A.

An Introduction to analytical methods

1. What is analytical chemistry? Analytical chemistry is a branch of chemistry which involves the study of matter in order to reveal its composition, structure, and extent. It is used to obtain information and solve problems in many different areas related to chemicals and their use. Analytical chemistry deals with the separation, identification, and quantification of chemical components of natural and artificial materials (drugs, chemicals, pharmaceuticals and biological materials). For the final analysis of components, sample of the analyte has to be prepared. his may require the use of some separation techniques. In the past analytical techniques were relatively simple but not very sensitive. he classical methods used separations such as precipitation, extraction, and distillation etc. he modern separation processes include various !inds of chromatography (paper, gas, and liquid" electrophoresis" crystallography" microfiltration etc he analysis of a component involves both qualitative (identification i.e. focused on determining what elements and#or compounds are present) and quantitative (assessment that aims to establish the precise quantity of an element or compound in the given sample). he components of interest are measured by spectrometers (atomic absorption spectrometers, infrared spectrometers, and mass spectrometers etc.). $ethods used in analytical chemistry are being constantly developed and revised to ma!e the analysis accurate, valid, reliable, reproducible and precise even for measuring a very low concentration of the component. he classical methods of analysis were based on analysis by color, odor, melting point, boiling point etc. $odern quantitative analysis use sophisticated instruments to measure the components. 2. Scope hese days, Analytical chemistry has very wide application in different types of industries. % &hemical (obtain information, insure safety, and solve problems in many different chemical areas) % Agricultural % Forestry % &eramics % Food and beverage % &osmetics % 'ynthetic materials such as polymers % (atural materials, such as minerals and water % )harmaceutical etc.

Analytical chemistry has wide scope in industrial quality assurance (+A), health care, forensic, bioanalysis, clinical and environmental applications. It plays an increasingly important role in the pharmaceutical industry, pharmaceutical research and health services (quality assurance, discovery of new drugs and in clinical applications) In pharmacy, the quality control approach is mainly base on analytical chemistry. ,very manufacturing process is controlled by the analytical approach. he pharmacopeial standards have set different parameters to perform tests based on chemical analysis. Importance of analytical chemistry in quality assurance of pharmaceutical products he main aim of quality assurance is to establish or maintain % 'afety % ,fficacy % +uality In all of these aspects, analytical techniques play ma-or role in decision ma!ing. In good pharmaceutical practice, different aspects can be managed, controlled and ensured by adopting and using the analytical techniques. In order to identify and minimi.e the source of impurities, the quality of materials, processes and conditions have to be maintained. 'ome of these are/ 0aw materials $anufacturing process 'tarting materials and intermediate process 0eagents, solvents and catalysts &hemical and physical instability 'torage condition )hysical changes 0eaction with container materials emperature effect $anufacturing ha.ards (particulate contamination, contamination due to certain gases e.g. sulphur dioxide, hydrogen sulphide etc, cross%contamination, microbial contamination, process errors, pac!ing errors )roduct specification

3. Classification of analytical methods Analytical methods can be classified by different ways and approaches. A. hysical! chemical and physiochemical methods ". #ualitati$e and quantitati$e methods C. Classical and instrumental methods %A& hysical! chemical and physiochemical methods'

)hysical methods ($elting point, Free.ing point, 2oiling point, 3ensity, weight per m4, 'pecific gravity, 5iscosity, 0efractive Index, 6ptical rotation etc.) &hemical methods (acid%base reactions, titrations including volumetric methods, colorimetric method, gravimetric methods, hydrogen ion concentration, gasometric assays, nephelometric methods etc.) )hysical and chemical methods ('pectroscopic methods, thermal analysis, chromatographic methods, radio chemical methods, electro%analytical methods etc) #ualitati$e and quantitati$e methods

