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© 2002 Dionex Corporation
Document No. 034461 January 2002
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Table of Contents
1 • Introduction
What is Chromatography? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . History . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 2
2 • The Process of Ion Chromatography
2.1 Eluent . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1.1 2.2 6 Functions of the Eluent . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6 9
The Chromatographic Separation . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2.1 2.2.2 2.2.3
Resolution . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9 Thermodynamic Factors of Chromatography . . . . . . . . . . . . . 12 Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 26
3 • The Chromatography System
3.1 Eluent . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 27 3.1.1 3.1.2 3.1.3 3.2 3.3 Function of the Eluent . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 27 Preparation of Eluent . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 28 Troubleshooting Eluents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 28
Pump . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29 Injection Valve . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30
Principles of Ion Chromatography 1/2002
. . . . . . . . . . . . . .4. . . .4 Headspace . . . . . . . . . . . . 55 4. . . . . . . . . . . . . 53 Other Detection Techniques . . . . . . . . . . . . . .3 3. . . . . . . 58 Principles of Ion Chromatography 1/2002 iv . . . . . . . . . .4 Columns . . . . . . . . . . . . .5. . . . . .4. . . . . . . . . . . . . . . . . .5. . . . . . . . . . . . . . . . 37 3. . . . . . . . . . . . . . . . . .5 Conductivity Detection . . 53 4 • Method Development 4. . . .3 Ion Exclusion . . . . . . . . . . . . . . . . . .2. . . .1 4. . 33 Channels . . . . 39 3. . .5. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .2. 57 Detection . . . . . . . . . . . . . . . . . . . . . . 56 Reverse Phase Chromatography . . . . . . . . . . . . . . . . . . . . . . . . . . 55 Selecting the Appropriate Separation Mode . 52 Fluorescence Detection . . . . . . . .2 Define Goals of Analysis . . . . . . .1 3. . . . . . . . . . . . . 51 Absorbance Detection . . . . . . .2 3. .5. . . . . . . . . . . . . . . . . . .4. . . . . . . . . . . . 35 Column Cleaning . . . . . . . . . . . . . . . . . . . . . .5. . . . . . . . . . . . . 57 Column Selection . . . . . . . . . . . . . . 34 Contamination . . . . . . . . . . . . . .1 4. . . . . . . . . .4. . . . . . . . . . . . . . . . . . . . . . . . . . . . .2 3. . . . . . . . . . 32 3. . . . . . . . . . . . . . . . . . . . . . . . . . . 39 Amperometry . . . . . . . . . . . .2. . .Contents 3. .3 4. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .2 4. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .4 3. . . . . . . . . . .3 3. . . . . .5 Detection . . . . . . . . . . . . . . .1 3. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
is used to transport the analytes through the stationary phase. • • Principles of Ion Chromatography 1/2002 1 . or stationary phase. Among these characteristics are size. By altering the qualities of the stationary phase and/or the mobile phase it is possible to separate compounds based various physiochemical characteristics.1 What is Chromatography? • Chromatography is the separation of a mixture of compounds into its individual components based on their relative interactions with an inert matrix.1 • Introduction Course Objectives • Outline the basic concepts involved in chromatography and develop them with respect to Ion Chromatography – – Define chomatography Overview the basic chromatographic process • • Discuss the factors affecting chromatographic separation Discuss the components of a chromatography system and their roles in separation and detection. which interact with the analyte(s) of interest. from single elements to large molecular complexes. The matrix . polarity. is generally an inert solid or gel and may be associated with various moieties. Components exhibiting fewer interactions with the stationary phase pass through the column more quickly than those that interact to a greater degree. 1. usually a liquid or gas. Various forms of chromatography can be used to separate a wide variety of compounds. A mobile phase. ionic strength. Interactions between the analytes and stationary phase are non-covalent and can be either ionic or non-ionic in nature depending on the type of chromatography being used. Separation results from the differential migration of the compounds contained in a mobile phase through a column uniformly packed with the stationary matrix. and affinity to other compounds.
or its ability to absorb light in the UV spectrum. discovered that he could separate colored leaf pigments by passing a solution through a column packed with adsorbent particles.Principles and Troubleshooting Techniques of Ion Chromatography Figure 1. Separation resulting from differential migration of compounds Chromatography is the separation of the components of a mixture by differential partitioning between a mobile and stationary phase 1. graphy – writing). technological advances have been limited to a great extent by the ability to detect and measure the analytes of interest. Compounds not inherently possessing any of these characteristics could sometimes be subjected to post-separation reactions that rendered directly 2 Principles of Ion Chromatography 1/2002 . Since the pigments separated into distinctly colored bands. Tswett’s initial experiments involved direct visual detection and did not require a means of quantitation. Tswett named the new method “chromatography” (chroma – color. fluorescence. a Russian botanist. due mainly to the difficulties involved with the detection of ionic species in an ionic mobile phase. Other detection methods were developed that exploited a compound’s radioactivity.2 History The development of chromatography as an analytical tool began in 1903 when Michael Tswett (1872-1919). Several developments were made over the next few decades but it wasn’t until the early 1970’s that ion chromatography began to be seen as a viable process for ion separation and analysis. Throughout the development of chromatography.
at best. Eluent suppression . The early 1970’s saw the introduction of a process that could allow direct conductivity of ions. impractical. allowing a higher degree of sensitivity.1 • Introduction measurable products. The separation of ions. This technology utilized a second ion exchange column after the separator column that reduced the overall conductivity of the mobile phase without adversely affecting that of the analyte. as it came to be known. the significant background signal generated by the mobile phase caused their detection and quantitation to be either impossible or. Although adequate separation of these species was attainable. relies on the use of an ionic mobile phase that bears the same characteristic (the capacity to act as a conductor) as the analytes of interest. These characteristics were easily discernable from the general levels of background noise contributed by the mobile phase. Principles of Ion Chromatography 1/2002 3 . however. allowed low levels of common inorganic ions to be separated and detected using a standard ionic mobile phase.
Principles and Troubleshooting Techniques of Ion Chromatography
Principles of Ion Chromatography 1/2002
2 • The Process of Ion Chromatography
Overview The basic process of ion chromatography involves introducing the sample into a moving stream of mobile phase. This mixture passes into a column that is uniformly packed with particles coupled to an active site with an opposite charge than that of the analyte. Thus, for cation analysis a column is used that has negatively charged active sites. The mobile phase, or eluent, is made up of an aqueous solution of ion salts and serves several functions in the separatory process. Following the column, the mixture proceeds through a suppressor (suppressed ion chromatography) and to the detector (typically conductivity detection for ion chromatography). All ion chromatography systems consist of the same basic components:
• • • • • • •
Eluent Pump Injection Valve Columns Suppressor Detector Data Collection System
Figure 2. Components of an Ion Chromatograph
Principles of Ion Chromatography 12/2001
Principles and Troubleshooting Techniques of Ion Chromatography
2.1.1 Functions of the Eluent • Stabilize sample ions in a solution
Provide kinetic flow of sample ions through a system Provide counter-ions to compete with analytes for active site on a stationary phase
Different analytes in the sample mixture will pass through the column at different rates depending on their relative interactions with either the mobile (eluent) or stationary phases. The rates of analyte migration can be affected by altering eluent composition and/or using different formulations of stationary phase (Figures 3 and 4).
