experiment produces a huge amount of data. II. B. In commercially available MS detectors.4. slit separator.50 mm. meaning that for 1 minute of a chromatographic run.4. The whole process is illustrated in Figure II.54).000 times per second.4.55. Consequently. Accordingly. one concludes that each m/z channel is sampled with a 800 μs rate for generating the MS response. L2=0. L1=0. the instrument will 228 .8.

008 seconds for each mass channel ∼ 20 seconds 3 4 5 6 Time (min. Data production algorithm in an MS detector coupled to a GC system. Figure II. Thus.0e4 2. At least 0.2e5 1.) At least 1 MS spectra every 2 seconds Figure II.0e4 4.produce up to 6. most data systems find m/z peaks in real time and convert them into mass / intensity pairs. Each spectrum stored on the hard disk of a computing system is associated to a retention time value.0e4 4.4.0e4 0 0 50 100 150 200 250 m/z 1.0e4 2.0e4 6. 219 220 213 214 215 216 217 218 229 .0e5 8.0e4 Intensity 6.0e5 8. to avoid saturation of bulk storage devices.4.000.55.0e4 0 0 1 2 6 2/250 = 0.54. Ability of reproducing Gauss shaped profiles according to the sampling data frequency.000 numbers.8 mseconds for each data 212 (m/z) 1.

5. comparison between the MS spectra considered as “characteristic” for a given chromatographic peak with a spectra library. Comparing normalized MS spectra acquired during peak elution lead to a spectral purity check tool. examining MS spectra acquired during peak elution.6.8. because the intensity of chosen specific lines varies for each chemical species. useful for evaluation of an eventual chromatographic co elution phenomenon.II. having a comparative look on all mass spectra characterizing compounds separated as integrated peaks within the chromatogram. a MS instrument coupled to a GC system produces a 3D data set. Chromatograms obtained in the SIM or MID modes are in fact TIC. Note that EIC is related to the functioning of the MS detector in the scan mode (see chapter referring to Mass Spectrometry). The quality of the structural information strongly depends on the quality of the mass spectrum considered as characteristic for an eluted chromatographic peak. the sensitivity is enhanced by means of a higher Signal to Noise ratio (S/N) and by the inherent selectivity induced to the processes. Each point obtained in the xOy plane (abundance as function of retention time) is obtained by addition of all individual intensities existing on the m/z scanned channels over the collected mass spectrum (placed in the yOz plane). 2. the assumption that the MS response is a universal one does not apply anymore. II. Qualitative Information in GC / MS Qualitative information in GC / MS is obtained only if the MS instrument runs in the scan mode. 3. the data processing software is able to retrieve only narrow intervals or specific m/z values. 230 . going through the chromatogram and inspecting individually the MS spectra obtained at the apex of each of the chromatographic peaks. On operator request. The Selected Ion Monitoring (SIM) or Multiple Ion Detection (MID) modes are related to the operation of the MS instrument and not to the data processing mode.4.8. Data Interpretation Modes in GC / MS Unlike traditional GC detectors. Chromatograms obtained by considering only the intensities recorded on specific m/z channels or limited m/z intervals are usually referred as EIC (Extracted Ion Chromatograms).4. because each point in the chromatogram represents the intensities addition on all available m/z channels. However. MID mode is generally associated to spectral confirmation. Four approaches of examining GC / MS data exist: 1. Such manner of data interpretation is named TIC (Total Ion Current Chromatograms). For EIC. 4.

If low intensity averaged spectrum is obtained. e. PBM allows calculation of the relative uniqueness of the spectral lines (higher m/z lines exhibit higher relative uniqueness compared to lower m/z lines). this is representing the “reduced” spectrum. library spectra are also represented in this format.5 ng reaching the ion source).Conditions for getting “good” characteristic MS spectrum for a chromatographic peak are: a.4. avoid spectral saturation (a lot of MS signals having 100% relative abundance within the spectrum – see Figure II.1% relative abundance – illustration in Figure II. eliminate background interference by means of background subtraction (make subtraction of the averaged MS spectrum of the baseline close to the considered chromatographic peak). the software further considers only the 10 most abundant lines separated by at least 14 Da.56). their relative uniqueness. If spectral saturation is observed during peak elution. enough analyte (at least 0. d. m/z scale large enough to observe the molecular ion as well as its intrinsic isotopic profile.56). Another library searching algorithm is the PBM (Probability Based Matching) initiated by F.4. it is advisable to consider only scans around peak apex. is advisable to consider only MS scans at the beginning or at the end of the peak. c. 231 . b. Comparison in PBM considers three factors: m/z values. match quality is based on the recovery of the 10 lines in the sample reduced spectrum within the reference reduced spectra (line is present or not and line intensity is comparable or not). The most common one is the 10 peaks algorithm: from the characteristic MS peak spectrum. McLafferty from Cornell University. their relative intensities. avoid elimination of low intensity MS signals by means of an inappropriate choice of the intensity threshold (generally equal to or lower than 0. There are basically two library searching algorithms.

232 . Specific instrumental conditions may not be known. It is worthwhile to note that spectra libraries are commonly containing Electron Impact (EI) mass spectra produced with a 70 eV striking energy. Distinguishing “good” characteristic spectrum from saturated or high threshold computed ones. Custom made libraries can be obtained on specific ion production and / or mass analysis.56.100 90 “Good” characteristic spectrum 200 Saturated spectrum 173 200 215 Relative Abundance (%) 80 70 60 50 40 30 20 27 10 0 180 190 100 110 120 130 150 170 200 140 160 210 100 130 150 160 180 190 110 120 140 170 200 200 Too high threshold 210 20 40 60 70 90 30 50 80 20 30 50 60 80 90 40 70 43 58 173 68 71 96 104 122 132 158 145 27 104 96 122 132 145 215 43 58 71 68 158 100 90 Relative Abundance (%) 80 70 60 50 40 30 20 10 0 160 170 180 190 200 100 110 130 150 120 140 220 210 20 30 40 50 60 70 80 90 43 68 132 158 58 173 215 m/z Figure II. the library search can be made as a forward search (is the characteristic spectrum of the peak retrievable within the library? – faster approach) or as reverse search (is the library reference spectra fitting on the characteristic spectrum of the chromatographic peak? – slower approach).4. and obtained with Quadrupole Mass Analyzers. Does not matter which algorithm is used. Matching for spectra obtained with Ion Trap Mass Analyzers may lead to altered results.

8. Using isotopic labeled internal standard and the SIM operating mode. Quantitative Information in GC / MS Quantitation in GC / MS is based on peak area measurement in TIC or EIC chromatograms resulting either from full scan or SIM / MID operating modes of the MS instrument. The best internal standard should be chemically very similar to the analyte itself. This results in important acquisition timesavings. the open tubular columns used with silicone polymer bonded phases are from 100 % methyl silicone or 95 % methyl and 5% phenyl (nonpolar ones) to 50 % methyl and 50 % phenyl. Applications GC / MS should be considered as the principal primary screening method for pesticide analysis in food. II.4. The use of an external standard supposes the availability of the target analyte as a reference standard substance. 2.4. With SIM / MID operating modes.8. sensitivity characterizing GC / MS technique falls in the low pg range. increased S/N ratio (the addition of multiple measurements amplifies the signal and self-annihilation of the background due to its random character). SIM / MID modes allow data sampling with an increased frequency only on a given m/z channel or narrow channels interval. Instead of scanning over the whole m/z range. Consequently. environmental and clinical samples. Due to the broad range of polarity characterizing the analytes. The control software of modern MS instruments allows the selection of different ions during the same chromatographic run. The two main calibration strategies are based on external and internal standards. 86 % methyl and 14 % phenylcyanopropyl or 50 % methyl and 50 % phenylcyanopropyl (medium polar or polar ones).000 folds interval. The very best internal standard is an isotopically labeled version of the analyte. The primary choice concerning the stationary phase for screening residues of pesticides is a low polarity one. either 100 % methyl silicone or 95 % methyl and 5 % phenyl. 233 .8. separations are achieved on stationary phases exhibiting different polar character. The conversion of the peak area to analyte amount imposes calibration. the signal intensity is taken only on a single m/z channel (or a limited number of m/z channels). Such achievement leads to the highest sensitivity obtained for all separated compounds in a single chromatographic run. 50 % methyl and 50 % trifluoromethylpropyl. Enhanced sensitivity is produced due to: 1.II. any losses of the analyte during the sample preparation procedure are duplicated by losses of the internal standard. Consequently. increased accuracy for peak integration (peak shape is better reproduced due to a larger number of points generated during peak elution).7. The overall gain in sensitivity for SIM / MID operating modes ranges in a 100 to 1.

precise temperature control during injection (achieved by PTV) and the presence of suitable chemical additives. The use of the selective ion-monitoring (SIM) feature drastically enhance on sensitivity although structural confirmation is lost. Studies achieved for organophosphoric and carbamate pesticides. corresponding to cis and trans isomers.1 µm should be avoided for use. helium.33 µm film thickness should be considered as a basic starting choice. baseline separated. Thus. Some other phenomena are associated to injection systems.20 – 0. leading to the split of the chromatographic peak in two equally intense peaks. when vaporized in packed liner fitted PTVs. The latter. Hydrogen is not suitable for the MS detector. the ion 234 . The presence of acetic acid. Nitrogen. is quantitatively transformed to deltamethrin in HSIs. for example. as well as the (+) or (-) ion monitoring modes are encountered. low molecular mass amines. especially for OCI. hot splitless injector (HSI) and programmed temperature vaporizer (PTV) are mainly used. phenylureas lead in conventional hot splitless injectors to extensive and irreproducible formation of isocyanates and amines. As an example. especially for separation of labile pesticides. while nitrogen exhibits less flat Van Deemter curves for gas velocities higher than optimal. and organic anhydrides during injection determines a minimization of the thermal decomposition effect or reproducible conversion to the corresponding isocyanates. pulsed split less and on column injectors lead to the following hierarchical tolerance: OCI < pulsed HIS < PTV splitless < PTV solvent split. The use of a retention gap between the injection port and the capillary column has been widely discussed in literature. The influence of the solvent. Checking for each particular case should be more advisable. differentiation between parent compounds and the N-demethylated metabolites is possible.4. helium remains the best choice.Considering the deactivation degree of the column wall in a close relationship with the deposited layer of stationary phase. readily isomerizes. Injection of thermally unstable analytes can be controlled via pressure pulses (based on electronic pressure controlled systems – EPC). Derivatization may also enhance on thermal stability. the gap geometry. 25 – 30 m length fused silica open tubular (FSOT) columns. on PTV splitless.15 – 0. In Table II. in parallel. Phenylurea pesticides are alkylated with iodoethane and sodium hydride to yield thermostable compounds. As injectors for capillary GC separation of pesticides and related residues. with 0.9. and the analyte concentration on peak efficiency and symmetry do not lead to a general accepted recommendation considering the use of retention gaps. cold on column (OCI). films thinner than 0. When using iodoethane instead of iodomethane. and hydrogen are carrier gases commonly used in GC.25 mm internal diameter and 0. Tralomethrin. PTV solvent split. the injection volume. Both electron impact (EI) and chemical ionization (CI) are currently used as ionization techniques in MS detection.

125. 281 97.9. 242 125. 185 127 235 235 195. 153. 323 197. 219 171. 241.p’) DDT (p.fragments frequently monitored by MS for some pesticides separated by GC are enlisted. 132. 171 109. 231. 183 123. 329 235 . 263 123. # 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 Pesticide Azinfos Ethyl Chlorfenvifos Chlorpyrifos Chlordane Coumafos Dichlorvos Dicrotofos DDD (p. Major m/e signals in the mass spectra of some pesticides separated in gas-chromatography with mass spectrometric detection (ionization by electron impact). 298 373 226. 377 209. 125. 277 100. 242. 298 73 227 291 109.4. Organochlorine Cyclodiene Organothiophosphate (aliphatic) Organothiophosphate (aliphatic) Organothiophosphate (phenyl) Organothiophosphate (phenyl) Cyclodiene. 125. Bridged biphenyl Organochlorine. 384 158. Organochlorine Organochlorine Phosphonothioate Phosphonothioate Ester Organochlorine Organothiophosphate (phenyl) Organothiophosphate (phenyl) Pyrethroid ester Pyrethroid ester Organophosphate Major m/z signals in MS 77. Bridged biphenyl Cyclodiene. 272 109. 181. 260. 362 109. 143. 160 267. 200.p’) Endosulfan Endrin Ethion Ethoprop Fenchlorfos Fenitrothion Heptachlor Lindane Leptophos Merphos Methoprene Methoxychlor Parathion Parathion (methyl) Phenothrin Resmethrin Stirofos Chemical Classification Organothiophosphate Organophosphate Organothiophosphate (pyridine) Cyclodiene Organothiophosphate (heterocyclic) Organophosphate Organophosphate Organochlorine. 285 109. 339 263. 270. 265. Table II.

New York (1998). 3. Antibodies belong to the family of glycoproteins known as immunoglobulins. Niessen. Mc Master.W. F. R.4. Principles & Techniques. R. Handbook of Mass Spectra Environmental Contaminants .edu/spectral/massspec/ II. Boca Raton (1992).E. Wiley & Sons.9. Practical Introduction to GC-MS Analysis with Quadrupoles.accustandard.A.W. www. The macromolecules able to develop such an immunological response are called antigens. C.REFERENCES 1. J. Karasek. 5. Gaithersburg. 2nd Ed. MD:NIST Standard Reference Data (1995). Amsterdam (1988). Wiley – VCH. 2. M. 6. herbicides. Oehme. Useful Net sites 1.A. Basic Gas Chromatography-Mass Spectrometry: 2.washington. Registry of Mass Spectral Data with Structures . Series Chromatographic Science. Certain analytes may be quantitatively determined as well. Clement. F. They are synthesized by animals as response to the presence of a foreign substance. Weinheim (1999). Vol. McMaster. polychlorinated biphenyls (PCBs). 86. Current Practice of Gas Chromatography – Mass Spectrometry .com/asi/.. FL Lewis Publishers. Mihaela BADEA Enzyme immunoassay kits are now available for qualitative field-testing or for laboratory screening and semiquantitative analysis of pesticides. W. mononuclear and polynuclear aromatic hydrocarbons. 4. Antibodies are known to show affinity for specific compounds. M.edu/gcms. Elsevier Press.C. and many other compounds in aqueous and soil samples. 7. 3. http://ull. Marcel Dekker. http://depts. GC-MS: A practical User’s Guide. nitroorganics. New York (1989).chemistry.M. Hites. 5th Ed. New York (2001). The method is rapid and inexpensive.uakron. IMMUNOASSAY Giuseppe PALLESCHI. Wiley – VCH. 236 . McLafferty. with a degree of accuracy comparable to gas chromatography or high performance liquid chromatography determination. pentachlorophenol.. NIST / EPA / NIH Mass Spectral Library for WindowsTM.

substrates and cofactors. metals.9. After the binding reaction the amount of label associated with the solid phase will be inversely related to the concentration of analyte. etc) or function of the label on the formation of the antibody-antigen complex A more sensitive approach that is less prone to interference problems is the heterogeneous assay format in which there is a separation step. The first major distinction is made between homogeneous and heterogeneous immunoassays. the extent of this displacement will be dependent upon the amount of analyte added. b) Displacement immunoassay in which the antibody is immobilized on a solid phase and the antigen is labeled. electrochemically active compounds. When the unlabelled antigen (the analyte) is added there is a displacement of the labeled material and. After the binding reaction a second labeled antibody against a different epitope is added in excess and the amount of labeled antibody associated with the solid phase is directly related to the amount of analyte. At the beginning of the assay all the available binding sites on the immobilized antibodies are occupied by labeled antigen. A homogeneous system does not require separation of free and bound antigen. electrochemical. The classification of the immunoassays is somewhat variable. Immunoassay Principle An immunoassay may be defined as a technique based on the reaction between an antigen and an antibody for measuring the concentration of the desired analyte.4. In this format a competition between the labeled and unlabelled antigen (the analyte) for the available antibody binding sites takes place. 237 . An antibody (Ab) recognizes specifically the corresponding antigen (Ag) and forms a complex antibody-antigen (Ab-Ag). There are many types of labels used to monitor the antibody-antigen binding reaction including: particles. Within each of these classes there are different ways for designing the assay. II. the assay relies upon the alteration of a property (optical. radionuclides. dyes. but generally reflects the assay principle and the nature of the label used to monitor the immunoaffinity reaction. A capture antibody is attached directly or indirectly to a solid phase. solid-phase immunoassay in which antibody is immobilized on a solid phase and the antigen is labeled. enzymes. Some examples of immunoassay configuration are described below: a) Competitive. c) Sandwich immunoassay is suitable to use only for high molecular weight antigens which posses at least two antigenic sites (epitopes).1.The specificity of an antibody is directed against a particular site on the antigen known as epitope. under appropriate condition.