%"&

he main scope of analytical chemistry is to deal with the quality control activities of raw materials, finished products and quality evaluation of a drug product in the mar!et. he nature of raw material varies widely/ crude drugs, active ingredients, solvents, water, essential oils and additives li!e starch, lactose, gums, disintegrating agents, emulsifying agents, preservatives, essential oils, thic!eners etc. )articularly, in the pharmaceutical industry, the analyst#analytical chemist play a !ey role in determining composition, purity and quality of different raw materials and drug products. Analytical chemistry is concerned with determining either the identity of the drug product (an ingredient) or the amount of the product (ingredient). he former type of the methods of analysis is a qualitative and the latter is quantitative analysis. In quality assessment, both qualitative and quantitative methods have to be employed to ensure the stated specification and standard of both raw material and the final product. he following are the important areas for the application of both methods. a) +uality control b) $onitoring and control of pollutants c) &linical and biological studies d) 0esearch #ualitati$e and quantitati$e methods with e(amples/ #ualitati$e methods/ A qualitative analysis determines the presence or absence of a particular compound, but not the mass or concentration. hat is, it is not related to quantity. +ualitative analysis is the identification of elements, species and#or compounds present in the sample. hey are also employed in structural analysis of a compound (spatial arrangements of atoms in a molecule or the identification of functional groups). he following are the examples of some qualitative methods frequently employed in analysis of drugs and pharmaceuticals. i. est for solubility ii. 3ensity#specific gravity

iii. 2oiling point#free.ing point iv. $elting point v. Identification tests of the substance based on colour tests e.). identification of ascorbic acid/ About 18 solution in water 9 * drop of freshly prepared :8 solution of 'odium nitroprusside 9 1 ml of *;8 (a6<. he solution becomes yellow. o this solution, add ;. : ml of <&l (conc.) drop wise and stir, the yellow solution turns blue. ests for impurities e.g. test for the presence of =%aminophenol in paracetamol, and presence of oxalic acid in ascorbic acid. vii. 4imit tests for the presence of toxic heavy metals (lead, mercury, arsenic etc.) In many cases instrumental method of analysis are also employed for qualitative purposes. Infrared spectrophotometer and 0aman spectrometry are employed to identify a specific drug molecule. In these methods, the recorded spectrum of the sample is compared with the spectra of a !nown reference substance. hese methods also identify the presence of different functional group. )otentiometeric method can also be used for identification of a metal in solution. In <)4& and >4& methods, the retention time (t 0) can be used for identification of different compounds e.g. identification of methyl alcohol, ethyl alcohol and isopropyl alcohol, amyl alcohol and other related substances. hin layer chromatographic>enerally people compare compounds based on their 43:;?s and ma!e decision about the safety of the compounds based on this number. method also identifies a particular compound )olarometric methods provide the information on optical isomer of a compound, e.g. laevo (%l), dextro (9d) or @ (dl) form. he use of uv%visible spectrophotometric method provides the estimation of the wave length for maximum absorption (Amax) which is specific quality to identify a compound. e.g. Amax of some drugs/ % e.g. paracetamol at 1=Bnm % cortisone actate at 1=;nm % adrenaline in (#*;; <&l at 1C; but adrenalone (an important impurity) exhibits it at 7*;nm. #uantitati$e methods/ +uantitative analysis is the determination of the absolute or relative amounts of elements, or compounds present in a sample. +uantitative data are required in wide range of situations including raw materials, final product and mar!eted products. he main aim of the analysis is the determination of the quantity of a component (analyte) in the sample. In quantitative analysis the following are the most important steps. 'ampling/ 'election of sample and preparation of sample )reparation of standards containing !nown amounts of the analyte vi.