Principles of Ion Chromatography 12/2001
2 • The Process of Ion Chromatography Comparison of Anion Analysis With Varying Eluent Concentrations Figure 3. Effect of Eluent Concentration on an AS14A separation Principles of Ion Chromatography 12/2001 7 .
7 mM NaH CO3 Flow Rate: 2.8 mM Na2CO 3 /1.0 ml/min (a) AS4A-SC (b) AS4A Figure 4. Effect of varying column (stationary phase) on anion separation 8 Principles of Ion Chromatography 12/2001 .Principles and Troubleshooting Techniques of Ion Chromatography Comparison of Anion Analysis with Varying Stationary Phase Eluent: 1.
1 Resolution Resolution is the measure of separation of any two given solutes and can be defined by the equation: R = (2)(flowrate)(T2 .2 • The Process of Ion Chromatography 2.T1 ) (W1 + W 2) where: V = the elution volume of the peak W = the width of the peak at the baseline where: T1 = retention time of peak 1 T2 = retention time of peak 2 W 1 = peak Width at baselie of peak 1 W 2 = peak width at baseline of peak 2 Figure 5.2.2 The Chromatographic Separation This process of separation can result in three possible outcomes: • • • The solutes will be completely resolved (Figure 6a) The solutes will be partially resolved or (Figure 6b) No resolution will take place (Figure 6c) 2. Resolution Principles of Ion Chromatography 12/2001 9 .
indicated by an R value near 1. Complete Resolution 10 Principles of Ion Chromatography 12/2001 . Figure 6a.5 (Figure 6a).Principles and Troubleshooting Techniques of Ion Chromatography Two peaks are considered to be completely resolved when a distinct baseline can be observed between the peaks.
Partial Resolution Total Eluent 12.4%. Poor Resolution Principles of Ion Chromatography 12/2001 11 . Resolution 0.56mM Carbonate Ratio: 90.090 Bulanesulfonate Propanesulfonate Pentanesulfonate Figure 6c.2 • The Process of Ion Chromatography Figure 6b.
which are. Thermodynamic and Kinetic Factors determining resolution 2. This equilibrium can be described as the distribution coefficient K D and is defined by the equation: 12 Principles of Ion Chromatography 12/2001 .Principles and Troubleshooting Techniques of Ion Chromatography The resolution of any two solutes is dependent on their respective retention profiles and peak shapes.2 Thermodynamic Factors of Chromatography 2. affected in a composite manner by the kinetic and thermodynamic factors inherent in the chromatographic system.2. known as capacity (retention characteristics). flow rate. temperature. etc.e. These factors.). In a system without flow.1 Distribution Coefficient (K D) The flow rate of the eluent and the distribution of the solute between the mobile and stationary phases determine a solute’s retention time. in turn. a solute will achieve equilibrium between the two phases.2. Figure 7.2. and efficiency will be unique for every combination of mobile/stationary phase and will vary based on the physical conditions of separation (i. selectivity.
although a solute favors the stationary phase. An analyte’s retention time is determined by the eluent flow rate and by the distribution of solute between the mobile and stationary phases. We see. The KD describes the ratio of sample in either phase at equilibrium under a given set of conditions. Given a particular combination of mobile and stationary phases. A solute with a high K D is more likely to be found associated with the stationary phase at any given moment. Thus. This analyte will be pushed through the column more quickly than one with a higher KD An analyte with a higher K D favors distribution towards the stationary phase.2 • The Process of Ion Chromatography K D = CS/CM where : C S = the concentration of solute in the stationary phase C M = the concentration in the mobile phase. any two analytes will generally have distinct distribution coefficients. that this only true for a small minority of molecules. The distribution is influenced by the ionic attraction to the active sites on the column packing. This analyte elutes at a slower rate. • • Under ideal conditions the K D of a molecule within a system composed of a stationary and mobile phase at a constant temperature will be constant. A a low K D indicates a solute that favors the mobile phase. This difference in K D’s is the basis for the differential migration of various components. It is observed that in most systems a molecule’s K D will vary over a range of solute concentrations. The relationship between the K D of a molecule and its concentration can be described by a function called an isotherm. Principles of Ion Chromatography 12/2001 13 . it is still present to an extent in the mobile phase and can flow through the column. • An analyte with a relatively low K D favors distribution in the mobile phase of the system where it is subject to the influence of eluent flow. in fact.
• Isotherm A represents an ideal state where K D remains constant throughout the concentration range. • Isotherm B is a more accurate representation of most molecules in ion chromatography. 14 Principles of Ion Chromatography 12/2001 . • Isotherm C is a less common situation in which lower concentrations of solute actually favor the stationary phase over the eluent. Here we see that as the concentration of component in the sample increases its K D will decrease.Principles and Troubleshooting Techniques of Ion Chromatography Figure 8. Isotherms Figure 8 depicts the three types of isotherms with KD represented by the slopes of the lines. resulting in an increased distribution of the solute into the mobile phase. The importance of isotherms will be established in later sections when we discuss the kinetic factors influencing peak shape.
The capacity factor is a comparison of the elution time of the solute with the void volume of the column. The capacity factor gives us a measure of the time the analytes spends in the stationary phase versus the mobile phase. K’ = (Te .2 • The Process of Ion Chromatography 2. This may improve separation.2. which is a comparison of the elution time of the solute with the void volume of the column.2 Capacity Factor Another way to describe the retention characteristic of an individual component is by its capacity factor. The equation for K’ is K’ = (Ve-Vm )/Vm where: Ve = the elution volume of the solute V m = the void volume of the column.2. Given a constant flow rate we can substitute the times into this equation to yield k’ = (Te-T0)/T0 where: T0 = the time needed to flush one column volume (this is the duration of time from the injection to the water dip). Principles of Ion Chromatography 12/2001 15 .T0)/T 0 Figure 9. but it will also lengthen analysis time and lead to increased peak broadening. K’. Te = the resolution time of the solute Analytes with higher capacity factors will elute farther from the void volume.
Increasing values of α indicate analytes that would be more thoroughly resolved. 16 Principles of Ion Chromatography 12/2001 .Principles and Troubleshooting Techniques of Ion Chromatography 2. eluting later than the larger ones. Selectivity determines analyte elution order • • The elution order of a mixture of analytes is determined by the selectivity of a stationary phase to each analyte in that mixture under a given set of conditions (mobile phase composition. The selectivity factor. with the smaller hydrated ions maintaining more stable associations with the stationary phase and.3 Selectivity Selectivity is described as the ratio of two analytes’ capacity factors. α. thus. however.2. This theory.). did not explain the tendency for ions to change elution order when structural changes were made to the stationary phase (no alteration to the active site). etc.2. is defined by the equation: α = (T2-T0)/ (T 1-T0) • • If α = 1. Figure 10. Early theorists postulated that the size of the hydrated analyte determined its relative attraction to the stationary phase. is equal to one there is no resolution between the analytes.