In other words. a spectrophotometer should be used to read the absorbance to plot a calibration standard curve. anchored onto the antibody sites. During this period. antibodies are immobilized to the walls of the test tubes.5’tetramethylbenzidine if horseradish peroxidase is used as label) is added to the mixture. Then a solution of a chromogenic substrate (i. In the ELISA one of the reagents is bound to a solid phase. For qualitative screening. Thus. horseradish peroxidase. which usually takes the form of a colored dye. The enzyme conjugate. In ELISA an enzyme is used as label linked either to the antigen or the antibody. After the incubation. this solid phase has the form of a 96-well microtitre plate. the lesser the amount of the analyte in the sample. Each enzyme molecule can rapidly catalyze the conversion of thousands of substrate molecules into product molecules that react with chromogen. A measured amount (between 10 and 50 μL) of sample or sample extract is added to one such test tube containing an assay diluent (a phosphate buffer). For semiquantitative determination. many enzymes may be used: urease. alkaline phosphatase. etc.9. The required period for incubation varies from substance to substance but can range from 5 238 . This label in conjunction with a suitable substrate produces the assay signal. ELISA techniques that involve electrochemical measurements and the screen-printed electrode technology were developed. The enzyme catalyses the transformation of the substrate into a product that reacts with the chromogen causing a colored product. Usually. As labels.e. ELISA Technique The enzyme-linked immunosorbent assay (ELISA). Such test tubes and plates are commercially available and supplied in the test kit.2. or microwells. Each of these can be employed with a number of substrates to generate a signal. however. In the last years. the color should be read as soon as possible because it becomes unstable after 30 min.II. the darker the color. a visual comparison of color with standards can be made. The solution mixture is incubated or allowed to stand for a specific amount of time. The enzyme conjugate is a solution containing the same analytes labeled with an enzyme.4. leaving behind the bound ones. inversely proportional to the analyte concentration in the sample.. bound to antibody sites on the wall. remains the most popular immunoassay technique. An equal volume of analyte-enzyme conjugate (commercially available and supplied in the kit) is then added to the test tube. In the classical spectrophotometric ELISA. plates. Usually. conversely. the unbound molecules are washed away. introduced for the first time in 1971. the color intensity is directly proportional to enzyme conjugate concentration and therefore. the enzyme conjugate competes with the analyte molecules for a limited number of antibody binding sites in the test tube. the greater the amount of bound enzyme conjugate or. reacts with the chromogenic substrate forming a blue color. 3’. This plate gives the possibility to handle many samples at one time and it facilitates the automation of the assay. β-galactosidase.

in the sandwich format. and the developed color turn is read by a spectrophotometer. The enzyme conjugate and the antibody-coupled magnetic particles suspension are then combined with the sample extract. Antibodies specific to the analyte of interest are covalently bound to paramagnetic particles rather than test tubes. The reaction is then stopped by adding HCl. For example. 2. Also a sandwich immunoassay format can be used for different applications. miniaturization of 239 .4.to 10 min to 1 or 2 hours.6-trichlorophenol may interfere in the test for pentachlorophenol. the immunochemical reaction must be stopped after certain time. The more common coated-tube method is simple and rapid and does not involve centrifugation. Immunosensors are very attractive due to their high specificity and the signal achieved is related to a single analyte or small number of related compounds. Detection limits in the range of low parts per billion (ppb) can be achieved by immunoassay testing for certain parameters in aqueous samples. detection limits of <10 ppm can be achieved for many contaminants. have both advantages and disadvantages when compared with each other. which are then washed and treated with a chromogenic reagent. using different agents (such as 1 N HCl). Immunochemical Sensors (Immunosensors) Immunosensors are analytical devices incorporating an antibody-based biorecognition molecule utilized in conjunction with or integrated within a physicochemical transducer or transducer microsystem and yielding a digital electronic signal.3. which increases the analysis time. The particles are suspended in a buffered saline solution with preservative and stabilizers. including those discussed above. there may be loss of antibody because of absorption onto the solid surface. For soil samples. II. Such interference effect may. The method. decrease of analysis time.9. be reduced by using an antibody that is most selective for the target analyte. For example. requires the use of a magnetic field for separation. Also. These assay methods. The presence of substances having the same functional groups can interfere in the test. An alternative assay procedure involves the use of antibody-coupled magnetic particles. The mixture is incubated. A magnetic field is applied to separate the magnetic particles. however. Particulate systems require centrifugation to separate the bound particles.3. In certain analysis. However. an additional incubation period is required for the second antibody. however. giving a false positive value. which is proportional to the concentration of a specific analyte or group of analytes. The main advantages of immunosensors over immunoassays are simplification of the analysis procedures. An advantage of the magnetic particle method is that the antibody is covalently bound to the solid particles of uniform size (1 µm) giving even distribution throughout the reaction mixture. its disadvantage is that the surface of the tube or plate limits the number of antibody for the reaction.

Different amperometric immunosensors have been developed for pesticide detection. herbicides. UK) brought a significant improvement in optical sensor technology. toxins.equipment and automation. 240 . The sensor is based on an indirect competitive assay with the antigen conjugate adsorbed directly onto the working electrode surface. capacitive. Optical sensors based on surface plasmon resonance (SPR) such as BIAcore™ equipment developed by Pharmacia (Uppsala. usually employing enzyme labeling and amplification techniques. These devices can be classified depending on the analysis place and time. portable bench-top instruments and one-shot disposable sensors. conductimetric and amperometric detection for environmental analysis. A range of immunosensors for pesticides. A range of immunosensors has been developed using this type of transducer where the antibody has been immobilized on an ion-selective electrode.4-dichlorophenoxyacetic acid (2. The BIAcore developed immunosensors for a range of analytes in water and soil extracts. and enables to detect ppm of 2. Optical Immunosensors Advances in optical fibers and laser technology have contributed to the wide use of this kind of transducers.4-D) analysis in soil was developed. Sweden) and IAsys using resonant mirror from Affinity Sensors Ltd (Cambridge. and can include large multi-analyzers. sensors based on ion-sensitive field-effective transducers (ISFETs). A screen-printed immunosensor for 2. Also. A detection limit of 1 μg L -1 for atrazine with this kind of immunosensors was achieved. Electrochemical Immunosensors Electrochemical immunosensors are based on the use of an electroactive label or substrate. These devices have been adapted to be used in flow injection analysis. endocrine disrupters and microorganisms has been developed for environmental analysis. chemically sensitive field-effective transistor (CHEMFET) and light-addressable potentiometric sensors (LAPS) have been proposed. especially for online analysis of waters. Most are based on screen-printing electrodes for one-shot analysis. Different types of electrochemical immunosensors have been developed based on potentiometric.4-D in soil extracts. Potentiometric immunosensors are based on the measurement of changes in potential that arise from the reaction of the analyte with the specific receptor.

Brian Law. London. Rapid Detection Assays for Food and Water. Immunoassay.Piezoelectric / Acoustic Immunosensors Piezoelectric / acoustic immunosensors are based on quartz crystal microbalance (QCM) and.G. Marcel Dekker. atrazine and 2. The Royal Society of Chemistry. Ed. Boca Raton. 1991. Tothill. USA 3. Joseph Wang and Marco Mascini. 1996. USA 241 . immunosensors for parathion. Loïc J. Clive Thompson. New York. Coulet.. Netherlands 2. Ibtisam E. UK 4. By immobilizing the antibody for target analytes on the crystal. Ed. Ulrich J. Piezoelectric sensors have been used for the detection of pollutants such as pesticides. Ed. Ed. Biosensors. Oxford University Press. 1990. UK 5. Cass. respectively source acoustic transducers can also be classified as direct immunosensors when immunoreagents are used as receptors. Biosensors for Direct Monitoring of Environmental Pollutants in Field. Inc. Blum and Pierre R. REFERENCES 1. 2003. Kluwer Academic Publishers.E.4-D have been developed. Clark. Ed. 2001. K. Cambridge. USA 6. Krull. A. Nikolelis.. A Practical Guide. Biosensor Principles and Applications. A Practical Approach. William Keevil and Mark S. CRC Press. New York. C. Ed. Smith. 1997. Stuart A. Taylor & Francis Ltd. Rapid and on-line Instrumentation for Food Quality Assurance. Dimitrios P.

g. The presence of interfering materials in the sample. as well as other constituents of water.5. have grown in the last few years due to the need to develop fast and cost effective technologies suitable for application in the field and as a screening method prior to chromatographic analysis. METHODS FOR MONITORING IMPORTANT POLLUTANTS II. Directive 75 /440/EEC states that maximum levels of phenolic compounds in surface water for drinking purposes should be in the l-10 pg/L range depending on the required treatment [4]. capillary electrophoresis (CE) is a powerful alternative to classical chromatographic techniques for the separation of polar analytes. Future trends will focus on biosensors because of their faster response and lower cost. e. Furthermore. there is a general trend to change these procedures to liquid solid extraction (LSE) and liquid chromatography (LC) methods to avoid manipulation of large amounts of toxic organic solvents and because the derivatization of phenols is not straightforward. However.1. 6]. with atmospheric pressure (API) liquid chromatography-mass spectrometry (LC-MS) interfaces structural information similar to chemical ionization can be obtained and the disadvantages of other LC-MS interfacing devices such as thermospray (TSP) have been overcome. clean up and final determination will be reviewed. should always be taken into consideration because they will notably affect analytical performance [5. US-EPA 604 [2] and 625 (acid extractable section) [3] and the recently introduced 8041 [5] are based on liquid-liquid extraction (LLE) followed by gas chromatography using different detection devices such as electron capture or mass spectrometry (MS).. Current official analytical methods.II. mainly humic and fulvic acids in river water. In addition. PHENOLS Jenny EMNEUS THE MOST A number of phenolic compounds are listed in the European Community (EC) Directive 76/464/ EEC concerning dangerous substances discharged into the aquatic environment [1] and in the US-EPA list of priority pollutants [2. 3] due to their toxicity and persistence in the environment. An overview of the current analytical methods for the determination of priority phenolic compounds in water and wastewater is presented here and various aspects such as sample extraction. mainly immunoassay and biosensors. Biological techniques. Several immunoassays are commercially available for the detection of various organic contaminants. Other emerging approaches are the use of solid-phase microextraction (SPME) and the combination of LSE with supercritical fluid chromatography (SFE).5. 242 .

has been reported by several authors. which recommends the derivatization to methylated phenols instead of to pentaflouorobenzoyl ether derivatives. This approach has the advantage of high sensitivity and selectivity. such as liquid-liquid extraction (LLE). The analytical approaches for phenols and phenol derivatives determination from environmental samples are almost based on chromatographic techniques (e. have been used in order to eliminate the sample matrix influence on the analysis.2 µg/L with FID and ECD. g. heptaflourobutyric anhydride [10] or diazomethane [11]. GC of underivatized phenols using capillary columns with conventional phases is difficult as phenols. pentaflouorobenzoyl bromide [2]. When using this methodology LODs of phenols included in US-EPA list range from 0. acetic anhydride [10]. this method requires the use of diazomethane. respectively 0. capillary electrophoresis (CE)). but also biological techniques ( e. Various derivatization reagents have been reported. ECD [8] or MS [9]. are available. However.g. Gas chromatography in conjunction with various detection devices. e. mainly FID [2]. It is currently being used in US-EPA official methods.1 µg/L to 13 µg/L and from 0. in the last few years it was pointed out that US-EPA official methods for phenols may often lead to incorrect results because derivatization of phenols. liquid-solid extraction (LSE) and solid-phase micro-extraction (SPME).5 µg/L to 2. in this case adsorption of humic material into the sorbent is increased so a large interfering peak appears somewhere in the chromatogram [7]. explosive). However. However. which allow the unequivocal identification of phenols. associated with its use (carcinogenic. even when using highly deactivated columns [8]. and in particular nitrophenols. Derivatization using acetic anhydride is more straightforward but dinitrophenols gave problems. but in general derivatization is required prior to analysis. reagents such as pentaflouorobenzoyl chloride are preferred because they introduce electronegative substituents into the molecule. immunoassay and biosensors). thus non-nitrated and non-halogenated phenols can be detected using ECD although the method fails for nitrophenols. liquid chromatography (LC). The US-EPA method 625 provides conditions for GC-MS analysis [3]. Mass spectrometry (MS) is by far the most suitable detection device when analyzing unknown samples because mass spectra libraries. g. However. The current US-EPA methodology for phenols (method 604) involves derivatization using pentafluorobenzyl bromide prior to GC separation. Recently. Various sample handling strategies for the determination of phenolic compounds in water. gas chromatography (GC). is not straightforward. and the existence of mass spectra libraries for screening of unknown samples. followed by either FID or ECD although the use of GC-MS for identification purposes is also recommended [2]. which has potential hazards.Acidification of the sample can help to overcome this drawback and can avoid the deprotonation of the most acidic phenols. the US EPA has reported a new protocol (method 8041). especially nitrophenols. tend to tail. 243 .

UV detectors are the most common detectors and detection is usually carried out at 280 nm. and LODs ranging from 0. EC detection is a good alternative for monitoring relatively clean samples. 4-methylphenol and 2. for instance drinking water. With on-line LSE using Lichrolut EN.7 µg/L were obtained for all phenols using UV detection [6] but values below 0. Different interfaces are currently available for LC-MS experiments. coulometric sensors convert 100% of the analyte because oxidation of phenols occurs in a high porosity electrode. where electrode fouling is not a critical problem. The thermospray interface (TSP) provides a good response in the negative ion mode for the listed phenols with the exception of phenol. LODs below the ng/L level were obtained but derivatization to dansyl derivatives was necessary and a tubular flowthrough reactor was required for indirect detection.25 pg/L using off-line LSE were found. Recently coulometric array detectors were introduced.02 µg/L were obtained [14]. The high sensitivity of EC allows a reduction in the sample volume. and when processing only 10 mL of water LODs of 0. The particle beam interface was reported for pentachlorophenol analysis but its use in the environmental field is prevented because it gives lower sensitivity compared with other available devices 244 .38 ng/L were obtained when combined with LSE [15]. which show a better signal at 310 nm [6. the more robust UV will be preferred. spectral libraries can be used for confirmation purposes [12]. Diode array detectors are recommended because even though little decrease in sensitivity occurs. An increase in sensitivity of up to three orders of magnitude can be obtained compared to diode array detectors.The absence of derivatization requirements and the possibility of on-line coupling with LSE without sophisticated instrumental requirements has made LC with C18 or C8 columns the most commonly applied separation technique for the analysis of phenolic compounds. thus increasing the risk of exceeding the breakthrough volume of the more polar phenols. Unlike common electrochemical detectors in which electrodes typically react to 10% or less of the injected sample.4dimethylphenol because current buffers cannot deprotonate them even when working at a high buffer concentration level [12. In their most sophisticated approach. 7]. However. although EC may be required to detect the low breakthrough volume analytes.03 ng/L to 0. 14. For the analysis of complex samples such as river or wastewater. a serial array of electrodes at increasing potentials provides 3-dimensional chromatograms (similarly to diode array detectors) where the analyte voltammogram facilitates peak identification. Several authors have reported the monitoring of phenolic compounds using direct [16] or indirect [17] fluorescence detection. an important decrease in LODs was obtained when using EC detection instead of UV [13] and values in the low pg/L range for most of the compounds studied were obtained using on-line LSE [14]. 16]. except for nitrophenols and pentachlorophenol. because of the lower sensitivity. They cannot be used for multi-screening purposes because the gradient elution is not suitable. However. higher sample volumes are required for the LSE step. In summary. UV shows better performance with regard to signal stability although. LODs below 0.