&alibration of instruments to determine the responses $easurement of the instrumental#apparatus response for each sample under the same condition as for the standards All measurements may be replicated to improve the reliability of the data &alculation and statistical evaluation.

he main techniques employed in quantitative analysis are based on the following/ &hemical reactions and formation of products e.g. 5olumetric and gravimetric methods )otentiometry (measurement of potential of an electrode) 5oltametry (measurement of current at specific voltage) &oulometry (measurement of current and time to complete reaction) 'pectrophotometric method (measurement of the absorption of monochromatic radiation between 1;; to D:;nm) Atomic absorption spectroscopy (determination of trace elements/ it involves the spraying a solution of metal in flame and measurement of the absorption of radiation) <igh )erformance or <igh pressure 4iquid &hromatography (It involves the separation and quantification of an analyte from other compounds) >ass liquid &hromatography (It involves the separation and quantification of an analyte from other compounds 2ased upon the si.e of the sample, +uantitative methods can be further classified into/ % macro % 'emimiocro % Eltramicro

%C& Classical and instrumental methods &hemical analyses (both qualitative and quantitative) can be divided into classical (non% instrumental) and instrumental methods. i. Classical methods' Although modern analytical chemistry is dominated by sophisticated instrumentation, the roots of analytical chemistry and some of the principles used in modern instruments are from traditional techniques. In classical analytical methods the measurement of the analyte can be carried out by chemical and physical means. hese means can be used for both qualitative and quantitative analysis. hese methods only require the use of/ &hemicals 2alance &alibrated glassware (pipettes, burettes, volumetric flas!s, funnel, measuring cylinder, burner, hot plate, water bath etc.) he following are the examples of classical methods/ a) *olumetric methods (titrametric methods)/ hese methods involve measurement of the volume of a solution of !nown concentration which is required to react with the analyte. In this method the solution of one of the chemical reactants !ept in the burette (mostly titrant) is added to a solution of a second chemical reactant (analyte) !ept in a conical flas!. he volume of the titrant added at the end point of the titration is measured and used to calculate the amount of the sample. he endpoint of the reaction is located by using a suitable indicator. he Fbalanced equationG of the reaction may be represented as/ aA 9 t H )roducts, where FaG is the number of molecules of analyte A, and FtG is the number of molecules of titrant ( ). In such situation, for the calculation, we have to use the following relation. t Imol of A J a Imol of KKK(i) or, t I mmol of A J a I ml of titrant consumed I molarity of titrant. 'ome examples of volumetric methods/ 3etermination of calcium in calcium gluconate in-. (complexometric titration), determination of ephedrine hydrochloride by non%aqueous titration etc. b& +ra$imetric methods In gravimetric method, the measurement is one of weight. his method is also dependent on chemical reaction. he classical method usually consists of precipitation of a salt of the analyte (assayed substance). he precipitate is collected, filtered, dried and weighed. he use of sintered glass crucibles are widely used for the filtration, collection, drying and weighing.

c& Separati$e methods 'ome of the non%instrumental separative methods used in chemical analysis are/ distillation, precipitation, filtration, osmosis, reverse osmosis, paper chromatography, column chromatography etc. ii. Instrumental ,ethods he first instrumental analysis was flame emissive spectrometry developed by 0obert 2unsen and >ustav Mirchhoff. hese days, most quantitative analyses are carried by instrumental methods. Instrumental methods use devices that measure a physical or chemical property of the analyte (assayed substance)). ,ssentially all analytical instruments are electrically operated.

he main -ob of an instrument is/ >eneration of signal from the chemical#physical property of the analyte ransduction(conversion) of original signal to measurable response#signal Amplification of the transformed signal )resentation of signal into meaningful numbers, charts, graphs etc. he growth of instrumental methods of analysis is related to the developments in the field of electronics. hese days, instrumental methods have become the bac!bone of pharmaceutical analysis#analytical and experimental chemistry. Instrumental methods find application in both qualitative and quantitative analysis. Instrumental methods of analysis are divided into three broad categories based on the property of the analyte. 'pectroscopic methods ,lectro analytical methods 'eparative methods a& Spectroscopic methods hese methods use an instrument to measure the amount of radiation that is absorbed, emitted, or scattered by the sample (analyte). D