) Temperature of mobile phase Structure of stationary phase/active site Chemical composition of active site Principles of Ion Chromatography 12/2001 17 . There is some thought that the higher hydration energy exhibited by small ions enables them to enter the highly structured water matrix of the mobile phase. • Factors Controlling Selectivity: – – – – – Counterion composition/concentration Nonionic modifiers in mobile phase (isopropanol. It stands to reason that an increase in the ionic strength of the mobile phase would cause more of its water to be tied up in the hydration of eluent salts. As the larger ions approach the stationary phase they are more subject to the electrostatic attraction with the active sites. lowering the concentration of the eluent would cause the ions to become less stable in the mobile phase. thus enhancing the retentive effects on the ion’s travel through the system. are not as able to reorient water molecules within the eluent in a manner that permits stability and are displaced toward the stationary phase. with lower energies.2 • The Process of Ion Chromatography Elution order most likely results from a combination of analyte hydration energy and electrostatic attraction Further research suggested that selectivity is influenced by the relative hydration energy of the ions as well as by electrostatic attraction between the analyte and the active site on the column packing. Larger ions. etc. Conversely. resulting in an increased retention time. thus allowing the later eluting components more freedom to enter the mobile phase.
2.2. Separation in an ideal system 1. Figure 11. This phenomenon is known as band broadening and can lead to the loss of resolution between closely eluting peaks. • For solutes with a type A isotherm (KD is constant throughout the concentration range) the concentration distribution varies such that the eluted peak is Gaussian in nature.Principles and Troubleshooting Techniques of Ion Chromatography 2. (Figure 12) 18 Principles of Ion Chromatography 12/2001 . In actuality it is observed that the concentration of an analyte varies throughout its region of occupation in the column.4 Efficiency In an ideal system each component would travel through the column in discrete band with a constant concentration and it would be possible to completely resolve compounds with very little differences in their K D’s (Figure 11).
0. The Craig Distribution model illustrates this process. Efficiency is measured by calculating the number of theoretical plates in the column. (Figure 13) Principles of Ion Chromatography 12/2001 19 . Band broadening can lead to loss of resolution 0. Efficiency • Efficiency is the ability of a column to separate a component without spreading it out. Band Broadening • Under a given set of conditions peak width is found to be directly proportional to both the length of the column and the particle size of the stationary phase. – A theoretical plate is an abstract term describing a complete step of equilibrium exchange of a solute between the mobile and stationary phases. • Peak width will tend to vary directly with changes in the eluent flow rate.2 • The Process of Ion Chromatography Figure 12.
with the amount in each phase determined by the K D. we can expect more broadening to occur. or Gaussian. 20 Principles of Ion Chromatography 12/2001 . Likewise. While we do see a broadening of peaks. Craig Distribution Model of Theoretical Plates Consider a series of squares each representing a site of exchange between two phases. Solute is introduced into the mobile phase of the first compartment and achieves an equilibrium between the two phases. distribution. increasing the plate number is often beneficial in that it can allow better separation between components with closely related distribution coefficients.Principles and Troubleshooting Techniques of Ion Chromatography Figure 13. solute remaining in the first stationary compartment is free to establish an equilibrium with fresh eluent entering its associated mobile compartment and the process is repeated. over many stages the concentration will assume a binomial. Solute remaining in the mobile compartment is transferred to the next stage by eluent flow where it undergoes the same equilibrium process. The most common method of increasing the number of plates is to increase the length of the column. Because the quantity of solute transferred to any successive stage is dependent on the amount remaining in the mobile compartment under equilibrium conditions. • • As the number of theoretical plates increases.
This can be done by optimizing the composition of mobile and/or stationary phases for a particular application.2 • The Process of Ion Chromatography • The term Height Equivalent Theoretical Plate (HETP) is used to describe the efficiency of different columns and is calculated by dividing the column’s length by the number of theoretical plates. Principles of Ion Chromatography 12/2001 21 . Maximizing the number of theoretical plates (better separation) in the shortest length possible will maximize the efficiency of a column.5 mM Bicarbonate 2520 3060 3120 2660 3140 Standard AS4A Eluent 1050 2850 3130 2130 4050 Increasing the number of theoretical plates without increasing the length of the column will allow a more efficient separation to the stationary phase active site.5 mM Carbonate 1480 2220 1900 2930 3960 3. Plates per Column Solute Fluoride Chloride Nitrate Phosphate Sulfate 3. The amount of band broadening is found to be proportional to the square root of the column length. HETP = L/N • Lower values of HETP indicate more efficient separation.
2. The kinetics of mass transfer lead to band broading As noted earlier most applications deal with analytes with a K D value that is concentration dependent. Figure 15 depicts such a peak under normal conditions. Given a situation with no flow.2. 22 Principles of Ion Chromatography 12/2001 . (Figure 14) This is the predominant kinetic cause of band broadening.Principles and Troubleshooting Techniques of Ion Chromatography 2. the portion of solute in the mobile phase will be advanced ahead of the portion remaining in the stationary phase. Figure 14. Consider a compound with a type B isotherm (K D decreases as the concentration increases). When we introduce directional flow of the eluent.5 Flow It is important to stress the effect of eluent flow rate on loss of efficiency. We know that solute advancement through a zone is dependent on its concentration within that zone. This shift from an ideal condition further influences the shape of the eluting peak. causing a longitudinal expansion of the solute zone within the system. an analyte will assume an equilibrium distribution between the mobile and stationary phases determined by its distribution coefficient. and that the concentration of the solute is not constant throughout its region of occupation.
we note that the solute in this portion of the peak will tend to favor the stationary phase and. represented in the figure by the dashed line. where it is pushed ahead due to eluent flow. the K D of the solute in zone 1 is higher than what might be expected. If we focus on discrete bands within various portions of the peak. the distribution coefficient will shift to allow more solute in the mobile phase. Thus. will lag behind solute contained in the eluent. The opposite effect is observed once we have passed the peak maximum. with the solute tending to lag behind the eluent front as its concentration Principles of Ion Chromatography 12/2001 23 . for example.K D’s Solute in the mobile phase will be advancing ahead of that retained by the stationary phase. In zone 1. Since the K D at this point is higher. The peak shape of a compound is affected by its isotherm. we find that the changes in concentration caused by this shift of analyte influences the shape of the peak. thus. Most compounds exhibit a type B isotherm. Different zones of peak exhibit pseudo .2 • The Process of Ion Chromatography Figure 15. As the solute’s concentration increases. the concentration of solute in the mobile phase is actually lower than in a zone immediately downstream.
2. This creates a non-equilibrium distribution of solute that is proportional to the rate of eluent flow and which leads to further broadening of the peak. thereby broadening while maintaining a Gaussian profile. The molecules in the mobile phase contribute to the progress of the solute through the system and that molecules retained in the stationary phase will lag behind the peak’s center of mass. the changes in solute mobility caused by fluctuations of K D result in a skewing of the peak. 24 Principles of Ion Chromatography 12/2001 . (Figure 16) Figure 16. Dispersion of the peak can be minimized by choosing conditions such that the equilibrium conditions are maintained and that the rate of mass transfer between the two stages is maximized. with a more abrupt decline in the tailing shoulder compared with what we would expect from a Guassian distribution.6 Effects of Stationary Phase on Efficiency Particle size and the uniformity of packing also influence a column’s efficiency. however.Principles and Troubleshooting Techniques of Ion Chromatography diminishes. Effects Isotherm on Peak shape 2. For type B compounds.2. zones 1 and 2 would simply “drift” farther apart. A solute with a type C isotherm exhibits the opposite behavior. For a compound with a type A isotherm (where K D is constant). with a sharper leading edge.