For this reason. 245 . and ease of use. cyclohexylaminoetansulfonic acid. which is in the range of few nanoliters. Even though this approach is just emerging. post-column addition of buffer is necessary in order to generate ions in solution when per-forming LC-ISP-MS experiments. The main advantage of atmospheric pressure interfaces (API interfaces) are the resulting higher sensitivity (especially when using atmospheric pressure chemical ionization. 4-methylphenol and 2. it offers the possibility of carrying out sample enrichment in the capillary using isotachophoresis and shows compatibility with mass spectrometer devices such ISP. the basic pH requirement for CZE can be a limitation because it can induce hydrolysis of some analytes such as nitrophenols.3 pg/L to 1 µg/L [21]. although they can be improved by using EC or fluorescence detection [22]. Interest in developing fast. 21]. robustness. APCI).[18]. or the polymerization of catechol. has high resolution. Also. The EPA’s Office of Research and Development is leading efforts to develop this promise and to extend the range of analytes. some authors prefer the use of micellar electrokinetic chromatography (MECC) 2 [23]. thus overcoming their major drawback. Since an acidic pH is normally required for the chromatographic analysis of phenolic compounds. Standard operating protocols are written in formats such as those found in EPA solid waste (SW-846) protocols [25]. CZE has been applied at basic pH using electrolytes such as sodium borate. However. thus allowing the quantification of the most polar phenols such as phenol or cathecol. 14. mainly capillary zone electrophoresis (CZE). and phosphate buffer [20. although methanol percentages above 85% are required [19]. Moreover. Deprotonated humic and fulvic acids can be effectively separated from other contaminants due to their different migration kinetics. By far the most interesting feature of the ISP interface is the detection of phenol. it is really promising because it has the advantage that trace enrichment is carried out on the top of the capillary and the LODs can be lowered. Several reports dealing with the separation of phenols have been published. which can be monitored [24]. Only a few of the reports refer to the determination of pollutants in environmental matrices. Biosensors and the more established immunoassays (ELISA) are really promising in this area.4-dinitrophenol determination). The major drawback of CE is its low loading capacity. and is suitable in the analysis of polar and ionic compounds as well as the more nonpolar ones. most of them dedicated to demonstrate the technique’s high-resolution applications to specific pollutants. has grown in the environmental field mainly because it is fast. by raising the extraction voltage structural information can be obtained via CID using a simple quadrupole instrument (2. A few papers have dealt with the analysis of phenols using the ionspray (ISP) interface [12.4-dimethylphenol. Typical LODs when CZE is combined with off-line LSE range from 0. portable and cost-effective field analytical methods that are capable of effectively screening for the presence of target analytes in environmental samples has grown in the last few years. In the last few years the application of CE. In most cases UV detection is used. 18].

. 304. W. 733. 13. P.. W.4-dinitrophenol in urine and water samples. (6) Puig. Efer. F.C. Other authors have reported the development of immunoassays for the determination of 4-nitropheno1 and 2. Anal. 145. the signal transducer and the detection system. pp 1-28. Chromatogr. J. J.4. G. Similarly to immunoassay techniques.. Fresenius J. M. J. I. O. 361. D. biosensors take advantage of the intrinsic specificity enzyme-substrate reactions for selectively monitoring pollutants in environmental samples.. 246 . Hence.. (4) Hennion. 371. 1995. 1993... Engewald. Co-immobilization of laccase can increase the number of detected phenols (e. 435. 1995. One of the commercially available immunoassay for phenolic compounds is for pentachlorophenol. K. K. Barcélo. 343. 1991... only 2. Bjorseth. REFERENCES (1) Vincent. Acta 1995. G. E. The detection system is usually based on amperometric principle because of its higher sensitivity and selectivity. respectively) and furthermore matrix interferences did not affect immunoassay performance [26]. A.980) and soil (0. Most of the experiments are done with tyrosinases. D. E.: Kluwer. (12) Ruana. pp 58-66. Chromatogr. (5) 8041.. Lukasewycz. (13) Galcerán. 304. phenol.1% and 15. 654. 40. 767.. Chromatogr. S. 1984. V. D. K. (8) Mubmann. 4-chlorophenol and 4-methylphenol [29].5. Cross reactivity studies showed the good specificity of the method. Phenol oxidizes (tyrosinases and laccases) or peroxidases are the enzymes currently used for this propose [28]. in the latter case with LODs of 7 µg /L [27].. which show a response mainly for catechol. Pichon. 348.4. 217. pp 153-174. The method compares favorably with GC-MS and HPLC measurements in water (~0. 1994.6-tetarchlorophenol gave significant values (54. P. T. H.. Wom Wasser 1992. hydroquinone and 2-amino-4-chlorophenol)[30]. Baker...They take advantage of the high specificity of biological recognition to selectively monitor target analytes in complex samples. (11) Nick. E.. D. which depends on the chosen transducer. Chromatogr. Radeck. F. Chem.1%. C. Angeletti. Anal. Barcélo. (10) Mubmann. m. m. an electrical transducer and a potentiostat for detection are used. g. which characterizes the method. Preib. Wunsch. A.: Washington D.3.. Levsen. Barcélo. D. Urbe. G. A 1996. (3) 625. M.. 79. E. Chim. Fresenius L. Jáuregui. (7) Puig. (2) 604. Environmental Protection Agency. (9) Durhan. 655. Any biosensing device can be divided into three parts: the biological recognition unit.996) and LODs of 60 ng /L are obtained [26].. Anal. m. Borrull. Levsen. J.. 75. Scholer.6-tetrachlorophenol and 2. M.. Peroxidase enzyme (horseradish peroxidase) can be also applied for a large range of phenols [31]. A 1993. 629. Chem. 1992. Environmental Protection Agency. TrAC 1994. J. 1984..

. Barcélo. Jarskog. E. Nitrite. P. 14. nitrate (NO3 ) and ammonia (NH3). m. B.. Emnéus. Hayes. Hensel. D.. 271. Acta 1995. N. Hammock.. Seiber. Anal. Berloni. K. S. Williams. G. Chromatogr. Herzog. M. F. Gee. P. G. pp 1-17. U. 1995. 1995. A.. Chromatogr.. J. A. Chem. Varga. L.. Nitrogen can also be a part of an organic compound and is then referred to as organic nitrogen.. 133. Marti.. Haag. R. 8.. Chim. Bohle. (30) Yaropolov. F. Pathak. S. Q... The most common inorganic forms of molecules containing nitrogen are nitrite ( NO2). (15) Achilli. Environ. J. Kharybin. C. J. J.. G. Acta 2000. R. D. C.. (21) Aguilar. G.. Chim. D. Brikman. (26) Hottenstein.. Lawruk. M. 60.. J. J. 29. C. S. Sci.. Zhao. D. M. 63. S. 311. Anal. 1988. S. J. 229. (29) Lutz. Total Environ. 29.. Chem. Acta 1995. 1685. D. 663. (20) Bachmann. C. Eril. G. V. G. Emnéus. 47.5. J. 247 . (25) 4010A. 2754. A. S. V. Acta 1993. Emnéus. Nitrogen Nitrogen is an important eutrophic element that can exist in various forms. Gorton. TrAC 1996. D. Ruzgas. X. Anal. Agric.. B. L. 1991. T. P. In the environment microorganisms have the ability to convert one form of nitrogen to another form.. Acta 1993. J..5.. Lin. E. Chim. Dombek. 716. 283..2. 666. Environ. I. M. Technol... L. Food Chem. K. 305. 53. A 1995.. Chem. M. F. 39. 132. W.. T. Chim. M. (31) Wang. 1994. 289. 362-375.2. (24) Rogers. Gobhadi.. (28) Varga. Rubio. 15... J. 917. Cellerino.. Huber. P. A. 1995. Chromatogr. Anal.. Sci. NITROGEN (Nitrate. (19) Puig. (22) Gaitonde. Y. M. V. G.. Anal. 319. A 1994. Burestedt. G. II. TrAC 1995.. Bruner. R. 1995. J.. The determination of the inorganic forms is usually made on filtered samples whereas it could be valuable to determine organic nitrogen for both filtered and total samples. Frei. F... A. G. Chim.. C. Farran. 1994.(14) Puig.. (23) Praus. H. de Jong. M... W.. 350. Anal. T.. Varga. J. A. Ammonia) Tomas ALEXANDERSSON II. Anal. A. P. (17) Lamprecht. Anal. Sci. Kurth. Y. W. (27) Li. M. G. 14. K. Barcélo..1. J. Jourdan. (16) Ruiter. 697. 667. N. D. 357. D. 1993. Gottlicher. 2295. (18) Capiello.. Famiglini. S. Gorton. Technol. Palma. EPA: Washington.. Bird. M.

such as sodium hydroxide. where ammonia is converted to 5-aminosalicylate. indophenol.2. If a measurement is made with an ion selective electrode the sample is mixed with a strong base. which diffuses through the hydrophobic gas-permeable membrane of the electrode into the internal solution. the instrument gives a direct reading of the ammonia concentration. Nitrite and Nitrate Both nitrite and nitrate are easily soluble in water. In the standard method EN 11732 for determination of ammonia in a flow injection system the sample is injected into a continuous carrier stream and mixed with an alkaline solution. gas sensitive electrode and wet chemistry.g. The result after an analysis of a water solution is usually expressed in units of nitrite or nitrate nitrogen. Samples with a high concentration of ions may affect the measurement. This color change is measured in a spectrophotometer and is proportional to the ammonia concentration in the sample. ammonia reacts with hypochlorite and phenol with nitroprusside as a catalyst to an intensively blue compound. The incoming ammonia will change the pH in the internal solution and this change is detected by measuring the potential between a pH-electrode and a chloride-reference electrode immersed into the internal solution. With an electrode it is possible to do determinations directly on turbid and colored samples. 248 . Usually when an analysis of a water solution is expressed in units of ammonia. By calibrating the electrode. It is also possible to convert ammonia into a colored compound and measure the color with a spectrophotometer.5. There is also another method. which is determined by absorption at 660 nm. The raise in pH will convert all ammonium to ammonia. The indicator changes its color due to the change in pH caused by the diffusing ammonia.2. the term ammonia refers to the sum of ammonia and ammonium. Dissolved ammonia acts as a base with ammonium as the conjugating acid. After oxidation and oxidative coupling a green complex is formed. e. There the solution passes a hydrophobic gaspermeable membrane and the ammonia diffuses into a continuous flowing indicator solution. The developed color is determined by absorption at 640 nm.II.3. II. The equilibrium between the acid/base pair is determined by the pH of the water solution. Ammonia Ammonia is a gas at room temperature and it is soluble in water. the modified Berthelot method. Ammonia can be analyzed by several different methods. Determination of nitrite can be done with wet chemistry methods whereas determination of nitrate also can be done with ion selective electrode and UV spectrophotometric method. The ammonium in the sample is transformed to ammonia and is separated in a diffusion cell. In the phenate method.5.2.

When it is immersed into a solution. which then is determined according to the previously described procedure. It is a very rapid and convenient method since there is no need for reagent solutions and pumps and tubes for sample transport.1 mg/L to 1400 mg/L. There is though a possibility of interference from organic matter.0 and 2. cyanide. if the content of organic matter in the sample is low or constant the accuracy of the method is improved. Cyanides are metal salts or complexes that contain the cyanide ion (CN –). By calibrating the electrode it is possible to determine the nitrate in the range from 0. especially for metal cleaning and electroplating. Many on-line instruments utilize the fact that nitrate is absorbing light at 220 nm. Cyanide is widely used in certain mineral-processing operations. [Fe(CN) 6]4-. However. The cyanide ion has a strong affinity for many metal ions. forming relatively less-toxic ferrocyanide. which also could absorb at this wavelength. Several different anions such as nitrite. It is also important to have the same pH and ion strength in both sample and calibration solutions. The normal procedure is to make an additional measurement at 275 nm where nitrate does not absorb and use this for correction of the amount of organic matter.5. II. Ka of 6 x 10-10. for example.S. Cyanide is widely used in industry.5. which therefore may be regarded more as a tool for process control than an accurate analytical determination.3. with iron(II). The amount of color is then determined by measuring the absorbance at 543 nm. This will of course introduce a small uncertainty in the result. These cyanides could be subdivided into two categories: (1) simple cyanides such as 249 . a weak acid. CYANIDES Giuseppe PALLESCHI. a deadly poisonous substance. In order to get a measure of only nitrate a correction for the amount of nitrite must be done. a potential develops across an inert membrane that holds a liquid ion exchanger. exists in water as HCN. This method could also be used for the determination of nitrate if the sample is pretreated in glass column filled with cadmium granules treated with copper sulfate. and bromide may interfere with the measurement.In the determination of nitrite a reddish purple azo-compound is produced by reaction of diazotized sulfanilamid and N-(1-naphtyl)-ethylenediaminedihydrochloride (NED dihydrochloride) at a pH between 2. sulfide. Mihaela BADEA Cyanide. When the sample passes through the column the nitrate is reduced into nitrite. Volatile HCN is very toxic and has been used in gas chamber executions in the U. Use of the cadmium column will determine the samples content of the sum of nitrite and nitrate. It is also one of the main gas and coke scrubber effluent pollutants from gas works and coke ovens. since the method depends rather on the nitrate activity than on the quantity of nitrate. The nitrate could also be determined with an ion selective electrode.

affect colorimetric.3. and (2) complex cyanides such as K4Ce(CN)6 or NaAg(CN)2 containing two different metals in their formula unit. Then. the absorption liquid is analyzed using one the following methods: Silver nitrate titrimetric method Colorimetric method Ion-selective electrode method Ion chromatography II. If presence of S 2. these complexes are subject to extensive and rapid photolysis. NH4CN. fatty acids. the metal cyanide is converted to HCN gas. Analytical distinction between HCN and other cyanide species present as complex cyanides is possible [1]. animals. and oxidizing agents need to be removed by special procedures. Sulfides will distil over with cyanides and. Sulfides.5. The fatty acids are removed by extraction with iso-octane. NaAsO 2 or Na2S2O3 should be added if the presence of the oxidizing agents is identified.is less than that of HCN. including the nature of the metal. yielding toxic HCN. Samples Pretreatment The nature of the preliminary treatment varies according to the interfering substances present.NaCN. The zinc. In equally dilute solutions there is much less dissociation for the nickel-cyanide complex and the more stable cyanide complexes formed with copper(I) and silver. and dilution. The latter may further dissociate to CN – which forms HCN. but in case of the water pollution. and aquatic life. However. usually one is an alkali metal and the other a heavy metal. after removal of the interfering substances. which result in acute toxicity to fish at any ordinary pH. hexane or CHCl 3.and cadmium-cyanide complexes are dissociated almost totally in very dilute solutions. therefore. Most other interfering substances are removed by distillation. 250 . which is distilled and adsorbed in a sodium hydroxide solution. as the pH of most waters is substantially lower than the pK a of HCN. For avoiding this. The iron-cyanide complex ions are very stable and are not toxic. The complex cyanide dissociates to metal and polycyanide ions. For total cyanide determination. titrimetric and electrochemical procedures. The cyanide ion and HCN are highly toxic to human beings. Oxidizing agents (such chlorine) decompose most of the cyanides during storage and manipulation.1. or Ca(CN)2 containing one metal ion (usually an alkaline or alkalineearth metal or ammonium ion) in its formula unit. this is not important because most of the free cyanide exists as HCN. pH of the solution. The degree of dissociation of the various metallocyanide complexes at equilibrium increases at decreased concentration and low pH.is identified lead acetate or lead carbonate is added. The toxicity of CN. when diluted solutions are exposed to direct sunlight. The degree and rate of dissociation of complex cyanides depend on several factors.