he following are the examples of main spectroscopic methods" Atomic absorption method Flame photometric method/ It has been observed that light (radiation) is emitted when certain metallic salts are vaporised in flame (e.g. sodium produce golden yellow flame). he thermal energy of the flame excites the metal atoms, which then return to the ground state by emitting radiation. he intensity of the emitted light is used to calculate the concentration of the analyte. he instrument used formeasurement of the emitted radiation is called flame photometer. he presence of two or more metal radiations under the same condition would result errors. herefore, filters are used between the flame and detectors to measure the selected metal. Eltraviolet% visible spectrophotometry (If the absorbed radiation is measured, the method is absorption spectrophotometry. In this method absorbance is measured which is directly proportional to the concentration of the analyte in the solution. 'uch measurement can be made at any point in the electromagnetic spectrum (1;;%D:;nm). his method can also be used for the characterisation of organic compounds and for the determination of related compounds as impurities. Infrared absorption spectrometry 'pectroflourocence method (If the absorbed radiation is emitted in the form of fluorescence and if such fluorescence is measured it is called spectrofluorometry) 0efractrometric method )olarometric method (uclear magnetic resonance N%ray methods ,lectron spectroscopy 0adiochemical methods (e.g. 0adio%immunoassay assay of digoxin) hermal analysis b& -lectroanalytical methods In these methods, either an electrical signal is applied to one of the electrodes dipped into the solution of the analyte or an electrical property of the solution is measured. Following are the examples of different categories of electro analytical methods. Amperometry &onductometry &oulometry ,lectrogravity )otentiometry 5oltametry iii. Separati$e methods

In separative methods, different components (constituents) of a mixture are separated by using the physical or chemical properties of the analyte. hese methods can be further classified into the following methods a) &hromatographic methods b) ,lectrophoresis methods

c)

$ass spectroscopy.

Chromato)raphic methods hese days, chromatographic methods have widest scope of application in pharmaceutical analysis. &hromatography is the process by which the components of a mixture can be separated, identified and quantified from a liquid or gas phase. he principle is based on the concentration equilibrium of a component of the analyte between two phases, one stationary (pac!ed in a column), the other being mobile (a transporting medium moving through the stationary phase). he mobile phase may be gas or liquid. he differential migration of compounds through the column results separation. hus, inside the column, partition, solubility, adsorption etc. processes affect the components, and therefore, different compounds will have difference in migration based on the nature of the molecule. Ohen a component comes out the column, its concentration is detected by a suitable detector. >as liquid chromatography and <igh )ressure liquid &hromatography have been used for the direct quantitative measurement of different components of a mixture. >as 4iquid chromatography (substance is put on the head of the column, pac!ed with suitable inert solid adsorbent supporting a thin film of non%volatile material usually a liquid. he compound gets volatised by heating and an inert gas used as mobile phase carries the compound with it. hus, the components of a mixture are volatised, eluted individually and are detected. <igh )erformance &hromatography uses a liquid mobile phase (eluent) pumped at high speed through a narrow column containing a support material of uniform si.e and shape. he sample is placed on the head of the column and it becomes distributed as a narrow band in the mobile phase. 3uring progress through the column, separation of different components occurs and each compound is eluted from the column. he individual compounds are monitored by the detector and are recorded as pea!s in a chart recorder. ,ass spectrometry he material under examination is vaporised using a high vacuum and the vapour is bombarded with electrons of high%energy ((P*; e5). First the compound may loose an electron and so yield a positive ion. $ 9 e H$991e $9 is often unstable, having an energy excess, and in the mass spectrometer it fragments in a specific manner. 'cience a compound contains different groups#atoms -oined through different bonds, excess energy cleaves the molecule into different fragments producing ions of varying si.e. hese ions can be sub-ected in a magnetic where they follow a path dictated by their mass#charge ratio. he mass#charge ratio for each fraction detected and recorded.