Molecules travel through the column at a rate Principles of Ion Chromatography 12/2001 25 .2 • The Process of Ion Chromatography 2. – If particle size is decreased without reducing the volume of the stationary phase.2. at some junctures a molecule may choose a lateral path. there will be more “decisions” against forward progress. resulting in less dispersion due to eluent flow kinetics. resulting in a loss of forward motion.2.7 Particle Size A molecule’s travel through the column can be considered as a series of steps at which it must make a “decision” on which path to follow through the system. – • • • The molecules will tend to “re-bunch” around the center of mass of the peak. Inconsistency in the size of column packing can also lead to loss of efficiency. (Figure 17) Although net movement will be in the direction of eluent flow. Figure 17. Increasing the number of particles generates a greater surface area of interaction between solute and the stationary phase. This event is repeated over many stages. Flow of molecules through column packing • Decreasing the particle size increases the number of “decision” steps.
2. and determine the differential migration of solutes through a given system. the ability of a column to retain a component without spreading it out. • • 26 Principles of Ion Chromatography 12/2001 . or.Principles and Troubleshooting Techniques of Ion Chromatography determined by the eluent flow and the size of the column packing. Solute molecules will progress through the column at different rates depending on the size of the particles they encounter in their zone of travel.3 Summary • The goal of chromatography is the separation. leading to a dispersion of molecules away from the center of mass. These factors describe the phenomena associated with the establishment of an analyte’s equilibrium distribution between the mobile and stationary phases. This is achieved through the unequal partitioning between mobile and stationary phases under the influence of eluent flow. 2. or resolution. The predominant kinetic factors associated with resolution are those which contribute to the efficiency of separation. and the flow rate of the eluent. • The thermodynamic factors that influence peak resolution are the capacity factor and selectivity. uniformity of column packing. Efficiency is determined mainly by the physical components of a system such as the size of the stationary phase particle. of the individual components of a mixture of analytes.
Stabilizing the sample in solution.1 Eluent 3. resulting in a loss of retention of the sample. There are several characteristics of the eluent which affect its interactions with the column and analyte. Counter ions with a high affinity for the stationary phase and to the active sites. — The selectivity of a column for the counter ion in the eluent will affect the equilibrium distribution of sample ions in the system.1 Function of the Eluent The function of the eluent in a chromatography system includes: • • • Establishing the basic ionic condition of the separation environment.1. — Counter ions in the eluent will preferentially elute sample ions of the same valence.3 • The Chromatography System Objectives • • Discuss the functions of each component of the chromatography system Overview basic troubleshooting of each component Introduction Although there are several configurations of ion and liquid chromatographs. they share many components including: • • • • • • Eluent Pump Injection valve Column Suppressor (ion chromatography) Detector 3. Principles of Ion Chromatography 1/2002 27 . Promoting progression of the analytes through the system.
Ionic contaminants in the eluent may also generate analysis problems. preferably from a concentrated stock solution. • • • • Eluents should be made from dry. Since eluent is continually flowing through the system it is constantly generating a background signal. Eluent reservoirs and lines should be kept clean and free of contamination or particulate matter. Because the eluent flow is constant.2 Preparation of Eluent Eluent Preparation • Use reagent grade chemicals and at least 18 M Ω deionized water • Thoroughly degas eluent (for hydroxide eluents degas water prior to adding sodium hydroxide) • Dilute running strength eluents from concentrated stock solutions 3.Principles and Troubleshooting Techniques of Ion Chromatography — Counter ion valence and selectivity are affected by the pH of the eluent.3 Troubleshooting Eluents Problems associated with the eluent may manifest in a shift in analyte retention time due to changes in eluent concentration. Any contamination in the eluent is subject to the same separation process as the sample and will generate a signal response.1. 3. 28 Principles of Ion Chromatography 1/2002 . high purity reagents using the highest quality 18Ω m or higher deionized water. ionic contamination usually results in series of random peaks or an increase in background conductivity.1. Eluents should be made in a consistent manner.
02 0.065 0.3 • The Chromatography System Eluent Troubleshooting • • • • Changes in retention time Loss of peak efficiency Loss of sensitivity Increased background conductivity 3.2 Pump Different types of pumps include isocratic and gradient versions of serial and parallel pumps.32 17249 Figure 18.01 0. especially when running high salt eluents. is recommended to help ensure continuous smooth operation. retention time changes.88 6 7 8 Minutes 9 10 11. A pulse-free pump is essential for optimum chromatography.04 µS 0.0 4. and/or irregular peak shapes. Changes in retention times can also occur when the eluent proportioning valves used in gradient analysis malfunction. Inconsistencies in flow rate and pressure may result in noisy baseline. Routine pump flushing and maintenance. Pump Noise Principles of Ion Chromatography 1/2002 29 .03 0.05 0. 0.
usually consisting of a two-position valve.60 0. eluent flows through an injection device.75 17270 Figure 19.Principles and Troubleshooting Techniques of Ion Chromatography 3. A malfunctioning injection valve may lead to: • • • • • Reduced peak height No response Excessive pressure Poor reproducibility (Figure 19) Sample carryover between runs (Figure 21) 45 30 µS 20 10 -5 0.50 Minutes 0.40 0.13 0.30 0. Poor run-to-run reproducibility 30 Principles of Ion Chromatography 1/2002 .20 0. The valve serves as a means of directing eluent flow and introducing sample into the system.3 Injection Valve Following the pump.
60 Minutes 0.80 1. Excellent Reproducibility (overlay of 6 runs) Figure 21. Sample Carryover Principles of Ion Chromatography 1/2002 31 .20 0.40 0.3 • The Chromatography System 50 40 µS 30 20 10 -5 0.00 1.00 0.25 17269 Figure 20.
great care is taken to ensure that the particles are distributed uniformly throughout the entire column volume. Structural changes in the column packing generally result in changes in the shape of the analyte peaks. 32 Principles of Ion Chromatography 1/2002 . it is possible to generate and optimize stationary phases for a wide range of analytes under diverse conditions.Principles and Troubleshooting Techniques of Ion Chromatography 3. In general. Columns are the site of chemical activity in the separation process. Polymerization of Polystyrene and Divinylbenzene to form Column Substrate By varying the amounts of cross-linker and/or modifier used in the formulation. the column packing is constructed of an inert core composed of polystyrene molecules that have been cross-linked with divinylbenzene to form a bead of uniform size.4 Columns The flow path continues from the injection valve to the column(s). (Figure 22 and 23) Figure 22. Anything that alters the structural or chemical makeup of the stationary phase (column) has the capacity to affect resolution. The beads are then modified with an ionic moiety that provides the appropriate functionality for separation. When packing a column.