Colorimetric Method Cyanide is converted to cyanogen chloride. On addition of pyridine-barbituric acid or pyridine-pyrazolone reagent. which hydrolyzes to glutaconaldehyde. Alternately I– may be used as an indicator. Thus.5. Ion-Selective Electrode Method The CN. When no more free CN– is left. by treatment with chloramine-T at pH < 8 without hydrolyzing to cyanate. This method can be used to determine CN. Cyanide concentrations higher than 1 mg/L can be determined by titrimetry. produces a distinct color with an indicator that can determine the end point of the titration.4. CNCl. II. p-dimethylaminobenzilidenerhodanine. Ag(CN)2. the little excess of Ag + added reacts with the indicator.+ NO3- When all the CN– ions in the sample are complexed by Ag + ions. 251 .5..the intensity of which is proportional to the concentration of cyanide in the sample.in the concentration range of 10-5 to 10-2 M CN-. The latter reacts with barbituric acid or pyralozone to give a blue color (absorbance reading at 578 nm). The titration method is suitable for cyanide concentration above 1 mg/L. in the presence of a silversensitive indicator. 2 CN. Such samples should be preserved using calcium hydroxide.3. CNCl.3. Silver Nitrate Titrimetric Method Cyanide reacts with silver nitrate as shown below forming the soluble cyanide complex.+ AgNO3 Ag(CN)2. The colorimetric method can be used to determine CN . reacts with pyridine to form an intermediate nitrile.2.concentration in the range of 1 – 5 µg/L. turning the color of the solution from yellow to salmon.3.selective electrode in combination with a double-junction calomel reference electrode and a pH meter having a resolution of 1 mV.5.in the alkaline distillate from the preliminary treatment procedures can be determined potentiometrically by using a CN . any further addition of a few drops of titrant. Ag+ ions at first combine preferentially with CN–. CNO –.3.The carbonate in high concentration may affect the distillation process by causing a violent release of carbon dioxide with excessive foaming when acid is added before distillation. II. AgNO 3. Titrate to first appearance of turbidity (due to the formation of AgI). II.

HEAVY METALS Giuseppe PALLESCHI. Jacques Buffle and George Horvai. because of their widespread use. Cyanide in Aerosol and Gas Samples Hydrogen cyanide and cyanide salts in aerosol and gas may be analyzed by NIOSH Method 7904 (NIOSH. REFERENCES 1.5. Arnold E. Determination of Hydrocyanic Acid and Free Cyanide in Aqueous Solution. Chemical Analysis and Speciation .5. Mihaela BADEA. USA II. United Book Press. Ed. Chem.J.5.5 to 1 L/min is passed through a filter-bubbler assembly of a 0. 20th Edition. Broderius. In Situ Monitoring of Aquatic Systems. chromium. West Sussex. The KOH extract and the bubbler KOH solution are analyzed for cyanide by selective-ion electrode technique using KCN standards. John Wiley & Sons. S..4. 1998. Cyanide analysis is then performed on the leachate. 2000. Standard Methods for the Examination of Water and Wastewater . Baltimore. Ed Leonore S. mercury. II. Ltd. HCN is trapped over the KOH solution in the bubbler. Andrei Florin DĂNEŢ Although many of the metals are toxic.II. lead. The membrane filter is then placed in 25 mL of 0. The European Pollutant Emission Register has classified seven metals as priority pollutants: cadmium. only some metals are major environmental pollutants.1 N KOH solution for 30 min to extract the cyanide particulates deposited on it. Cyanide in Solid Samples The determination of soluble cyanides requires sample leaching with distilled water until the solubility equilibrium is established. Inc. Between 10 and 180 L of air at a flow rate of 0.5.3.. nickel and zinc.6.3. Andrew D. Greenberg.8-µm cellulose ester membrane and 10 mL of 0. 53 (1981) 1472 2. Eaton. copper. 252 . While cyanide particulates retain over the filter membrane. 1984). High levels of cyanide indicate soluble cyanide in the solid sample. Anal. Clesceri.1 N KOH solution. Low cyanide concentration in the leachate may indicate the presence of soluble metal cyanides. England 3.

mercury (30 %) and zinc (84 %). II. they are time consuming and a detection limit below 10 µg/L is difficult to achieve for most metals. . convenient. voltammetry. The pollution with nickel is due to the mineral oils and gas refinery (41 %) and combustion installations (33 %). Some metals are essential to plant and animal growth while others may have toxic effect.4. and loss of metals by adsorption and /or precipitation in the sample container. Atomic absorption or emission spectrometry is often chosen. ion chromatography. including ionselective electrodes. Although colorimetry methods can give accurate results. or the sum of the concentrations of metals in the dissolved and suspended fractions. electrophoresis. copper (63 %). air.The metal industry and metal ore industry or sintering installations. lead (83 %). because it is rapid. . Important errors may be introduced during sampling and storage because of contamination from the sampling device. Preliminary treatment is often required to present the metals to the analytical methodology in an appropriate form.total metals – the concentration of metals determined in an unfiltered sample after vigorous digestion.suspended metals – metals that in an un-acidified sample are retained by a 0. . chromium (57 %). neutron activation analysis. decide what fraction is to be analyzed (dissolved. In addition.5. suspended. Metals in general can be analyzed by Colorimetry and Atomic Absorption Spectrometry (AAS) or Atomic Emission Spectrometry (AES).45 µm membrane filter. This decision will determine in part whether the sample is acidified with or without filtration and the type of digestion required. and gravimetry. soil) ranges from beneficial through troublesome to dangerously toxic. redox titration. Before collecting a sample. The effect of metals in the environment (water. The benefits versus toxicity of some metals depend on their concentration in the environment. total or acid-extractable).dissolved metals – metals that in an un-acidified sample pass through in a 0.45 µm membrane filter. failure to remove residues of previous samples from the sample container. some metals may be determined by other methods. installations for the production of ferrous or non-ferrous metals are the most important pollutant sources for cadmium (62 %).1. The choice of method depends on the precision and sensitivity required. 253 .acid-extractable metals – the concentration of metals in solution after treatment of an unfiltered sample with hot dilute mineral acid. and gives the low detection levels as required in the environmental analysis. Sampling and Treatment Metals can be classified as: .

sulfuric acid. II. a monochromator or a filter to isolate the characteristic absorption wavelength.e.3. Therefore. the samples are filtrated before preserving. For metal concentrations of μg/L.4. After acidifying the sample. the latter should not be allowed to dry. The heat source is an air-acetylene or air-nitrous oxide flame or a graphite furnace. Sample Digestion Aqueous and non-aqueous samples must be digested with an acid before their analysis by atomic absorption or atomic emission spectrophotometry.2. can be used. but these materials are too expensive. Samples must be acidified immediately by sampling with concentrated nitric acid to pH<2. Also. a laboratory-grade microwave unit. for which the limit is five weeks). The preferred containers are made of polypropylene or polyethylene. 254 . however. The acid extract after boiling and cooling is diluted with water to a measured final volume for analysis.5. When the sample is boiled with acid. The metals and their salts present in the sample are converted into their nitrates due to the fact that the nitrates of all metals are soluble in water. samples with several mg/L are stable up to 6 months (except mercury. II.4. Nitric acid alone is. perchloric acid. the samples should be analyzed as soon as possible after sampling. adequate for digestion of most metals.The best sample containers are made of quartz or Teflon. The light source usually is a hollow cathode lamp or an electrode-less discharge lamp composed of the element to be measured..5. specifically designed for hot acid digestion. the samples are stored in a refrigerator at approximately 4 °C to prevent change in volume due to evaporation. or hydrofluoric acid is used in sample digestion for the determination of total metals. Atomic Absorption Spectrometry for Heavy Metals Determination An atomic absorption spectrophotometer consists primarily of a light source to emit the line spectrum of an element (i. for mercury analysis the samples may be preserved by adding 2 mL/L 20 % K2Cr2O7 solution (prepared in HNO3). It is strongly recommended to use containers and filters that have been acid rinsed. The acid digestion is performed using a small volume (5 to 10 mL) of nitric acid alone or in conjunction with one of the previously mentioned acids on a hot plate. Alternatively. For dissolved metals. the element to be analyzed). concentrated nitric acid by itself or in conjunction with hydrochloric acid. and a photoelectric detector associated with a microprocessor and a digital readout device for measuring the absorbance due to the metal at its corresponding concentration. In these conditions. a heat source to vaporize the sample and dissociate the metal salts into atoms.

it is charred at an intermediate temperature to destroy the organic matters and volatilize the compounds. the sample is dried by low current heating.acetylene Air . A small volume of sample is aspirated into a graphite tube.acetylene Air .acetylene N2O .7 324.9 An atomic absorption spectrometer equipped with a graphite furnace or an electrically heated atomizer instead of the standard burner head gives better sensitivity and much lower detection limit than what is obtained with the flame technique. Chelation extraction Direct aspiration Direct aspiration.acetylene Technique Direct aspiration. Metal Cadmium Chromium Cobalt Copper Iron Lead Manganese Wavelength (nm) 228.5.0 Titanium Tin Vanadium Zinc 365.acetylene Air . and Technique for Flame Atomic Absorption Analysis. The sample is aspirated into the flame and atomized.3 283.In flame atomic absorption spectrometry. 217.4 213.acetylene N2O . The energy absorbed is proportional to the concentration of the element in the sample. Chelation extraction Direct aspiration. Chelation extraction Direct aspiration.acetylene N2O . The metal atoms absorb energy at their own characteristic wavelength (Table II.0 279.1. Chelation extraction Direct aspiration.acetylene Air .3. Chelation extraction Direct aspiration. Then. The light beam is directed through the flame. First.6 318. Chelation extraction Direct aspiration. Chelation extraction Direct aspiration Direct aspiration Direct aspiration Direct aspiration.3 224.acetylene Air .5. the heat source is a flame. which is heated in several stages. The principle of this technique is the same as for the flame method.acetylene Air . Chelation extraction Molybdenum 313.acetylene Air .7 248.8 357. Chelation extraction Direct aspiration. Finally.acetylene Air .3 Nickel 232. Table II. Flame Type.1). Recommended Wavelength.5 Flame Air . it is heated to incandescence by a high current in an inert atmosphere to atomize the 255 .9 240.acetylene Air .

forming the metal chelate. (NH4)2S2O8. which is then extracted with methyl isobutyl ketone (MIBK). Such interference may arise from molecular absorption or from chemical or matrix effects. The chelate is extracted with MIBK by shaking the solution vigorously with the solvent for 1 min. chromium. To prevent the formation of metallic oxides and minimize the oxidation of furnace tubes.e. however. the metal must be oxidized with KMnO4 under boiling and the excess KMnO4 is destroyed by hydroxylamine hydrochloride prior to chelation and extraction. and silicon react with graphite at high temperatures.. Atoms in their ground state absorb monochromatic radiation from the source. In order to determine the total chromium. vanadium. argon should be used as a purge gas. (NH4)2SO4. This can be reduced or eliminated by correcting for background absorbance and by adding a matrix modifier into the sample. and zinc. Many metals can be determined at a concentration of 1 µg/L (1 ppb). the less the amount of light transmitted through). The intensity of the transmitted light is measured by a photoelectric detector. the greater the quantity of ground state atoms in the optical path. APDC chelates of certain metals such as manganese are not very stable at room temperature. nickel. The primary advantage of the graphite furnace technique over the conventional flame method is that the former requires a smaller volume of sample and the detection limit is much lower. copper. The calibration standards of the metal are similarly chelated and extracted in the same manner and the absorbances are plotted against concentrations. ascorbic acid. The extract is aspirated directly into the air acetylene flame. NH4NO3. A disadvantage of the graphite furnace technique. interference due to other substances present in the sample can cause a problem. These metals include cadmium. If an emulsion formation occurs at the interface of water and MIBK. Such chemical interaction may be prevented by using pyrolytically coated tubes. Some common matrix modifiers are: Mg(NO3)2. The intensity is inversely proportional to the number of ground state atoms in the optical path. Therefore. Chelation-Extraction Method Many metals at low concentrations can be determined by chelation-extraction technique. i.element. barium. manganese. use anhydrous Na2SO4. oxalic acid. is that because of high sensitivity. nickel. The chelation-extraction method determines the chromium metal in hexavalent state. A 100-mL aliquot of aqueous sample is acidified to pH 2 to 3 and mixed with 1 mL APDC solution (4% strength). Certain metals such as molybdenum. iron. cobalt. the analysis should be commenced immediately after the extraction. the greater the absorbance (in other words. lead. thus forming carbides. Low concentrations of aluminum and 256 . silver. A chelating agent such as ammonium pyrrolidine dithiocarbamate (APDC) reacts with the metal.

and heated. Sample digestion with nitric acid.5 mL NaBH 4 solution (5% in 0. HCl. To this solution. let the solution stand for 15 min. The hydrides formed are purged by nitrogen or argon into the atomizer for conversion into the gas-phase atoms. Heat the mixture to boiling for 2 h in a water bath. The absorbance is measured at the wavelength 253. respectively. Standard solutions of these metals are treated with NaBH4 in the same manner for the preparation of the standard calibration curve. oxidizes these metals to their higher oxidation states. Hydride Generation Method Arsenic and selenium can be determined using the hydride generation method. Add to this solution 0. The digested sample is then further acidified with conc.1 N NaOH. After the sample is acid digested with conc. The commercially available continuous hydride generator units make the operation simpler than the manual method outlined above. These metals are reduced to As(III) and Se(IV) by boiling with 6 N HCl for 15 min. or a 10% hydroxylamine hydrochloride solution instead of (NH2OH)2 · H2SO4).7 nm. prepared fresh daily) and stir. which it volatilizes to vapors. treated with sodium iodide (for As determination only). Cold Vapor Method for Mercury Determination Cold vapor atomic absorption spectrophotometric method is applicable only for the mercury analysis. The principle of this method is described below. however. producing As(V) and Se(VI). The presence of other substances in the samples causes little interference because hydrides are selectively formed and are removed from the solution. After acid digestion with nitric acid. 257 . the extract is treated with two strong oxidizing agents: KMnO 4 and potassium persulfate (K2S2O8). mercury and its salts are converted into mercury nitrate. add about 10 mL of 5% K 2S2O8 solution. The hydride generated (arsine or selenium hydride) is purged with the carrier gas such as argon and transported into the atomizer. Free chlorine produced from chloride is removed by treatment with hydroxylamine sulfate reagent and by sweeping the sample gently with air. H 2SO4 and HNO3. any interference from sulfide and chloride are removed by oxidizing the extract with potassium permanganate. Under aeration the vapors of mercury are carried by air into the absorption cell. The reaction with NaBH4 is rapid when the metals are in their lower oxidation states as As(III) and Se(IV). After adding 15 mL of KMnO4 solution (5%) to the acid extract.beryllium can be determined by chelating with 8-hydroxyquinoline and extracting the chelates into MIBK and aspirating into a N2O-acetylene flame. These metals in HCl medium can be converted to their hydrides by treatment with sodium borohydride. Prior to reduction. The excess of KMnO 4 is destroyed by adding NaCl-hydroxylamine sulfate solution (12% concentration of each. Treatment with stannous chloride reduces mercury into its elemental form.

i. The sampling and analysis of atmospheric Hg is often made as Total Gaseous Mercury (TGM) which is an operationally defined fraction that includes species passing through a 0. The standard solutions are analyzed first prior to the sample.4. Mercury concentrations in precipitation samples depends on a number of factors primarily related to emission sources type. typical concentrations observed in different European sites were in the range of 5 to 80 ng L -1 for total mercury.e.6 ng m -3 for elemental mercury. location of the monitoring station and meteorological conditions (i. their chemical and physical structure cannot be exactly identified by experimental methods but are instead characterized by their properties and capability to be collected by different sampling equipment. and reduction. Another species of particular interest is methylmercury (MeHg) due to the high capacity of this species to bioaccumulate in aquatic foodchains and to its high toxicity. 5 to 50 pg m-3 for RGM and TPM. following acid digestion. Of these three forms. Extensive research efforts have been put into the identification and quantification of these species over the last decades. RGM is defined as a water.5. Reactive Gaseous Mercury (RGM) and Total Particulate Mercury (TPM).0 to 3. A standard calibration curve is constructed by plotting absorbance vs. as described above.The calibration standards are made from a soluble mercury salt.soluble mercury species with sufficiently high vapor pressure to exist in the gas phase. The concentration of Hg in the sample is then determined by comparing the absorbance with that in the calibration curve. The reactive term refers to the capability of stannous chloride to reduce these species in aqueous solutions without pre-treatment.e.. frequency and intensity of precipitation events).45 mm filter or some other simple filtration devices such as quartz wool plugs and which are collected on gold. such as. whereas MeHg levels were between 0. only Hg0 has been tentatively identified with spectroscopic methods while the other two are operationally defined species. TPM consists of mercury bound or strongly adsorbed to atmospheric particulate matter. II. mercuric chloride. from 1 to 50 pg m -3 for RGM and TPM whereas MeHg has been found in the range of 1 to 20 pg m -3.005 and 0.4. however. the main three forms of Hg are: elemental Hg vapor (Hg 0). TGM is mainly composed of elemental 258 . The most likely candidate for RGM species is HgCl 2. Specific Methods for Determination the Most Important Heavy Metals Pollutants Mercury A particular characteristic of mercury is that it exists in the environment in a number of different chemical and physical forms each with different behavior in terms of transport and environmental effects. Ambient concentrations of mercury in air may range between 1.5 ng L -1. oxidation. concentrations of Hg (or mg Hg). In the atmosphere.