(Figure 24) Principles of Ion Chromatography 1/2002 33 . broadening the peak by prolonging the introduction of sample in continually decreasing amounts over a short period of time. Under normal circumstances the volume of mobile phase “before” the column packing is negligible and the sample is transferred into the column as a “slug” of fairly uniform concentration (variation in concentration resulting from laminar flow through the tubing will not significantly affect peak shape). 3. The formation of headspace creates a small void volume of mobile phase that allows the sample to diffuse before it enters the stationary phase.3 • The Chromatography System sulfonation (or amination) of inert bead substrate surface of activated substrate Figure 23. in effect.4.1 Headspace Headspace occurs when a gap is formed between the column bed support and the column packing. • Two predominant changes that can occur within the column packing are the generation of headspace and the formation of channels. This results in a phenomenon known as peak tailing. This causes the concentration of the latter portions of the slug to be less than the leading portions and. Modification of Column Substrate to Generate Active Sites This ensures that the analytes will have consistent chemical and physical interactions with the stationary phase as they migrate through the column.
Principles and Troubleshooting Techniques of Ion Chromatography Figure 24. This results in the advancement of a small amount of sample ahead of the rest of the peak. (Figure 25) 34 Principles of Ion Chromatography 1/2002 . It is then carried by the mobile phase to the end of the void where it re-enters the solid phase. The effects of a channel. which can range from a slight amount of peak fronting to the appearance of ghost peaks before each analyte.2 Channels Channels are tiny void spaces within the column packing. depend on its severity and location in the column. changes in the direction of eluent flow. a small portion of the band will pass out of the solid phase and into the void space. or as a result of the column packing drying out. As an analyte passes through a region containing a channel. A small amount may occur under normal operating conditions due to compaction of the column matrix over a long period of time. Peak Tailing resulting from Headspace • Headspace is generally caused by excessive back pressure or by mishandling the column during routine maintenance. 3. This does not usually affect peak shape as long as the direction of eluent flow is not changed.4. The formation of channels can occur following excessive spikes in pressure.
Columns are frequently contaminated with ionic components that bind so strongly to the stationary phase that they can not be released under normal operating conditions. fewer active sites are available for analyte separation. Peak Disruption due to Column Channeling 3. primarily affects the capacity of the column. This type of contamination. causing blockages and high system back pressure. thus shortening retention times and decreasing peak resolution. Contaminants may consist of strongly retained ions that do not elute under normal operating conditions or non-ionic molecules or particles that lead to column blockage. • Principles of Ion Chromatography 1/2002 35 . • Particulate matter and larger non-ionic contaminants may collect in the bed supports located at the upstream end of the columns.4.3 • The Chromatography System Figure 25. Large. polyvalent ions and metals are frequent culprits and may come from impurities in chemicals used to make up the mobile phase or may arise from the sample itself. As the level of contamination increases. which can result from either organic or inorganic species.3 Contamination • The prevalent cause of loss of column performance is contamination. Bed supports can be changed although care must be taken not to compromise the integrity of the column packing.
with efficiency steadily growing steadily worse. many of which can be reduced or eliminated by various methods of sample pre-treatment • Various forms of contamination may also cause loss of efficiency.Principles and Troubleshooting Techniques of Ion Chromatography • It is possible for certain contaminants to preferentially affect particular ions by inhibiting their passage through the column. Similarly. depending on the nature and amount of the contamination. retain the capacity to bind to the analytes of interest. a sample analyte will. in effect. after associating with the stationary phase. Some contaminants. If the source of contamination continues over time the entire column will become affected. we can expect different effects on the elution of the sample. Indications of Contamination Changes in retention time High back pressure Irregular peak shapes (fronting or tailing) Loss of efficiency Loss of sample recovery Changes in analyte selectivity • • • • • • 36 Principles of Ion Chromatography 1/2002 . In anion chromatography. for example. Sample matrices often contain a wide range of contaminants. This can cause either fronting or tailing of the peaks. thus. and. pass through a stationary phase for which it exhibits different capacity factors. in effect serving as alternate active sites. aluminum contamination may cause lower recoveries of phosphate and sulfate but will leave monovalent anions relatively unaffected. This may result in loss of efficiency or in loss of recovery. iron contamination will tend to decrease phosphate recovery before changes in the other analytes are noticed. These pseudo-sites function with the same thermodynamic and kinetic principles as the actual sites. Since the contaminants do not initially reside throughout the full length of the column.
possibly causing irreversible damage. Columns should be cleaned when analyte retention times shift by 10 percent Column matrices come in a variety of structural and chemical formulations and can respond quite differently to different mixtures of eluents and/or solvents. non-ionic contaminants and monovalent ions will be retained.4 Column Cleaning It is often possible to clean a column that has become contaminated. is a useful solvent for removing hydrophobic moieties from some columns. The guard column also accounts for a certain portion of chromatographic separation and can therefore be used as an indicator of contamination by monitoring changes in analyte retention over time. An extensive list of cleaning solutions for Dionex columns is provided in the Dionex Consumables CD-ROM (p/n 053891). it is necessary to select a solution that will effectively dissociate the contaminant from the column without adversely affecting the physical structure of the stationary phase or the nature of the active site. for example. Therefore. organic ions. A thorough cleaning protocol will generally involve washing the columns with specific solutions for removing contaminants with different properties (i. For this reason it is strongly recommended that a guard column be placed ahead of the analytical column. Principles of Ion Chromatography 1/2002 37 . contaminants are often introduced via the sample matrix. Acetonitrile. thereby increasing the likelihood of headspace or channel formation. leaving the sample analytes to pass through to the separator.4. It may not be necessary to use multiple cleaning solutions if the nature of the contamination is known. 3. when selecting an appropriate cleaning medium. acid or base-soluble. but can cause excessive swelling in the resin beds of others.3 • The Chromatography System • Even if all necessary care is taken to ensure that all reagents used are of high purity. A guard column generally has the same or similar composition as its associated analytical column but is shorter and less expensive.). etc. Other solutions may be capable of chemically modifying the column packing or active site.e. As the sample passes through the guard column.
4. Pump cleaning solution through the column at the appropriate flow rate for 45-60 minutes (some columns may need to be rinsed with deionized water before and after exposure to cleaning solutions) Repeat with additional cleaning solutions if necessary. Select appropriate cleaning solution(s) – refer to Dionex Consumables CD ROM to specify column.Principles and Troubleshooting Techniques of Ion Chromatography 3. Replace columns in their proper order and equilibrate for 30 minutes with standard eluent.1 General Column Cleaning Procedure • • • • • • Determine the nature of contamination (if possible). over time the column packing will begin to deteriorate. Re-plumb the column set by placing the guard column after the analytical column in the flow path WITHOUT changing the flow direction. NOTE -.4. 38 Principles of Ion Chromatography 1/2002 . Under these circumstances the column must be replaced. This is evidenced by the irrecoverable loss of capacity or efficiency or by abnormal operating pressure.Cleaning procedures for some columns may vary. Regardless of the amount of care given to a column. Always consult a column care manual before proceeding with cleanup.
or V=IR Conductance. measured in Siemens. conductivity is found to be directly proportional to the concentration of an ion in solution. The detector must have sufficient sensitivity to detect low levels of analyte. It is preferable that the output signal over this range vary linearly with the concentration of the analyte being measured. is the reciprocal of the resistance. At the concentrations routinely encountered in ion chromatography.5 Detection The three common modes of detection used in chromatography include: • • • • • • Conductivity Amperometry Absorbance When selecting the mode of detection for the application: The detector must have an adequate dynamic range for the solute concentration.1 Conductivity Detection Ionic species. 3. will dissociate into their constituent components when dissolved into a solvent with a high dielectric constant. Principles of Ion Chromatography 1/2002 39 .3 • The Chromatography System 3. These components have the capacity to conduct an electric current when placed between two electrodes with opposite polarity. by nature. Ohm’s law states that the voltage of a circuit is a product of the current and the resistance across two points.5.