01 ng m -3. The air sample is pumped at a known flow rate through a filter and over the Amasil μtrap. The samples are manually analyzed using thermal desorption and cold-vapor atomic fluorescence spectrometry (CVAFS) detection. The Gardis instrument is based on gold amalgamation and Atomic Absorption Spectrometry (AAS) detection.Hg vapor with minor fractions of other volatile species such as HgCl 2. A 25 mm diameter PTFE membrane is used to protect the analyzer gas inlet from contamination caused by aerosol particles. These developments make it possible to determine both urban and background concentrations of RGM. Manual Methods for TGM The manual methods are based on gold (or silver) trap amalgamation.1 picograms. which enters into the detector where it produces a transient peak. The mercury can be reliably trapped even at elevated temperatures. With 24 h sampling time the detection limit is typically 0. Alternatively. The detection level is below 0. which allows maximum sensitivity. the adsorbing material may consist of gold-coated quartz glass grains. thereby becoming trapped. Any mercury (Hg0) or mercury compound present forms an amalgam with the gold. Automated Methods for TGM The Sir Galahad II System is used to determine the mercury concentrations as TGM. This removes any trace of the sample gas that could cause quenching and replaces it with argon. PM and TGM. With a sampling flow rate of 1. The Sir Galahad II is based on the Millennium Merlin fluorescence detector. new automated and manual methods have been developed to measure TGM. B. A. CH3HgCl or (CH3)2Hg. The Tekran Gas Phase Mercury Analyzers (Model 2537A) is suitable for TGM measurements. The instrument utilizes two gold cartridges in parallel. A rapid heating cycle is then activated converting all forms of mercury to the vapor. The airflow is normally > 0. RGM and PM. The sampling is run at about 1 L min -1 with sampling times of 10 minutes. On completion of the sampling phase the trap is flushed with argon. The mercury is then thermally desorbed and detected in an integrated Atomic Fluorescence Spectrophotometry (AFS).5 L min-1 a detection limit of 0. A 47mm diameter Teflon pre-filter protects the sampling cartridges against contamination by particulate matter.15 ng m -3 is achieved. 259 . Under these conditions. Samples are collected on 10-cm long traps consisting of a 6-mm diameter quartz glass tube containing a mixture of small pieces (1-2 mm) of gold wire and quartz glass grains.5 L min-1. The pre-filtered sample air stream is passed through gold cartridges where the mercury is collected.1 ng m-3 is achieved. The Gardis instrument operates with ambient air as carrier gas and does not require Argon or Helium for detection. In the last few years. a detection limit of about 0. with alternating operation modes (sampling and desorbing/analyzing) on a predefined time base of 10 min.

C. RGM Measurements The mist chamber (MC) technique has been developed by Lindberg and Stratton (1995).2 µm for glass-fiber filters. The denuders are heated to 450 oC and purged with N2. by a nebulizer inside the chamber. The annular denuders are suitable for automated applications. Mercury(II) species adsorbed in the MC solution are analyzed after reduction to elemental mercury by SnCl 2 using the CVAFS detection. The gold trap is then analyzed using the normal desorption and CVAFS detection procedure. The annular denuders for sampling of RGM consist of a 15 mm outer diameter quartz tube with an inner. which then is analyzed using CVAFS.10 L min -1. As in all sampling for suspended particulate matter. possibly less than 0. Part of the MC-solution is dispersed as a fine aerosol. which is pre-concentrated on a gold trap. cristobalite. 260 . The detection limit is of 5 pg m -3 for a 24 hintegrated sample. the accuracy of volume meters should be checked periodically. During sampling. Techniques for sampling water are less complex than for air. The detection limit for a measurement with 2 h sampling time is under 2-3 pg m -3. For most purposes at least. The air sampling flow rate is 10-15 L min-1 and the detection limit for a 6 h sample is 1 pg m-3 Tubular denuders consist of 6 mm quartz tubes coated with KCl. Lead In air sampling. Analysis is made using thermal desorption and CVAFS detection. Air is pulled through the space between the two tubes. the denuders are electrically heated to approximately 45 oC to avoid water vapor condensation. enclosed 8 mm tube. in the range of about 1 µg m-3 or less up to 10 µg m-3. Both the inner surface of the outer tube and the outer surface of the inner tube are coated with KCl. The mercury released from the denuder is collected on a gold trap. or iodine crystals may be used for sampling organic lead compounds in air. Air is drawn through a Pyrex glass chamber of 100 mL total volume containing 30 mL diluted HCl solution. The major question is whether or not the water should be filtered before analysis since it is known that lead occurs in water both in the particulate fraction and in solution. In the analysis step the denuder is heated to 500 °C converting the adsorbed RGM to elemental mercury vapor. Liquid scrubbers containing iodine monochloride and solid scrubbers with activated carbon. The size of the pores of filters for collecting leadcontaining particles should be small. The RGM is quantitatively collected in the annular denuder at a sampling flow rate of 5 . A hydrophobic filter at the top of the MC separates the droplets from the air and allows the liquid to drain back into the chamber. The sampling flow rate is of 1 L min -1. high-volume samplers are preferable for accuracy (when it is necessary). it is reasonable to sample water without any fractionation of the material collected. but the low-volume technique is also useful for obtaining extensive data.

2). homogenization by grinding. also by different types of electro-chemical methods such as classic polarographic methods or anodic stripping voltammetry and cadmium-selective electrodes. Two non-destructive methods for lead analysis have been under investigation in the last two decades. Another problem is the backscatter from the exciting source. Cadmium The most popular method for cadmium determination remains the atomic absorption spectrometry (see II. Anodic stripping voltammetry is gaining in popularity for lead analysis. In the former. more recently.However. Nondestructive methods are more recent and still too complicated for routine studies. Cadmium can be determined. Electroanalytical methods have also been found useful for lead determinations. A dropping mercury electrode is placed in a solution where 261 . consideration must be given to the cost of the equipment and the time involved in performing the analyses. Perhaps no method of instrumental analysis for lead has enjoyed such a rapid acceptance in recent years as atomic absorption spectroscopy ( see II. the sample is first oxidized to destroy all organic matter. The polarographic method found wide application until more effective masking procedures were developed to increase the specificity of the dithizone method. all elements in a substance. nondestructively. The preparation of soil and soil dust samples for lead analysis usually involves drying (at 100 °C). In selecting methods. Numerous specific procedures have been developed based on the spectrophotometric determination of lead dithizonate. The first of these is not likely to find wide application for lead analysis because of the cost and the need for access to a fast neutron source. either for further preparative steps or for direct instrumental analysis. anodic stripping voltammetry. The basic principle behind the electrochemical methods is the change in the electrochemical potentials formed when electrons are transferred from one metal to another. The analytical methods currently in use for the estimation of lead content are of two general types. destructive and non-destructive.4.4. and sieving.2). The ash is then usually dissolved in an aqueous medium. These are neutron activation and X-ray fluorescence. X-ray fluorescence is also theoretically capable of detecting.4. The oldest and best known of the general methods currently in wide use are those based on the formation of the red complex that lead forms with dithizone (diphenylthiocarbazone). They include X-ray fluorescence analysis and fast neutron activation. and in soil. These include polarography and. Its advantage is that the concentration of many elements can be determined simultaneously. in some cases it may be necessary to determine the biological availability for absorption of the various forms of lead that occur in water. The latter is a dust source and may be a food contamination source as well. A major obstacle to the wide application of this method is the profound matrix effect of the substances being analyzed.4.

262 . Furthermore. Different metals can be determined simultaneously in a liquid sample. but never as the free ion.g. Sometimes. CrO 4-2. Therefore. and so it is not normally used for screening. proton-induced X-ray emission (PIXE). Specific cadmium-selective electrodes are commercially available. During oxidation and release from the amalgam. Irradiation with neutrons yields new radioactive cadmium isotopes. but their sensitivity is insufficient for cadmium measurement in most biological materials. can also be used for activation analysis of cadmium. the release of metals that have already been reduced and are bound to the mercury electrode. However. Cadmium has a number of stable isotopes. The irradiated sample is usually digested before the radioactivity is measured. +2. where no sample treatment is necessary. and +6 states are common. which can be quantitatively measured on the basis of their specific energy and half-life. Only the trivalent and hexavalent oxidation states are important for human health. as it is easily oxidized to the trivalent form by air. Divalent chromium is unstable in most compounds. Anodic stripping voltammetry is based on the reverse process. The main advantage of the method is its ability to detect and quantify cadmium in very small volume samples.the metal concentration is to be determined. the complexes being generally octahedral. but only the 0 (elemental). Although a compound CrF 6 is well known. since they form amalgams at different charges. they must always be examined separately. and problems can easily arise from various contaminants in the solution used. the electrodes are not ion specific. +3. the stable forms of hexavalent chromium are almost always bound to oxygen (e. The most crucial aspects are complete destruction of all organic materials and the transfer of cadmium ions from the sample into a non-contaminated electrolyte. i. a peak current can be recorded at a potential that is characteristic for the particular metal. different metals will be reduced and form an amalgam (a solid solution of metal atoms and mercury) with the mercury electrode.. the method is expensive since the samples have to be irradiated in a reactor.e. Irradiation with protons. As a rule. Chromium Chromium occurs in each of the oxidation states from -2 to +6. The detection limit for neutron activation analysis is low. By changing the charge of the electrode. The method is especially suitable for water analysis. Polarographic waves can thus be recorded.1-1 mg L -1 sample. its coordination number is 6. Non-radioactive cadmium can also be added after irradiation to enable measurement of the recovery after digestion and various concentration steps. Cr2O72 ). it may be necessary to concentrate cadmium by chemical methods and to separate the cadmium ions from other isotopes that have an energy spectrum overlapping the one for cadmium before measurement can be carried out. Anodic stripping voltammetry is one of the most sensitive methods for cadmium determination available. The trivalent form exists in coordination compounds. of the order of 0. Several elements are measured at the same time.

cobalt and nickel.2'-bicinchoninic acid. and a number of techniques can furnish satisfactory precision and accuracy. For total Cr determination the X-ray fluorescence and neutron activation can be used. reagents used in sample dissolution. Finally. by carrying out the whole analysis. The collection of dust from air samples may introduce contamination from the chromium in the filters. Gravimetric and colorimetric methods were the earliest procedures used for the measurement of copper. Atomic absorption spectrometry and inductively coupled plasma emission spectrometry are the most used techniques for determination of the chromium content of water and sewage.As chromium is present in low concentrations. the divalent oxidation state readily adsorbs to a variety of hydrated metal oxides including those of iron. On the other hand. The trivalent form of copper is found in only a few compounds and is a strong oxidizing agent.10-phenanthroline) and more recently 1-(2-pyridylazo)-2-naphthol.e. The sensitivity of instrumental analysis for the determination of chromium does not present any problems for concentrations in the mg/kg range. Water samples may extract chromium from containers. Thus. or in a disproportionation reaction. i. almost all of the copper in natural samples is complexed with organic compounds. Many cupric compounds and complexes are soluble in water and have a characteristic aqua-blue-green color.7-diphenyl-1. aluminum and manganese. Thus. the sensitivity of the instrumentation for the determination of chromium in the ng or µg/kg range is severely limited. may contribute significant amounts of chromium.10-phenanthrolinedisulfonic acid). BPKQH (benzyl 2-pyridyl ketone 2-quinolylhydrazone) and 2. etc.. preparation. care must be taken to avoid contamination. Useful spectrophotometric reagents for copper include cuprizone (biscyclohexanoneoxalydihydrazone). unless it is stabilized by complex formation. Gas chromatography with spectrometric detection can be applied only if Cr is chelated and extracted. acid digestion. OH-. Copper The most important oxidation state in natural. and no one method is entirely satisfactory. 30 ng/L Cr(III) can be measured using chemiluminescence detection. and to organic ligands via phenolic and carboxylic groups. bathocuproine (dimethyl-4. In environmental and mineral environments. and digestion. chelation. separation.7diphenyl-1. including sampling. Gravimetric methods are non-specific and may precipitate other cations including zinc. The copper(II) ion binds preferentially via oxygen to inorganic ligands such as H2O. with a detection limit of 1 ng. 263 . aqueous environments is copper(II).9-dimethyl4. it is necessary to control for these influences by simultaneously performing a blank analysis. using all reagents. cadmium. Any copper(I) present is quickly oxidized by any oxidizing reagent present. SO42-. and other reactions. bathrocuproinedisulfonic acid (2. CO32-.

Nickel Nickel usually has an oxidation state of two.005 µg/L. A dramatic increase in sensitivity over that obtained by flame AAS is obtained with graphite furnace AAS. Field instruments are available for scans of contaminated sites to estimate the metal in the surface layer of the soil. no specialized equipment is required. which is especially useful if stable isotopes of copper are used. Prior to the determination of nickel in 264 .and tetravalent ions. Nickel forms complexes (chelates) that are insoluble in water. For example. with a limit of detection of 0. which is the obvious advantage over other spectroscopic techniques. A further increase in sensitivity is obtained through the coupling of the ICP to a mass spectrometer (ICP-MS). but soluble in organic solvents. Voltammetric / potentiometric analyses offer sensitivity in the parts per billion (µg/kg) range for copper.15 % for copper and cadmium in zinc ore and for copper and molybdenum in domestic sludge. which are non-destructive techniques.The bathocuproine method can achieve a limit of detection of 2 µg Cu/Litre in water samples. In addition. nickel dimethylglyoxime is the compound that makes possible the separation of nickel from cobalt. Ion-selective electrode and potentiometric methods have been used for copper speciation in soil and in seawater. easily taught and rapidly carried out. An isotope dilution ICP-MS method reported precision of less than 0. the methods are. Many X-ray fluorescence (XRF) methods. XRF has been used for a long time as a rapid and convenient method for trace element determination although its sensitivity is somewhat lower than anodic stripping voltammetry. The ICP-MS technique has the additional advantage that isotopic information can be obtained. An attraction of potentiometric methods is their ability to help in the speciation of copper and limited multielement detection. inexpensive. in general. Although colorimetric methods can suffer from lack of specificity. Beyond a spectrophotometer and an analytical balance. they are nevertheless useful. Cathodic stripping voltammetry (CSV) is an extremely sensitive method for copper in both seawater and fresh water. simple. have been published for the determination of trace elements including copper. The attraction of the ICP methods is the ability to do multielemental analysis. especially in laboratories where more sophisticated instrumentation is not available. Atomic absorption spectrophotometric (AAS) methods are the most widely used for the determination of copper in various matrices. but also occurs as relatively stable tri. Increasingly more common is the use of emission methods in which the sample is introduced into a high temperature inductively coupled argon plasma (ICP) where the element is rapidly vaporized and ionized. The element is detected and quantified by atomic emission spectroscopy (ICP-AES). which is similar in its chemical and analytical behavior. These compounds are often very stable and play an important role in trace analysis.

pre-concentration steps are introduced. An isotope dilution gas chromatography-mass spectrometric method permits the detection of nickel in environmental samples at the ng/L level.environmental materials. the organic constituents must be oxidized or removed to avoid interference during analysis.g. The introduction of a Zeeman-compensated system in electrothermal atomic absorption spectroscopy (EAAS) improved the background compensation and permitted a more rapid and direct determination of nickel levels with considerably lower detection limits (0. Though it requires time-consuming sample digestion procedures. Techniques very frequently employed include chelate extraction with dithiocarbamates. The progresses in voltammetry have made this method the most sensitive. dimethylglyoxime.0. or 8hydroxyquinoline into organic non-polar solvents. The analysis for nickel in natural water is frequently performed by EAAS following pre-concentration. As nickel concentrations are often low in relation to analytical detection limits. Another pre-concentration technique. and removal of interfering humic substances can be achieved by pre-concentrating nickel and other trace metals in natural waters on XAD-7 regions (cross-linked polymer of methylmethacrylate) in a two-step procedure at two different pH values. which was suitable for routine use. The method depends on the preparation of a thermally stable and volatile chelate (chelating agents: sodium diethyldithiocarbonate or lithium bis(trifluoroethyl) dithiocarbamate) followed by on-column injection into a gas chromatographic column and electron ionization of the eluted chelate in the mass spectrometer. A less time consuming method for the preconcentration of nickel in seawater involves complexation of the trace metals by 8hydroxyquinoline followed by adsorption on C 18 chemically bonded silica gel. is the use of chelating ionexchanged resins. The differential pulse voltammetry (DPV) may be used after prior interfacial accumulation by an adsorption layer of nickel-dimethylglyoxime chelate at the hanging mercury drop electrode (HMDE). 265 .09 .5 µg/L). Inductively-coupled plasma atomic emission spectroscopy (ICP-AES) has gained importance in simultaneous multi-element determination. The two most commonly used analytical methods for nickel are atomic absorption spectroscopy and voltammetry. smaller sample volume. prior to analysis of nickel in fresh and seawater. which may also separate nickel from substances interfering with analysis. and less costly than EAAS. The measurement of nickel concentrations as low as 1 ng/L was possible using this method.. Large concentration factors (200:1) provide detection limits as low as 10 ng/L in seawater analysis. more rapid. furildioxime. A greater enrichment factor. e. Chelex 100 ®. voltammetry is more sensitive.

Chemical Analysis and Speciation . D. REFERENCES 1. Seiler. mitigate. Sampling for Analytical Purposes. Astrid Sigel and Helmut Sigel. Pests are representing living organisms occurring in places where they are not wanted. Ed. England 4. Ltd. Ltd.5.. United Book Press.T. Clesceri. Jacques Buffle and George Horvai. 1998. The Chemical Analysis of Water. John Wiley & Sons. Inc. General Principles and Techniques.. chemical or biochemical (metabolization in living organisms) degradation of a pesticide. conservation. Monitoring of Pesticides Definitions Pesticides are substances.E. such as welding fumes and grinding dusts. Pierre Gy. repel. Greenberg. Oxford. A pesticide residue represents any compound resulting from the physical.. Hans G. 1998.Electron microscopy and X-ray microanalysis can be used for the determination of nickel in single dust particles. USA 3. 2000. Andrew D. In Situ Monitoring of Aquatic Systems. animals or crops. Inc. Standard Methods for the Examination of Water and Wastewater . USA II. Ed. John Wiley & Sons.5. and destroy any pest. 2 nd Edition. Baltimore. Hunt and A. 1994. Marcel Dekker. mixtures of substances or living (micro) organisms intended to prevent. Alden Press.. Great Britain 5. West Sussex. West Sussex. resulting in the enhancement. PESTICIDES Andrei Valentin MEDVEDOVICI II. England 2. Ed Leonore S. Arnold E. or reduction of the initial toxic potential of the parent compound. 266 . 1988. Handbook of Metals in Clinical and Analytical Chemistry . Eaton.L. New York. resulting in damages to humans.5. 20th Edition.1.Wilson.5.

beneficial predators that eat insect pest). Slow release formulations). Biochemicals. Algicides. b) wastes from manufacturing sites.g. d. Baits. Pesticide persistence: the period of time during which a pesticide remains active at the application area describes its persistence. according to International Agency for Research on Cancer : Carcinogenic. Bactericides. d. Most of these reactions are microsomal mixedfunction oxidase reactions catalyzed by the cytochrome P-450 enzyme system associated with the endoplasmatic reticulum of the cell and occurring most abundantly in the liver of vertebrates. c. Glossary of Terms Pesticide sources are either non-point sources or point sources. acetyl) resulting in conjugated species. glutathione. Growth regulator. The non-point sources are represented by a) accidental release on storage. Molluscicides.g. products containing low risk ingredients (e. Attractant. Rodenticides. Classification Pesticides are classified according the following criteria: a. Probably not carcinogenic). biological control agents (e. their mode of action (Antifeedant. Ovicides. Avicides. Possibly carcinogenic. Antifouling agents. d) leaching through soil. which have a higher polarity. Insecticides. loading and transportation.the followings are not regulated as pesticides. Fumigant.Exceptions . Defoliant. Highly. e. Chemosterilant. Viruscides). the target pest they are intended to destroy (Acaricides. drugs used to control human or animal diseases. Dusts. Probably carcinogenic. Safener. Organics. b. Microbials). despite their apparent fit to the previous given general definitions: a. Synergist). b) accidental drift during application. Repellent. The transport of pesticides in the environment can be achieved according to the following processes i) direct fallout from application points. Fungicides. The point sources are represented by a) direct application points. Desiccant. Not classified. Its carryover effect describes the presence of the pesticide at the application area once its mission has been 267 . Moderately and Slightly Hazardous. Nematicides. Herbicides. Coatings. garlic or mint oil). Plant incorporated protectant. Aerosols. c) surface run-off from land at application points. Mating disrupters. type of formulation in which a pesticide is included (Granulars. their nature (Inorganics. iii) overland and/or subsurface flow (erosion). iv) atmospheric deposition (dry or wet). ii) adsorption on carriers. their impact on health (according to World Health Organization: Extremely. Pesticide fate: most of the lipophilic pesticides tend to undergo enzymatic reactions that make them more water-soluble and reactive by the attachment of polar functional groups such as –OH. The final products are then involved in conjugation reactions with endogenous conjugating agents (glucuronide. fertilizers and nutrients (not considered plant growth regulators). c. greater water solubility and thus are more easily eliminated. b. sulfate. c) spills during mixing.

two or three successive sample preparation techniques need to be applied to a sample. The mechanisms for resistance depend on the pesticide’s mode of action (as example. reduced. or no risk effects on environment and human health. 268 . Sample Preparation As showed in Figure II. The half life represents a measure of the pesticide persistence and is quantified as the amount of time taken by a pesticide to decompose by 50% from the applied chemical to an inactive form. while organo-phosphorus compounds. its size and corresponding concentration levels of the target compounds. photosynthesis inhibition induced by triazines can be avoided by some weed biotypes by developing slight changes in the chloroplast protein structure). more or less complicated procedures are used. and may exhibit higher. Sampled quantities should be in accordance with the analytical method further considered and the inherent sensitivity required. Adsorption cartridges or discs are obtained on the collection site and transported to the laboratory for analysis. but not glyphosate.1. Traditionally. Sample Preservation and Storage The sampling action should be oriented towards representativity and homogeneity. etc. Freezing samples immediately after collection should provide preservation for thermally unstable extremely volatile compounds. Usually. Sampling. Decomposition products may exhibit higher. Mainly for water quality monitoring (environmental analysis) samples may be subjected to concentration at the point of collection. plasticizers contamination from container walls. Preserving sample integrity depends on the stability of the target analytes in a specific matrix.accomplished. polypropylene or polycarbonate) are often preferred for their robustness. sampling containers are made on glass. reduced or no pesticidal activity compared to the parent compound. are adsorbed on polyethylene). For some particular compounds. in order to demonstrate the suitability of the material of the sampling container.5. enough is taken for at least the application of a second full analysis procedure. Depending on the type of sample to be analyzed. Laboratory tests should be performed prior to the collecting action. sample preparation methods applied to pesticide analysis are of extreme variety. Pesticide resistance: developed resistance of the target to commonly used pesticides over a period of years by means of natural selection of the occurring biotypes. More often.). Contamination and cross contamination risks should be attentively considered (conditions for transportation and manipulation of empty sampling containers and collected samples. Plastic containers (polyethylene. adsorption on polymeric materials or even glass may be significant (captan adsorbs readily on glass. in order to achieve analyte concentration and to eliminate efficiently the matrix interferences. Specific chemical addition of stability enhancing compounds is used occasionally (controlling the pH of the media or blocking active sites of the target analytes by means of derivatization).

while important reduction of the process duration and saving of extractant are obtained. Solid – liquid Extraction For practical reasons. C2. Direct desorption of the analytes into the LC or SFC columns by the mobile phase are achievable using an on-line set-up. amides. Four types of adsorbents are usually shared by SPE applications to pesticides. B. classical procedures are continuously upgraded to get improvements in terms of reliability and performances. and their mixtures are among the preferred extraction solvents for phenylureas. Ethyl acetate. special attention should be paid to cartridge / disk drying (this operational step is required between sample loading/clean-up and analyte desorption). When using desorption solvents non-miscible with the solvent of the initial matrix. A. Solid Phase Extraction (SPE) SPE is used for isolation of the target compounds from liquid media by means of adsorption on a granular solid bed (cartridge) or on a porous solid membrane (disk). especially from water and biological fluid samples. 269 . benzimidazoles and chlorotriazines. Some continuous LLE extractors or steam distillators are also available. C8. extractant volume and number of cycles. irradiation time. After matrix removal.2. The main factors affecting extraction efficiency are microwave power. The classical way of performing LLE is the separation funnel extraction. serious problems related to analyte focusing and chromatographic efficiency loss may arise.5. dichloromethane. The extraction efficiency is modified by adjustment of pH and ionic strength in the aqueous phase. Such an approach relates with the conventional Soxhlet extraction assisted by focused microwaves (FMSE). chlorophenoxy acidic herbicides are derivatized with dimethyl sulphate prior to their extraction in n-hexane). in terms of extracted mass and repeatability is similar to the classical method. The extract is concentrated by solvent thermal vaporization or gas flush. it is important to estimate the desorption kinetics of the trapped analytes. When using on-line procedures. analytes are desorbed from the solid material using a specific solvent or mixture of solvents. Comparison with the ISO 659-1988 reference extraction method proved a better efficiency of FMSE. C. Organochlorine pesticides (OCPs) and related residues were successfully isolated from sunflower seeds by means of FMSE. In situ derivatization of the target analytes is also used as an effective tool (ex. The result. Contrarily. C1) are extensively used for a large variety of samples (biological samples such as serum and urine for atrazine. carbamates. Hydrophobic modified silica materials (C18.Sample preparation methods used for pesticide isolation arranged according to the sample type are summarized in Figure II. Liquid – liquid Extraction (LLE) LLE is the classical method used for pesticide isolation. triazoles.

sulfonyl ureas or environmental samples such as different types of waters for alachlor. prometryne. aldicarb. 270 .simazine. phenyl ureas). ametryn. methiocarb . medium polar. neutral and alkaline herbicides.with concomitant hydrolysis.

Steam Distilation (SD). Supercritical Fluid Chromatography (SFC). Solid Phase Extraction (SPE).1. Ultra-Sonication (US). Micellar Electrokinetic Chromatography (MEKC). Subcritical Fluid extraction (ScFE). Evaporative Light Scatering Detector (ELSD. MS/MS. 271 . Thermoionic Detector (NPD). Inductively Coupled Plasma – Mass Spectrometry (ICP-MS). Suported Liquid Membrane Extraction (SLME). Thin Layer Chromatography (TLC). Capillary Electrophoresis (CE). including condensation nucleation – CN). Solid Phase Microextraction (SPME).Sampling Sampling storage Sample preparation Rough isolation Cleanup Target analyte(s) isolation Concentration Derivatization (precolumn) Separation method Derivatization (post column) Detection Data analysis Quantitation Structural confirmation Accelerated Solvent Extraction (ASE). Mass Spectrometry (MSD). Single Drop Microextraction (SDME). Liquid Chromatography (LC) (including capillary µ LC and capillary electrochromatography CEC). Microwave Assisted Extraction (MAE). Liquid-Liquid Extraction (LLE). Supercritical Fluid Extraction (SFE). Atomic Emission Detector (AED). Vis. Liquid Chromatography – fraction collection (LC). MS(n). Low Temperature Lipid Precipitation (LTLP). Electron Capture Detection (ECD). DAD) Figure II. Stir Bar Sorptive Extraction (SBSE). Fluorescence Detector (FLD. General analytical process for pesticide-containing samples. Focused Microwave Soxhlet Extraction (FMSE). Size Exclusion Chromatography (SEC).5. Pressurized Fluid Extraction (PFE). including Laser induced fluorescence). Matrix solid phase dispersion (MSPD). Immuno Afinity Clean-up (ImCU). Nanofiltration (NF). Electrochemical Detection (ELCD). Spectrometric Detection (UV. Gas Chromatography (including Fast Temperature Programming and Low Pressure)(GC).

Solid Phase Extraction (SPE) a. 4. 272 . 2. on cartridges I. matrix oriented (clean up) on shielded materials on ion exchangers II.2. 3. 2. mixed (SPE2) SOLID SAMPLES Vapours 1. b.5. Supported Liquid Membarenes (SLM) or Semi-permeable Membrane devices (SPMDs) 2. 3. 6. off line. 5. 2. Filter retention. Laser desorption / ionization/MS. Gum Phase Extraction (GPE) on Open Tubular Traps 1. on discs. Aerosols 1. LIQUID SAMPLES Liquid – Liquid Extraction (LLE) Size Excluzion Chromatography (SEC) Supported Liquid Membarenes (SLM) or Semi-permeable Membrane devices (SPMDs) 4. Solid Phase Microextraction (SPME) 6. on line. analyte oriented (concentration) on silicagel hydrophobic modified materials on polymeric materials on porous & non-porous graphitized carbon blacks on molecular imprints on immunosorbents on ion exchangers III. Sample preparation methods for pesticides according to sample type. c. 8. Gum Phase Extraction (GPE) on Open Tubular Traps Soxhlet Extraction (SE) and Focussed Microwave (FMSE) Pressurized Fluid Extraction (PFE) Microwave Assisted Solvent Extraction (MASE) Supercritical Fluid Extraction (SFE) Soil Column Extraction (SCE) Head-space – Solid Phase Micro-extraction (HS-SPME) Supported Liquid Membarenes (SLM) or Semi-permeable Membrane devices (SPMDs) Matrix Solid Phase Dispersion (MSPD) Solid airborne particulates 1. 7. Figure II. Filter retention. 2.SAMPLE PREPARATION METHODS for Pesticides AIR SAMPLES 1. Stir Bar Sorptive Extraction (SBSE) 5. d.

Extraction on immunoaffinity sorbents (ISs) is based upon the molecular recognition using antibodies. 2. acetanilides. diquat. Desmetryn served for producing its own 273 . juices. phenyl ureas. Ethylene glycol dimethylacrylate – methacrylic acid copolymers can be imprinted with trialkylmelamines or dibuthylmelamine in order to generate templates selective to atrazine. Triazines. aryloxyphenoxypropionic acids and related metabolites are well isolated on styrene – divinyl benzene (SDVB) hydrophobic materials. Recently. benzimidazoles. The entrapment of esterases in a ceramic SiO 2 sol-gel matrix leads to isolation of organophosphates and carbamates. was introduced for extraction of chlorophenolic pesticides in water samples. 2. Glufosinate and related metabolites in hard water samples and chlormequat in pear. The extraction and clean-up of complex biological and environmental aqueous samples are achieved in a single step even for large volumes. triazoles. Chlorophenoxy herbicides (requiring on-line derivatization with tetraalkylammonium salts). and cereals require matrix clean-up on strong cation exchangers. ureas and amidic herbicides are better isolated on the more polar OASIS materials while other compounds including carbamates. phenoxyalkanoic acids and quats (paraquat.Polymeric materials (styrene – divinyl benzene based copolymers such as PLRP and Lichrolut EN) as well as vinyl pyrollidone – divinyl benzene copolymers (commercially named OASIS) are mainly in current use. MCPB. Molecular imprints are synthetic polymeric phases on which selective receptors complementary to the target analytes have been generated during preparation. were successfully used for isolation of analytes from complex matrices. Strong cation exchangers are moreover used for sample clean-up (matrix elimination). Anti-atrazine monoclonal antibodies and antidinitrophenyl polyclonal antiserum were also immobilized on the same purpose. triazines.4-D.4. Surface functionalization with molecular imprinted polymers was obtained also for porous polypropylene films. Few applications on pesticide isolation relate to the use of ion exchanging resins. aryloxypropionic acids. respectively) are also used for herbicide isolation from water samples. difenzoquat) are readily isolated on graphitized carbon beds.5-TP and benzoic acid derivative of Dicamba) was achieved on hydroxymethylmethacrylate MFE polymer containing quaternary ammonium functional groups. 2.4-DB. neutral diphenyl ethers. MCPA.4-DP. polyaniline. a new polymeric material. Immunosorbents (IMS) produced by covalent immobilization of the antibody generated against the target analyte on inert materials (silica gel or activated agarose gel). Thifluzamide in peanut samples and imazalil in citrus fruits were rapidly isolated using IMS. thiocarbamates. Non-porous and porous graphitized carbon black solid beds (NPGC and PGC. The extraction of chlorophenoxy acidic herbicides residues from green bean samples (2. Two new directions evolved in the last few years related to the SPE of herbicides: the synthesis of immunosorbents and polymeric molecular imprints.