A Dionex Micromembrane Suppressor (MMS) or Self Regenerating Suppressor (SRS) may be used in this configuration. minimum drift.5. Typical IC Setup Detector Cell Eluent SRSULTRA Waste Waste Chemical Regenerant Figure 26. This became possible with the advent of suppression technology in the 1970’s. pulse free pumping system will help ensure smooth baselines 3. Flow – A well maintained. 40 Principles of Ion Chromatography 1/2002 . and consistent retention times. Suppression increases the efficiency of analysis not only through reduction of background signal.Principles and Troubleshooting Techniques of Ion Chromatography Factors Affecting Conductivity • • Temperature – Thermal stabilization is important for low noise. thereby enhancing their conductance as much as 3 to 5 fold. but also by converting analyte ions into their acid forms. Advances in suppression technology have led to modern units utilizing ion exchange membranes with neutralizing ions supplied by a chemical solution or through the electrolysis of water. In order to achieve lower background levels.1. it was determined that the conductivity contributed by the mobile phase would have to be eliminated entirely or reduced to an acceptable level.1 Suppression For many years the use of conductivity detection was not considered to be practical for ion chromatography due to the previously mentioned restrictions involved with bulk detection. The flowpath of a typical ion chromatograph using chemical suppression.
3 • The Chromatography System Waste CationExchange Membrane CationExchange Membrane Waste Na+ HSO-4 Na + A Na+ OH Na+ . Principles of Ion Chromatography 1/2002 41 .OH H+ A H 2O HSO4H+ Na + HSO-4 HSO4H+ H + HSO4- Na + A- H+ HSO 4 - Regenerant To Detector Regenerant Figure 27. Anion Eluent Suppression SRS Self Regenerating Suppressor The SRS may be used in 3 modes: • Chemical Suppression • Recycle Mode • External Water Mode The best mode to use depend on the application. Figure 28 shows the SRS used in the recycle mode.
+ H2 Conductivity Cell 16209 Figure 28. The Atlas Electrolytic Suppressor. 42 Principles of Ion Chromatography 1/2002 .Principles and Troubleshooting Techniques of Ion Chromatography Ion-Exchange Membrane 2H 2O 2H+ +1/2 O 2 + 2eAnode Regenerant Suppressed Eluent Regenerant Eluent Perforated Ion-Exchange Material Cathode 2H2O + 2e2OH. Atlas Electrolytic Suppressor Ion Exchange MonoDisc™ Regen Out Regen In Eluent In Eluent Out Electrode Electrode 16180 Figure 29. Anion Eluent Suppression Diagram for Chemical Regeneration Atlas Electrolytic Suppressor The Atlas Electrolyic Suppressor (AES) is designed for use in the recycle mode.
The strong monodisc suppression bed enhances the suppressors ability to withstand backpressure. The Flow of the Eluent through the Monodisk of the Atlas Suppressor Principles of Ion Chromatography 1/2002 43 . Regen Chamber Regen Out Eluent In Eluent Chamber Cation-Exchange Monolith Regen Chamber Eluent Out Regen In _ + Cation-Exchange Membrane Flow Distributor Disk Disc 16771 Figure 30. Resonance time is increased as the eluent is routed around flow distribution disks.3 • The Chromatography System AES Flow Path Figure 30 shows the flowpath of the eluent and regenerant through the anion AES.
0 mL/min Benefits Anions – Ease of use – Moderately Low Noise – Versatile – Use with carbonate and hydroxide eluents – For solvent applications. Choosing the optimum suppressor for your application 44 Principles of Ion Chromatography 1/2002 .0 mL/min – Ease of use with DCR – Low Noise – Fastest Start-up – Use with carbonate eluents – Use with methanesulfonic acid and sulfuric acid eluents – No solvents – Columns: CS12.2 and DX-600 Series “A” detectors or existing systems with SC20 Power supply Anion and Cation: 25 mN at 1. use external water or chemical regeneration Applications Cations – Use with methanesulfonic acid and sulfuric acid eluents SRS-ULTRA 2-mm & 4-mm Formats 15 and 50µ L void volume – For solvent applications use external water or chemical regeneration. use chemical regeneration – Columns: all cation columns Atlas 1 format for 2-.0 mL/min Cation: 110 mN at 1. chloride or nitrate Figure 31.Principles and Troubleshooting Techniques of Ion Chromatography Selecting a Suppressor Suppressor Regeneration Mode Electrolyic or chemical Optional Requirements All existing systems except DX-80 Capacity Anion: 200 mN at 1.0 mL/min – Ease of use with DCR – Lowest Noise – Fastest Start-up Cation: 70 mN at 1. CS12A. 3& 4-mm formats 35 void volume µL Electrolyic Requires PeakNet 6. for – Columns: All anion eluents containing columns chloride or nitrate.0 mL/min – Use with carbonate and hydroxide eluents and for eluents containing solvents – Columns: All anion columns – Columns: all cation columns – Use with methanesulfonic acid and sulfuric acid eluents containing solvents. CS14 MMS III 2-mm & 4-mm formats Chemical All existing systems Required for DX-80 Anion: 150mN at 1.
Running at higher settings will shorten its lifetime.1.5. These procedures are described in the appropriate suppressor manual on the Dionex Consumables CD Rom. Cleaning procedures and recommended solutions are listed in the suppressor manual on the Dionex Consumables CD Rom.94 Anion factor = 2.3 • The Chromatography System 3.47 Concentration = mN Principles of Ion Chromatography 1/2002 45 . • Overcurrenting — Running applications at the appropriate current setting will help increase the suppressor lifetime. — Equation for calculating suppressor current settings: • SRS current settings = flow rate x eluent concentration x factor Cation factor = 2. — Hydrate and quick-start. • Contamination — Samples may have contaminated the suppressor — Clean and quick start.2 Suppressor Troubleshooting Symptoms of a suppressor failures may include: • • • • Alarms Spikes Increased Noise High Background Conductivity Alarms Common causes of suppressor alarms include: • Needs hydrating — The suppressor may have dried out due to a prolonged period without flow.
07 0.1 5 10 15 Minutes 20 25 28 17303 Figure 32.Principles and Troubleshooting Techniques of Ion Chromatography Spikes 0.05 0. Spiking • Spiking can indicate contamination • Spiking can indicate running at too high of a suppressor setting — Calculate appropriate suppressor setting using the equations given in Alarms section • Hydration and quick-start may help eliminate spikes • Cleaning may eliminate spikes 46 Principles of Ion Chromatography 1/2002 .15 ECD_1 0.10 µS 0.00 -0.