Parameters as sample pH. The nature of the polymeric film as well as its thickness should be related to the polarity characteristics of the extracted compounds. Some other materials were especially developed for specific matrix elimination. solvent desorbed directly to LC columns or on-line / off-line desorbed by supercritical state carbon dioxide for SFC separations.5 trichlorophenoxy acetic acid was used as template in a system of 4vinylpyridine as monomer. Trapped compounds can be further thermally desorbed directly into GC inlets. Solid Phase Microextraction (SPME) SPME consists in the adsorption of the target compounds on a thin polymeric film deposed on the surface of a capillary fiber. polydimethyl siloxane (PDMS).g. ethylendimethacrylate as reticulation agent and methanol/water mixture as porogen. carbowax – divinylbenzene and PDMS – divinylbenzene films having thickness between 30 and 100 µm. Dichlofluanid. Pressurized Fluid Extraction (PFE). while the other acts as a matrix remover (clean-up). followed by trapping of the resulting vapors in the coated fiber (procedure is known as “Head– Space” HD/SPME). Basically. Assisted Solvent Extraction (ASE) Combining properties of supercritical fluids and/or extraction solvents with pressure/temperature/density effects and automatically controlled static/dynamic setup results in an increased flexibility and efficiency of the previously cited methods. sulphamides. Chlorothalonil. Fluometuron. serum. Captan. the hydrophilic character of the external particle’s surface and the lipophilic character of the inner pore surfaces in these materials are responsible on their intrinsic capabilities of discrimination. Isoproturon. E. ionic strength. stirring rates. plasma). organic additives. sequential coupling of two SPE processes (SPE 2) is sometimes required. Supercritical Fluid Extraction (SFE). Owing to the increasing complexity of the sample matrices. and contact duration have to be individually considered. Vinclozolin. The mass transfer can be achieved either from liquid media in direct contact with the extracting coated fiber as well as from gaseous environments. Metobromuron and Monouron) are better isolated on polyacrylate fibers. 274 . combining size exclusion and polarity based interaction mechanisms. 2. Recoveries of the analytes ranged between 70 and 124% when determined in sea water samples. D. Chloroanilines. Organochlorine herbicides were monitored in landfill leachates using PDMS coated fibers while ureas (Chlorsulfuron. Restricted access or shielded materials eliminate selectively macromolecular compounds from pesticide containing samples (e.template. phtalimides and oxazolidones (Dichloran. one SPE procedure is addressed to the isolation of the target compounds. Folpet and Captofol) were extracted on polyacrylate. soil extracts.4. Volatile or semi-volatile pesticides existing in solid samples can be easily transferred in the gas phase on heating in closed vials. Linuron. Generally.

Gum Phase Extraction (GPE) GPE is an application of the sorption phenomena on polymeric materials used during the sampling process at temperatures above their glass transition point (T g).However. phenyltrimethyl ammonium hydroxide and trimethylsulfonium hydroxide for methylation. The method is applied for the isolation of pesticides in tissues (animal or human origin). All pesticides enlisted in the 608 EPA method were 100% recovered by means of such a practice at a concentration level of 10 pg x L-1.0.3 m lengths of fused silica columns with 0. and vegetables. derivatization with heptafluorobutyric anhydride (HFBA) and gas chromatography / electron capture detection (GC-ECD). linuron. chlozolinate and herbicides diuron. Pesticides containing carboxyl or phenol groups were readily derivatized simultaneously in PFE by using acetic anhydride for acetylation. are generated by the in situ hydrolysis simultaneously carried out with steam distillation (SD) removal . procymidone. respectively. Trapped analytes are thermally desorbed into the gas chromatographic column. Extra column cryo focusing may be necessary.15 µm film thickness.5 mm inner diameters and 10 . 275 . Cyanazine. Air samples should be sucked through the coated tubing. neburon and propanil. food.4 dichloroaniline and 3.3 . iprodione. fruits.5 dichloroaniline as markers for the exposure to fungicides vinclozolin. polymeric materials no longer behave as pure solids but enter a gumlike or even liquid-like state with properties similar to those of organic solvents (considering diffusion and distribution constants). Addition of silica onto solid vegetal materials (such as tobacco leaves) proved to induce a major matrix removal effect during analysis of OCP’s. Open tubular traps are more commonly coated in 1 . As an example. realizing simultaneous disruption and extraction of solid or semi solid samples. followed by liquidliquid extraction (LLE). Atrazine. The commonly used sorbent is polydimethylsiloxane (PDMS). In-situ degradation of the target compounds in order to generate class markers was also practiced for non-persistent pesticide analysis in biological samples. G. 3. Metribuzin and Simazine were studied to determine their toxicity in aquatic media (including correlations with environmental temperatures and dissolved oxygen content) by monitoring their concentrations in catfish muscle and liver using MSPD as selective extraction step. In such conditions. two practices related to SFE / PFE / ASE processes should be mentioned. The first one relates to the fixation effects in the extraction thimble. Such practice was discovered during development of SFE procedures for pesticide determination in fruit juices (diatomaceous earth was “solidifying” liquid samples). F. N-O-bis (timethylsilyl) trifluoroacetamide (BSTFA) for silylation and borontrifluoride / methanol. The other practice relates to in-situ derivatization of target analytes during SFE / PFE / ASE procedures. Matrix Solid Phase Dispersion (MSPD) MSPD is a sample preparation process dating from 1989.

Atrazine. Desisopropylatrazine and Desethylatrazine were poorly recovered (< 7%). Stir Bar Sorptive Extraction (SBSE) Stir bar sorptive extraction (SBSE) is based on the same principle as GPE but the polymer covers the external surface of a magnetic bar stirrer. A practical example is given by the determination of Thiofensulfuron methyl and Tribenuron methyl in cottonseeds and cotton gin trash. while the light fraction is eluted later and is collected for subsequent clean-up. Propazine and Sebuthylazine. sterols) are excluded from the stationary phase material. ca.extraction efficiency relationship. Thin Layer Chromatography 276 . re-dissolving and injection to a LC column. while the organic solvent was immobilized into the pores of the membrane. 80% for Terbutryn and Prometryn.H. Aromatic aminophosphonate isolation from water samples based on SLME allowed identification and study of the operational parameters (pH and ionic strength of the aqueous phase. Cyanazine. The same effect is obtained if PDMS gum is packed into a cartridge and the liquid sample is pushed through using HPLC pumps. Supported liquid membrane extraction (SLME) emerges also as a fast and efficient sample preparation alternative solution for pesticides. 75% for Simazine. Separation Techniques A. 110% for Terbuthylazine). reducing lipid co-extraction. Good recoveries were obtained for triazines analyzed in water samples when isolation was made as stated previously (ca. with subsequent solvent removal for concentration. Metribuzin. I. Size Exclusion Chromatography (SEC) SEC is generally required as an isolation procedure for pesticides in fat matrices. separation or analysis. Hollow fibber membranes are also used for pre-concentration of nitrophenolic pesticides in water samples. J. with the stirring capabilities of the sample phase together with the permanent pumping of the organic phase.g. The rod is inserted within the liquid sample and stirred for a defined period of time. Supported Liquid Membranes (SLM) Micro porous membrane liquid-liquid extraction (MMLLE) of lipophilic pesticides in biological fluids combines the size exclusion properties of the membrane. composition of the membrane phase and concentration of analytes) as well as the structure . Such liquid-liquid-liquid micro extraction device (LLLME) is suitable for interfacing even with micro column liquid chromatography. Analytes are thermally desorbed directly in the gas chromatographic column or back extracted into an appropriate solvent. The mechanism is based upon the pH difference inside and outside the hollow fibber. GC-MS was used for separation and detection. triglycerides. ca. High molecular mass compounds (e.

Medium polarity stationary phases are widely used (SE 52. Triazines and some phenyl ureas are separated also on nonpolar polydimethylsiloxanes (OV 1 or equivalent). Supelcowax RSL 300 or equivalent). more than 90% use capillary columns (25 to 30 m length. In fact. AMD-TLC equipments being commercially available. the entire procedure represents the application of three or even more different step composition gradients during a single run over TLC silica plates having thickness higher than 100 μm. Starting program temperatures for triazines and phenyl ureas are lower (40-60 oC) while higher values are used for phenoxycarboxylic esters (80-85 oC) and organophosphoric derivatives (100 oC). OV17. finally leading to extremely sharp lines. trichlorophenoxy acetic and phenoxy propionic herbicides. Associating densitometry detection. Organophosphorus herbicides and chlorotriazines are separated with increased selectivity on SPB-35 or polyethylene glycols (Carbowax 20 M.30 film thickness). Thin Layer Chromatography (TLC) is associated to the screening of pesticides. especially from water samples. Of the GC methods for pesticides reported in literature. 0. DB 5. Flash vaporization is carried out at ca. Some phenyl ureas. even considering the possibility to produce UV-VIS reflectance spectra of the separated spots.25 to 0. Temperature gradients of 15-30 oC min-1 are generally used for 277 .15 to 0. Hot splitless injection (HSI) should be considered a rugged technique suitable for most pesticides. 54. Standardization of AMD-TLC. B.32 mm inner diameter and 0. The AMD approach uses more than 25 different development steps (1 .3 mm migration distance each) with intermediate drying steps. 220 oC for most analytes. quantitative determinations are allowed. Both mobile phase change and drying are made in an automated way. carbamates and organophosphoric compounds are susceptible to thermal degradation with HSI. or nature changing continuously from step to step. The Automated Multiple Development (AMD) feature highly increases separation capacity and versatility of the method. Gas Chromatography (GC) GC is a powerful technique for pesticide separation. Some applications of Large Volume Injection (LVI) on PTV are also cited in the literature for phenoxyacetic. each new development generates focusing of the spots. The temperature gradient is the key for tuning selectivity in GC. Scratching spots from plates followed by analyte extraction from the solid material in appropriate solvents and subsequent introduction in a mass spectrometer make possible structural confirmation and sensitive quantitation. Electronic Pressure Control (EPC) with pulse programming reduces decomposition of carbamates on injection. Because of intermediary drying steps. as a DIN method for pesticide determination in ground and drinking water. Silanized glass wool inserts also enhance the decomposition of labile compounds. dates since 1993. the mobile phase composition. DB7 or equivalent).More often. Sample throughput of the method should be considered as satisfactory since 12 to 24 sample applications are feasible on a single TLC plate. On-column injection (OCI) or programmed temperature vaporization (PTV) are offering powerful alternatives.

Enantiomers of the organophosphoric pesticides crotoxyphos. Applications on triazines use temperature gradients ranging from sub ambient conditions up to 70 oC. some organophosphorous. The detection systems used for LC analysis of pesticides are: UV spectrometric detection (especially diode array detection . fonofos. methamidophos. azoles. crufomate. Tautomeric equilibrium. Low limits are also obtained in some cases with selective NPD or ELCD detection (1 ng). mainly at low concentration of the target compounds. The normal phase (NP) mechanism is used only for achieving enantioselective separations. methamidophos. fensulfothion. There are a relatively reduced number of classes of pesticides not suitable for GC separation and therefore requiring the use of liquid chromatography (LC). Among them. phenylurea. profenophos. Liquid Chromatography (LC) The main reasons for failure of GC methods for pesticide analysis are the thermal instability. competitive to chromatographic partition. carbamates. Halogen containing pesticides are detected at the pg level with the ECD ( 63Ni). monocrotophos. Mass spectrometric detection is widely used due to its universal character as well as for its intrinsic sensitivity and selectivity. benzimidazoles.less complex samples. isofenphos. dinitrophenols.g. low volatility. Both EI and CI (using methane or isobutane as reagent gas) are used for ionization. prothiophos and trichloronate were successfully separated on polysaccharide based chiral stationary phases (ChiralPak AD. However.allowing peak 278 . malathion. dialifor. oxydemeton-methyl and vamidothion). separation of water un-extractible organophosphoric pesticides as acephate. Two different steps are required for complex mixtures or crowded matrices (first gradient range in the interval 10-25 oC min-1. In LC. ion exchange (IE) and ion pair (IP) mechanisms are also used. OD and OJ) under NP conditions. and high polarity characteristic of some target analytes. the separation mechanism dedicated to pesticide separations is undoubtedly the reversed phase one (RP). the inherent selectivity of LC affords separation of the tautomeric structures. AS. The use of micro columns also enables temperature gradient as an additional selectivity / efficiency tuning factor. is strongly affecting peak shape and symmetry.DAD .g. fenamiphos. C. Micro LC columns are used for a better compatibility with specific sample preparation methods (e. leading to serious peak splitting. Sometimes. benzoylureas. the second one between 3-6 oC min-1). omethoate. on column focusing) or interfaces for MSD. pyrethroid and quaternary ammonium derivatives should be cited. in order to stabilize the analytes in a single form. More often. In RP applications. especially for quaternary ammonium derivatives (quats). polar end capped octadecyl silicagel stationary phases are especially recommended for separations requiring high content or even 100 % aqueous containing mobile phases (e. the addition to mobile phases of concentrated buffers or strong acids is needed. up to 260-290 oC and a single ramp.

fluvalinate and bifenthrin) with production of highly fluorescent photo degradation products. fluorescence detection (FLD). The particle condensation nucleation practices should also enhance ELSD sensitivity. On-line hyphenation of SFC with SPE or SPME (desorption is made by the supercritical mobile phase) achieves high extraction yields and sample throughput. lufenuron and flufenoxuron) or pyrethroids (namely phenpropathrin. triflumuron. high selectivity. particle beam (PBI) and matrix assisted post source decay LASER desorption / ionization (CID-PSD-MALDI). Supercritical Fluid Chromatography (SFC) Packed Column Supercritical Fluid Chromatography (P-SFC) offers an interesting alternative for pesticide separations based on the normal phase partition mechanism. generally introduced before the automated pressure relief valve (nozzle) of a downstream configuration. Although ESI and APCI are more often used. hexaflumuron. MS is undoubtedly the solution of the near future for LC detection. acrinathrin. Improvements made on interfacing devices together with a continuous and sensible diminution of instrumentation costs promote MS as a universal / selective tunable detection system. Against the cited detection systems only ELS and MS are universal detectors. This could be overcome by derivatization. due to the lack of suitable chromophoric / fluorophoric molecular sites. Two post column derivatization modes are noticeable for pesticide detection. fenvalerate. Multi dimensional techniques (MS / MS or (MS) n) enable to find out the right ratio between selectivity and sensitivity of the detection process. other LC / MS interfaces produce reliable results in pesticide applications: thermospray (TSI). Extremely selective. and good peak symmetry are obtained on applying both density / pressure and modifier gradients. Atmospheric pressure electrospray ionization (ESI) and atmospheric pressure chemical ionization (APCI) are the most robust and popular devices for interfacing MS to LC systems. Reproducible retention. allowing fast (automated) off-line bi-dimensional separation techniques. electrochemical detection (ED). reproducible and fast SFC applications relate to chiral separation 279 . the other one deals with the photo irradiation of benzoylurea insecticides (namely diflubenzuron. cyfluthrin. UV or FL detection of the target compounds is sometimes not achievable.confirmation by means of spectra comparison). even when using nonpolar modified stationary phases. deltamethrin. Their characteristic sensitivities can be considered to increase in the following order: ELSD < UV (DAD) < ED ~ FLD < MSD. evaporative light scattering detection (ELSD) and MSD. A similar set-up based on the introduction of an additional suitable solvent flow through the nozzle leads to a versatile SFC semi-preparative or even preparative fraction collection / isolation alternative. One relates with the fluorescent detection of carbamates based on their reaction with orto-phtalaldehyde (OPA). D. Interfacing with MSD (both ESI and APCI) does not require any modification of the commercialized interface designs but only an additional solvent flow.

of pesticide racemates on chemically modified polysaccharides or antibiotic modified stationary phases. metribuzin. fluorescence methods (FIAs. together with the corresponding detection limits. On-line coupling of MEKC with ESI-MS was recently found as achievable using partial filling (PF) or reverse migrating micelles (RMM) techniques. although accepted standardized methods are still missing. limited shelf life.2. triadimefon. terbuthrin. in situ measurement capabilities and selectivity. desethyl 2-hydroxyatrazine. Drawbacks are related to inherent matrix interferences (mainly on biosensors). The measuring principle of a biosensor designed for pesticide determination is mainly enzyme inhibition. prochloraz. even on use of the classical on column diode array detection. difficulties for coupling receptors to supports.5. identification of sensitive transducers and a relative delay in generating the response. Some of the immunoassays designed for pesticides are given in Table II. atrazine. including the polarized PFIA approach) or chemiluminescence methods (CLIAs). Bio. radiometric methods (RIAs).Ag). imazalil. On-line stacking procedure carried out on injection acts as a pre concentration step and together with SPE for sample preparation shifts detection limits in the low µg x L-1 range. chlorotoluron.and Immuno Sensors Bio and immunosensors are systems based on measurement of the results generated by means of the binding to a specific receptor of the target analyte. Pesticides are not ordinarily antigenic. thiabendazole. carbendazin. Immunochemical Assays Immunoassays (IAs) are based upon the selective interaction between a specific antibody (Ab) and a hapten (H or antigen . protoplasts. thylakoid membranes. ethofumesate. Main advantages related to bio and immuno sensors lies to the high sample throughput. 280 . isoproturon. 3-chloro-4-methylphenyl urea are only some of the pesticides being reported to be separated by means of MEKC and CEC. linuron. 2-hydroxyatrazine. desethyl atrazine. Micellar Electrokinetic and Capillary Electro Chromatography Micellar Electrokinetic Chromatography (MEKC) and Capillary Electrochromatography (CEC) are powerful techniques achieving efficient pesticide and pesticide residue separations. consequently they have to be conjugated to a carrier molecule (usually a protein) in order to induce an immune response. methylthiophanate. Cyanobacteria. Quantitative migration – toxicity relationship for phenoxy acid herbicides has been obtained with MEKC on using micellar Brij 35 based migration media. any or limited sample preparation procedures. The N-methylcarbamates. lenacil. E. The result of the binding reaction between the Ab and H can be measured by means of enzymatic methods (EIAs). procimidone.

Determination of alachlor. as an example.100 1.referred as absolute quantity.000 0. p – polyclonal antibody. the indirect monitoring of the Ag – Ab interaction should be preferred. Other physical sensors are optics.500 50. No.025 0. 281 .000 0.220 0.4. Bio and immuno sensors are complementary to high-resolution techniques and represent a stimulating border research area between chemical analysis and microelectronics. leading to competitive liposome immuno assay based sensor.070 0. urine Wheat grains.7(ng)* 0. the antigen – antibody selective interaction is monitored.000 0.08 (ng)* 10.000 0.4 .000 30. In an immunosensor. The use of piezoelectric crystals as physical sensors is a continuously developing research direction.2.060 0. interferometric or grating couplers.immobilized enzymes and labeled enzymes are receptor components.600 10 .5 . The analyte blocks the enzyme. crops Food Soil Water Soil. In order to enhance on sensitivity.5. The conjugation of a lipid to a pesticide allows its incorporation into a liposome structure. water Biological fluids Food Soil Cereal grains Water.050 5. flour Surface water Liver Food m – monoclonal antibody.000 Sample type Biological fluids Environmental Environmental Food.03 1. using competitive tracers or markers.000 0. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 Pesticide Aldrin Atrazine Benomyl Bioallethrin Captan Carbofuran Chlorothalonil Chlorpyrifos 2. such as surface plasmon resonance (SPR). * .D Dichlorprop Dieldrin Heptachlor Hexazinone Imazamethabenz Picloram Pyrimiphos methyl Simazine 2.T Thiabendazole Triadimefon Test format RIA CLIA PFIA RIA RIA EIA EIA EIA EIA RIA PFIA PFIA RIA EIA EIA EIA RIA EIA PFIA RIA EIA EIA Ag Type p p p p p p p p p p m p p m p p p p p p m p Detection limit µ g L-1) 0. Table II. Immunochemical assays for some pesticides.000 1. enzyme systems or electron transport systems of intact cells.500 2. can be achieved with increased sensitivity by using liposomeencapsulated markers in a competitive binding reaction instead of enzymes.250 0.000 3.

4. (Bio)sensors for measurement of analytes implicated in food safety: a review. Current use of pressurized liquid extraction and sub critical water extraction in environmental analysis. Microwave-assisted solvent extraction of environmental samples. Brinkman. Mallat and D. Journal of Chromatography A 999: 103-121 (2003).B. D. Analysis of Pesticides in Ground and Surface Water . Brinkman.D.A. L. L. Journal of Chromatography A 970: 65-93 (2002). H. C. Barceló. W. Solid-phase micro extraction for herbicide determination in environmental samples. 11. 3. UK (2000). E. Trends in Analytical Chemistry 22: 232-244 (2003). Senseman and A.Th.S. V. Trace level determination of pesticides in water by means of liquid and gas chromatography. New trends in sample preparation for clinical and pharmaceutical analysis. 6. Keith (ed) Compilation of EPA’s Sampling and Analysis Methods. H.J. E. 10. Hampshire. 7.J. Krutz.S.Th.A.M. Kataoka. Camel. Sciumbato. 2 nd Edition. Berlin: Springer Verlag (1995). British Crop Protection Council Publications. Kriestenson and U.REFERENCES 1. Boca Raton: CRC Press (1996).A. S.A. Immunosensors for pesticide determination in natural waters.D.H. 9. P. Trends in Analytical Chemistry 20: 124-132 (2001).B. 8. 5. 12th Edition. Trends in Analytical Chemistry 212: 96-115 (2002). Analytical methods for biological monitoring of exposure to pesticides: a review. Ramos. 2. Tomlin (ed) The Pesticide Manual. L. Journal of Chromatography A 975: 3-29 (2002). Barr. Journal of Chromatography A 778: 5-29 (2002). R. Niessen and U. 282 .M. Patel. Geerdink. Stan (ed). Trends in Analytical Chemistry 194: 229-248 (2000).

A distinction is commonly made between the generally more carcinogenic ‘dioxin-like’ PCBs (non. An oven temperature in the range of 200°C and detector and injector temperatures around 300°C and 250°C. World production of PCBs is minor at the present. Thus.II. The following steps should be taken for the qualitative determination: 1. long-lived animals at high trophic levels appear to be most at risk from PCBs.and mono-ortho chlorinated) and the more immunotoxic ‘bulky’ PCBs (chlorinated in the ortho positions).1 ng PCBs. sulfur or chlorinated pesticides. Mihaela BADEA Polychlorinated biphenyls (PCBs) are a group of theoretically 209 different compounds of which 150–160 are found in nature. Other halogen-specific detectors such as Hall electrolytic conductivity detector can also be used to analyze PCBs. exhibiting a response to an amount below 0.6. such as phthalate esters. both with regard to chemical-physical properties and with regard to their biological activity.5. All the major peaks of the reference Aroclor standard must be present in the GC chromatogram of the unknown sample extract. These substances have high boiling points. It should be noted that the range of PCBs are very different substances. and flame resistance. Because PCBs produce multiple peaks. PCBs are not found naturally.o. PCBs exist in mixtures composed by several components that produce multiple peaks when a chromatographic method is used. including EU countries and banned in others. have very low water solubility and are predominantly associated with particles in natural waters. and herbicides. sharpness of peaks. Since 1929 at least 1 million tones have been produced. More than half is still in the environment or in products. may be obtained. exhibiting high chemical and thermal stability. on a capillary column. POLYCHLORINATED BIPHENYLS (PCBs) Giuseppe PALLESCHI. a detection limit (l.d.o. PCBs are lipid soluble.d. 283 . Due to their extreme mobility and ability to bioaccumulate and magnify in marine food webs. Electron capture detector (ECD) is the most commonly used detector for trace level analysis of PCBs by gas chromatography (GC). should give good separation. and fast analysis time. With proper sample concentration steps. PCBs are ‘severely restricted’ in some countries.) in the range of 5 µg/L can be achieved. but are now found all over earth due to their persistence and relative volatility. extra care should be taken to identify the genuine peaks from any other contaminants. respectively. detection levels several-fold lower to l. to avoid any false positive inference.

the kinetics of oxidative or microbial degradation of chlorinated biphenyls can be different for each compound in the PCB mixture. measure the area or height ratio of largest to second largest peak in the sample extract and compare the same to that in the standard.2. as it can lead to erroneous rejection. The retention times of the sample peaks must closely match with that of the standard(s). For example. Often. One or more internal standards (such as dibutyl chlorendate. many environmental samples show the presence of only some but not all characteristic PCB peaks. A singlepoint calibration can be used if the peak areas of the PCBs are close to that of the standard. 284 . the presence of Aroclor found in the sample on the primary column must be re-determined and confirmed on an alternate column or by GC/MS using selective ion monitoring mode. 4. Some of the internal standards mentioned above may also be used as surrogates.6. as well as Aroclor standards. The area counts or the heights of all major PCB peaks in the sample are first added up and then compared against the total area or the heights of the same number of peaks at the same retention times in the standard. When the chromatogram of the unknown sample has a number of peaks (some of which show matching with an Aroclor standard). Similarly. because some of the same chlorobiphenyl components are common to most Aroclors. Quantitation Although GC/MS is the most reliable technique for qualitative determination of PCBs. should not always be strictly followed. Oil samples may be subjected to waste dilution. The comparison of peak ratios.5. II. For example. then the ratios of the areas or heights of two or three major peaks of the unknown should be compared with the corresponding peak ratios in the standard at the same retention times. Soils. however. and solid wastes are extracted by sonication or Soxhlet extraction.. its quantitative estimation in environmental samples is more accurately done from the analysis by GC-ECD. the presence of two or more Aroclors in the sample can alter the peakratio pattern.1.e. If only the PCBs are to be analyzed. sediments.6. Sample Extraction and Cleanup Aqueous samples are extracted with methylene chloride by liquid-liquid extraction. Samples should be spiked with one or more surrogate standard solution to determine the accuracy of analysis.2. tetrachloro-m-xylene or p-chlorobiphenyl) should be added into the sample extract.5. The extract is concentrated and then exchanged to hexane. 3. hexane instead of methylene chloride may be used throughout. i. II. to monitor any response time shifts. if a sample is found to contain any specific Aroclor. Finally.

3. If sulfur is known or suspected to be present in the sample. The method does not distinguish accurately one Aroclor from another.diluted with hexane or isooctane and injected onto the GC column for determination by ECD or HECD. Clesceri.New York. The samples can be tested at the calibration levels of 1 to 50 ppm. Ed Leonore S. Inc. and other gaseous products. United Book Press. 1998. Instrumentation.6. water. PCBs in soils and wastewaters can be rapidly screened on site or in the laboratory by immunoassay technique. Standard Methods for the Examination of Water and Wastewater . PCB-enzyme conjugate. Most interfering contaminants can be removed from the solvent extract by gel permeation chromatography or by florisil cleanup. II. PCBs can be measured semiquantitatively by comparing the optical density of the color formed in the sample against a set of calibration standards using a spectrophotometer. 20th Edition. Andrew D. and a solution to quench the reaction. USA 2. sludges.H2SO4 (KMnO4 in 1:1 H2SO4). assay diluent. Gas Chromatographic Environmental Analysis. A normal phase HPLC technique with column switching can separate PCBs from chlorinated pesticides. Eaton. Greenberg. 1993. In addition to these cleanup methods. leaving behind the PCBs in the hexane phase. Inc. and solid wastes. Arnold E. VCH Publishers.. a color-forming substance. The kit primarily contains antibodycoated test tubes or magnetic particles. Alternative Analytical Methods PCBs at high concentrations can be measured by GC-FID. Baltimore. REFERENCES 1. Most organics at trace levels are oxidized under these conditions forming carbon dioxide. and the moisture in hexane is removed by anhydrous Na2SO4. Fabrizio Bruner. Concentrations over 100 ppm can be determined by HPLC by UV detection at 254 nm. and HPLC. an aliquot of the cleaned extract after KMnO 4 treatment may be subjected to sulfur cleanup either by using mercury or copper powder. Sample cleanup often becomes necessary to remove interfering substances such as phthalate esters and many chlorinated compounds frequently found in wastewaters. USA 285 . Techniques.5. NMR. the sample extract should be further cleaned up by shaking 1 mL extract with an equal volume of KMnO 4 . Immunoassay test kits are now commercially available from many suppliers. The hexane phase is then washed with water. Principles.

5.5 the solution is inoculated with a seed. The final DO-concentration in the bottles should not be lower than 1 mg/L and there should be an oxygen consumption of at least 2 mg/L. any oxygen consumption due to the oxidation of ammonia will also be included in the BOD-value. The difference between the initial value and final value corrected for seed consumption is a measure on the samples BOD. There are several criteria that have to be fulfilled before a BOD-analysis can be regarded as valid. First of all should the DO-consumption for unseeded dilution water not exceed 0. When the appropriate time has elapsed the remaining oxygen concentration is measured in the two bottles. The solution is transferred to three incubation bottles. which should not exceed 1. Normal incubation periods are either 5 or 7 days. 286 . The seed usually originates from a municipal biological treatment plant. To prevent this from happening an inhibitor could be added that will make nitrification impossible. The time that the bottles are incubated is somewhat different in different countries. BOD Tomas ALEXANDERSSON BOD is an acronym for biological oxygen demand. One bottle is used for determination of the initial dissolved oxygen (DO) concentration. When using a seed from a municipal treatment plant with nitrification. There is also a criterion for the oxygen consumption for the seeded dilution water. The other two bottles are incubated in a dark space with constant temperature (20°C± 1°C). which is an empirical method for determination of how much oxygen is consumed during biological degradation of compounds present in a water sample.7. This could otherwise indicate a too poor quality on the dilution water.0 mg/L.II. The method is used for determination of the impact a wastewater stream will have on a recipient and to evaluate a wastewater plants BOD-removal efficiency. Nutrients such as nitrogen. phosphate and trace minerals are added to the diluted sample and after adjustment of pH to a value between 6.2 mg/L. Common inhibitors are allylthiourea (ATU) and 2chloro-6-(trichloromethyl) pyridine. BIOLOGICAL OXYGEN DEMAND. Both oxygen consumed by biological degradation of organic matter and oxygen consumed by oxidation of inorganic material such as sulfide and ferrous iron are measured in the method. First the water sample is diluted to a suitable concentration since normally the BOD concentration is larger than the available oxygen concentration in an airsaturated sample. If the final value is below the limit the test has to be repeated with a more diluted sample.5 and 7. The consumption could be too low if the sample is too diluted or if it is toxic to the seed.

The analysis is rather simple with theses test kits. • Boil the sample at the specified temperature and for specified time. In a inter laboratory study with a 200 mg COD/L solution the achieved standard deviation was 13 mg/L. In an inter laboratory study it was found that the average BOD 5 value for this control mixture was 198 mg/L with a standard deviation of 30. For evaluation of the method a solution of potassium hydrogen phthalate could be used. The oxidation is done in a boiling mixture of potassium dichromate (K2Cr2O7) and sulfuric acid. COD Tomas ALEXANDERSSON Chemical oxygen demand (COD) is a method for determination of the amount of organic matter that could be oxidized by a strong chemical oxidant and is expressed in oxygen equivalents.8.5 mg/L. Since halides are interfering with the measurement a complexing agent consisting of mercury sulfate (HgSO 4) is usually added. The procedure is as follows: • Shake the test tube in order to stir up the solid matter in the reagent. 287 . II. • Seal the tube and clean the outside of the tube carefully. • Add the specified volume of the sample to the tube. which usually means 2 hours at 148 °C. • Take out the hot tube and invert it two times.The method and procedure could be tested by including a control consisting of a mixture of 150 mg/L glucose and 150 mg/L glutamic acid. CHEMICAL OXYGEN DEMAND.5. clean the outside of the tube and evaluate in a spectrophotometer. • Let the tube cool to room temperature. Formerly was the reflux method with titration used for COD determination but today there are several manufacturer that offers commercial tubes filled with reagents and the reading is done with a spectrophotometer.