550 16. Noisy Baseline Possible cause: • Bubbles in the conductivity cell or a tubing connection Troubleshooting: • “Burping” the conductivity cell — Disconnect the backpressure coil from the suppressor REGEN IN port — Turn pump on and create a pressure difference momentarily by plugging and unplugging the outlet of the tubing (3 seconds) — Repeat 2-3 times — Turn pump off and reconnect backpressure coil to REGEN IN port Principles of Ion Chromatography 1/2002 47 .570 16.590 16.3 • The Chromatography System Noisy Baselines Conditions: GP50 pump.630 16. 4-mm AS14 column.610 µS 16. 1.7 mM NaHCO3. recycle mode.5 mM Na2CO3/1. 3.530 20 30 40 50 Minutes 60 70 80 17305 Figure 33. 32 mA.2 mL/min. 16.
61 µS 23. recycle mode.1240 After burping the DS3 cell 0.0 mL/min.170 0.160 0.63 23.150 40 44 48 52 Minutes 56 60 0. Before burping the DS3 cell 0. 60 mA. recycle mode. An example of noisy chromatography baseline due to trapped bubbles obtained using a Cation Atlas Suppressor 48 Principles of Ion Chromatography 1/2002 .1235 0.1230 40 44 Average noise: 0.12 nS 48 52 Minutes 56 60 17309 Figure 35.155 Average noise: 4.Principles and Troubleshooting Techniques of Ion Chromatography Conditions: GP40 pump.165 µS 0.12 ns after removing the trapped air.60 23. 20 mM MSA.96 ns to 0. 0. Conditions: GS50 pump. Noisy Baseline Effects of Trapped Bubbles on Baseline Noise The baseline noise was reduced from 4. 23.59 Burping the DS3 cell 23. 4-mm AS9HC column. 33 mA. 3-mm CS12A column.58 550 600 650 700 Minutes 17307 750 800 850 900 Figure 34.96 nS 0. cation Atlas.5 mL/min.0 mM Na2CO3 1.62 23. 9.
4-mm AS14 column.784 16. recycle mode.3 • The Chromatography System Effects of Backpressure on Baseline Noise The baseline noise was significantly reduced when excessive backpressure was removed from the suppressor.786 µS 400 psi 16. 16.790 16.780 820 16.610 820 825 830 Minutes 835 840 825 830 835 Minutes 840 17310 Figure 36. Conditions: GP50 pump.614 16. 3.620 16. 32 mA.2 mL/min.7 mM NaHCO3.618 16.782 16.616 µS 100 psi 16.612 16.788 16. 1. It is important to check for plugs in the backpressure tubing and fittings after the suppressor to ensure appropriate pressure is being applied to the suppressor. Effects of backpressure on the chromatographic baseline obtained using an Atlas Suppressor Possible cause: • Backpressure coil for conductivity cell generating incorrect backpressure Troubleshooting: • Determine the pressure drop across the backpressure coil • Replace or adjust length according to recommended values Principles of Ion Chromatography 1/2002 49 .5 mM Na2CO3/1.
Solvent use is limited. keep suppressors clean and hydrated to increase suppressor life. Suppressor Summary • Choose the most appropriate suppressor for your application. • Minimize current settings. Troubleshooting: • Be sure the correct suppressor type is selected on detector front panel • Apply the correct current setting for the application. increased ruggedness. • Confirm eluent preparation is to the correct concentration with chemicals of the required purity. • Atlas gives low baseline noise. • SRS is the most versatile. • MMS provides the fastest start-up. and may be operated in chemical or electrolytic moses. 50 Principles of Ion Chromatography 1/2002 . • Ensure the correct current is applied for the concentration and flow rate of the eluent. and fast equilibration in its more limited applications. and operates in the chemical regeneration mode. is compatible with most solvents. • Confirm eluent concentration is correct for intended application.Principles and Troubleshooting Techniques of Ion Chromatography High Background Conductivity Possible cause: • No current is applied to the suppressor • Wrong current setting is applied to the suppressor • Eluent is not properly prepared for the target application.
Amperometric detection takes advantage of some analytes’ capacity to undergo chemical reactions when subjected to an applied potential. (Figure 37) This technique. leads to better reproducibility and allows the detection of a broader range of analytes than fixed potential amperometry.5.3 • The Chromatography System 3. the use of a fixed potential may result in poor reproducibility and loss of sensitivity due to the plating of the electrodes with contaminants generated from the sample itself.2 Amperometry Not all analytes separated by ion chromatography are amenable to conductivity detection. An amperometric cell is composed of a small-volume channel flowing between a pair of electrodes. By cycling the electrodes through a repeating sequence of potentials over a set period of time it is possible to shift the redox state of the sample. A potential is applied across these electrodes and causes either the oxidation or reduction of the analyte. For some applications. This current is referenced to a separate electrode and the result is compared to a standardized value to generate a viable measurement. Figure 37. called pulsed amperometry. Example of Amperometric Waveform Principles of Ion Chromatography 1/2002 51 . resulting in the “electrochemical” cleaning of the electrode surfaces. thereby rendering it capable of conducting an electrical current.
(Figure 38) Filter/Grating Photodetector Beam Splitter Light Source Reference Flow Cell Figure 38. of the applied radiation. This radiation. 52 Principles of Ion Chromatography 1/2002 . This results in a change in energy.5.3 Absorbance Detection Absorbance detection relies on the ability of molecular bonds within an analyte to absorb electromagnetic radiation at specific wavelengths. which can then be detected with a photometer. or intensity. Schematic of an Absorbance Detector Beer’s Law states that. in a fixed cell path. for most analytes measured by this method.Principles and Troubleshooting Techniques of Ion Chromatography In the course of normal usage these electrodes may become plated with contaminants which will result in the loss of sensitivity. the absorbance of a solution will be proportional to its concentration. thereby significantly increasing sensitivity. 3. it is possible to determine an effective range of linear response. This can usually be remedied by polishing with a special polishing compound or a pencil eraser. This method can be extremely advantageous in that it is often possible to choose a wavelength that is not readily absorbed by the sample matrix or mobile phase. usually in the visible or ultraviolet spectrum. Although deviations from this law do occur. causes the promotion of outer valence electrons in certain bonds within the sample analyte to a higher energy state.
5.3 • The Chromatography System 3. 3. shifting to a lower energy level and releasing a portion of the energy at a different wavelength than that which was absorbed. This is a common method used in the analysis of transition metals and various amines. usually following separation. leading to a stable reaction product that passes into the detector cell. It is possible in some cases to treat such a compound with a reagent that can confer the ability to absorb light at various wavelengths.4 Fluorescence Detection Not all spectrophotometric methods rely solely on the absorption of radiation for sample detection. Principles of Ion Chromatography 1/2002 53 .5.5 Other Detection Techniques Some molecules do not inherently exhibit an absorptive capacity. the electrons of some molecular bonds will relax. Following excitation to a higher energy state. The intensity of this fluorescence is proportional to the concentration of absorbing species in the sample. The analyte is mixed with a chemical reagent.
Principles and Troubleshooting Techniques of Ion Chromatography 54 Principles of Ion Chromatography 1/2002 .
4. Occasionally. it is essential to consider the different aspects involved in identifying and/or quantifying analytes in a sample mixture. In choosing a column it is first necessary to determine what style of separation must be used for a given analyte or sample matrix.4 • Method Development Objective • To provide basic guidelines to aid in method development Much work has been done in liquid chromatography over the last several decades. such as organic acids or hydrophobic molecules.1 Define Goals of Analysis The first step in the development of a method is to define realistic goals for the analysis. 4. For instance. may not be suitable for separation using ion exchange chromatography. in most cases. however. either yield complete protocols or at least enough information to provide a significant head start. It is helpful to have as much information as possible about the analyte(s) in question. is the chemical structure known? Are the molecules inorganic or organic? In what types of solutions and at what pH’s are the molecules soluble and in their ionic form? In many cases this information will be readily available to the analyst and all that is necessary is to determine which type of column and mobile phase to use. Some research applications may require the analysis of compounds for which little information has been gathered. it will be necessary for the analyst to spend some time and effort in the development of a suitable method for the analysis in question. it is necessary to select an appropriate column and mobile phase. Although this may seem basic. Some sample types. In these circumstances it may be necessary to perform separate qualitative tests in order to determine how to proceed with chromatographic separation. a thorough search of chromatography literature will. This section will provide some basic steps that are useful in this process. Two other commonly used separation methodologies are ion exclusion and reversed phase chromatography. When trying to develop a method.2 Selecting the Appropriate Separation Mode Once enough information has been gleaned regarding the sample. Principles of Ion Chromatography 1/2002 55 .
(Figure 39) Strong acids in the sample. Weak acids become protonated and. a perimeter of water molecules will be established a short distance from the surface of the stationary phase. Active sites on the column packing are readily available to all sample ions. in their neutral state.1 Ion Exclusion Ion exclusion chromatography is useful in the separation of weak organic acids from strongly dissociated ions. known as the Donnan membrane. This perimeter. are allowed access to the active sites on the stationary phase. are prevented from passing through the Donnan membrane and are eluted in the void volume. An ion exclusion column is highly sulfonated throughout the resin structure. Separation in ion exclusion 56 Principles of Ion Chromatography 1/2002 . Separation in ion exclusion is achieved by a combination of Donnan exclusion. separation is achieved through differential interactions between the sample and stationary phase.Principles and Troubleshooting Techniques of Ion Chomatography 4. will be slightly polarized with a partial negative charge oriented away from the exchange resin. which remain negatively charged. In ion exchange. By using a dilute solution of a strong acid as the mobile phase.2. and classic exchange partitioning. steric exclusion. Donnan Membrane R C00H H 20 C 1– C 1– Figure 39.
Some analytes. for example. or by increasing the percentage of nonpolar solvents in the mobile phase. For some applications it may be necessary to incorporate elements of normal and reverse phase chromatography. a hydrophobic ion of an opposite charge to the analyte of interest is added to the mobile phase and forms a complex with the analyte. If there is no information pertaining to a specific analyte. The best source of information as to whether or not a particular column will be useful for a given application is the literature provided by the manufacturer. This facilitates the separation of samples containing mixtures of neutral and ionic hydrophobic analytes. It is possible to alter the capacity of this system. may be easily separated on several different columns. Organic ions may also be analyzed by a technique known as ion pairing. Columns capable of operating in mixed mode are able to accept ionic mobile phases containing higher levels of solvents than normal exchange columns.2. reverse phase chromatography separates compounds based on their relative hydrophobicity. Issues to consider when choosing a mobile phase include sample solubility.2. by changing the type or concentration of pairing agent. it can be helpful to choose a column that is compatible with a similar compound for which a method has been developed. and thus optimize separation.3 Column Selection Once the type of chromatography to use has been determined you must choose a specific column set.4 • Method Development 4. If a sample mixture has a strong contingent of divalent anions. Most columns are formulated for particular applications with a specific mobile phase. the valences of different compounds in the sample. In ion paring. and detection requirements. it would be beneficial to use an eluent that also contained Principles of Ion Chromatography 1/2002 57 . This complex is then able to associate more readily with the non-polar stationary phase. It is then necessary to consider what type of mobile phase is to be used. Many column manuals provide example applications with various types of samples commonly run on a given column. Column packings are generally composed of a porous. non-polar core that is capable of hydrophobic interactions with organic compounds. We know that. common anions or cations for example. eluent salts will preferentially elute solutes of like charge. in general.2 Reverse Phase Chromatography Whereas ion exchange chromatography exploits the polar characteristics of various compounds to bring about separation. 4.
Additionally. This raises a dilemma in that significant interferences will arise from the high salt concentration in the mobile phase (most liquid chromatography/mass spectrometry methods (LC/MS) utilize reverse phase columns with solvent/water mobile phases). the separation of carbohydrates can be achieved on an anion exchange column by using a sodium hydroxide/sodium acetate mobile phase with integrated amperometry as a detection method. In suppressed conductivity it is desirable to reduce the conductivity contributed by the mobile phase as much as possible in order to maximize the sample detection limits. For example. the detection method is not capable of allowing the identification of a compound. In many situations the analyst will be able to select an appropriate system by investigating what others have used to analyze the same or a similar molecule. Thus. More qualitative information could be obtained by using mass spectrometry for detection. it is necessary to use a certain mobile phase composition that is not compatible with various forms of detection. in most cases the analyst is faced with having to sacrifice functionality in either separation or detection. however. background conductivity can be reduced to negligible levels (consider the suppression products of hydroxide and carbonate/bicarbonate).3 Detection Additional care must be taken to combine a detection scheme that is compatible with the appropriate separation parameters. although both are monovalent salts.Principles and Troubleshooting Techniques of Ion Chomatography bivalent ions. By choosing sodium hydroxide as for the mobile phase. Even in these cases there may be significant difficulties in obtaining useful results. In some situations. Sodium hydroxide is commonly used in place of a sodium carbonate/sodium bicarbonate eluent in low level analysis of anions. While this might be a suitable process for separation and quantitation. While a given system may be capable of separating a variety of closely eluting organic acids. the conditions used in the analysis of such acids in wine 58 Principles of Ion Chromatography 1/2002 . such as sodium carbonate. While there are some mass spectrometers that can remedy this interference. The background signal from suppressed carbonate/bicarbonate systems will usually be 15 to 20-fold higher than that of sodium hydroxide. sodium hydroxide and sodium bicarbonate may not be equally suitable for the separation of various anions on a given column. changes in eluent salts can alter the capacity factors for a column. 4. Few difficulties are encountered for analysts performing common ion analysis in that interferences arising from the mobile phases used with standard columns can be eliminated before the detection process. Detection requirements also factor in eluent suitability.
). gradient profiles. will help the chromatographer to determine the appropriate conditions for their particular separation. Indeed. Careful consideration of the theory behind this separation. as well as an understanding of the strengths and limitation of a given system. with the myriad analytes and sample matrices available for investigation come a wide range of possible interferences that must be dealt with in order to acquire suitable data. temperature. the only way to optimize a particular separation is through a process of trial and error with minor variations in separation conditions (i. Principles of Ion Chromatography 1/2002 59 .e. modifiers.4 • Method Development may be quite distinct from those used in the analysis of beer. Often. etc.
Principles and Troubleshooting Techniques of Ion Chomatography 60 Principles of Ion Chromatography 1/2002 .