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.

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hydrogen sulphide etc. pharmaceutical research and health services (quality assurance. In order to identify and minimi. cross%contamination. he pharmacopeial standards have set different parameters to perform tests based on chemical analysis. solvents and catalysts  &hemical and physical instability  'torage condition  )hysical changes  0eaction with container materials  emperature effect  $anufacturing ha. different aspects can be managed. the quality control approach is mainly base on analytical chemistry. #ualitati$e and quantitati$e methods C.g. sulphur dioxide. health care. processes and conditions have to be maintained. the quality of materials. discovery of new drugs and in clinical applications) In pharmacy.e the source of impurities. contamination due to certain gases e. hysical! chemical and physiochemical methods ".ards (particulate contamination.fficacy % +uality In all of these aspects.very manufacturing process is controlled by the analytical approach. A. . It plays an increasingly important role in the pharmaceutical industry. 'ome of these are/  0aw materials  $anufacturing process  'tarting materials and intermediate process  0eagents. process errors. Classical and instrumental methods %A& hysical! chemical and physiochemical methods' 1 . clinical and environmental applications. controlled and ensured by adopting and using the analytical techniques. forensic. Classification of analytical methods Analytical methods can be classified by different ways and approaches. microbial contamination. analytical techniques play ma-or role in decision ma!ing. Importance of analytical chemistry in quality assurance of pharmaceutical products he main aim of quality assurance is to establish or maintain % 'afety % . In good pharmaceutical practice.Analytical chemistry has wide scope in industrial quality assurance (+A). bioanalysis. pac!ing errors  )roduct specification 3.

gasometric assays. finished products and quality evaluation of a drug product in the mar!et. colorimetric method. the analyst#analytical chemist play a !ey role in determining composition. solvents. it is not related to quantity. essential oils. gravimetric methods. titrations including volumetric methods. hydrogen ion concentration. active ingredients. thic!eners etc. in the pharmaceutical industry. In quality assessment. essential oils and additives li!e starch. he following are the important areas for the application of both methods. he former type of the methods of analysis is a qualitative and the latter is quantitative analysis. chromatographic methods.) &hemical methods (acid%base reactions. 0efractive Index. 6ptical rotation etc. weight per m4. but not the mass or concentration. preservatives. thermal analysis. he nature of raw material varies widely/ crude drugs. 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. purity and quality of different raw materials and drug products. lactose. species and#or compounds present in the sample. est for solubility ii. 2oiling point.) )hysical and chemical methods ('pectroscopic methods. 3ensity#specific gravity 7 . )articularly. nephelometric methods etc. 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. +ualitative analysis is the identification of elements. Free. water. radio chemical methods. he following are the examples of some qualitative methods frequently employed in analysis of drugs and pharmaceuticals. hey are also employed in structural analysis of a compound (spatial arrangements of atoms in a molecule or the identification of functional groups). gums. both qualitative and quantitative methods have to be employed to ensure the stated specification and standard of both raw material and the final product.ing point. emulsifying agents. 'pecific gravity. i. 3ensity. hat is.   )hysical methods ($elting point. 5iscosity. disintegrating agents. Analytical chemistry is concerned with determining either the identity of the drug product (an ingredient) or the amount of the product (ingredient).

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

he main techniques employed in quantitative analysis are based on the following/ • &hemical reactions and formation of products e.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. to D:.e of the sample. +uantitative methods can be further classified into/ % macro % 'emimiocro % Eltramicro : .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.• • • • &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..

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

instrumental methods have become the bac!bone of pharmaceutical analysis#analytical and experimental chemistry. osmosis. emitted. 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. Instrumental methods find application in both qualitative and quantitative analysis. D . filtration. column chromatography etc. precipitation. he growth of instrumental methods of analysis is related to the developments in the field of electronics. Instrumental methods of analysis are divided into three broad categories based on the property of the analyte. • 'pectroscopic methods • .ssentially all analytical instruments are electrically operated. graphs etc.ethods he first instrumental analysis was flame emissive spectrometry developed by 0obert 2unsen and >ustav Mirchhoff. Instrumental methods use devices that measure a physical or chemical property of the analyte (assayed substance)). .c& Separati$e methods 'ome of the non%instrumental separative methods used in chemical analysis are/ distillation.lectro analytical methods • 'eparative methods a& Spectroscopic methods hese methods use an instrument to measure the amount of radiation that is absorbed. most quantitative analyses are carried by instrumental methods. ii. paper chromatography. Instrumental . hese days. or scattered by the sample (analyte). hese days. charts. reverse osmosis.

'uch measurement can be made at any point in the electromagnetic spectrum (1. he presence of two or more metal radiations under the same condition would result errors. Following are the examples of different categories of electro analytical methods.%D:.lectron spectroscopy • 0adiochemical methods (e. 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. herefore. he thermal energy of the flame excites the metal atoms.lectrogravity • )otentiometry • 5oltametry iii. Separati$e methods In separative methods. filters are used between the flame and detectors to measure the selected metal.g.lectrophoresis methods C . he intensity of the emitted light is used to calculate the concentration of the analyte.nm). different components (constituents) of a mixture are separated by using the physical or chemical properties of the analyte. he instrument used formeasurement of the emitted radiation is called flame photometer. sodium produce golden yellow flame).g. which then return to the ground state by emitting radiation. • Eltraviolet% visible spectrophotometry (If the absorbed radiation is measured.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. his method can also be used for the characterisation of organic compounds and for the determination of related compounds as impurities. 0adio%immunoassay assay of digoxin) • hermal analysis b& -lectroanalytical methods In these methods. • Amperometry • &onductometry • &oulometry • .. the method is absorption spectrophotometry. In this method absorbance is measured which is directly proportional to the concentration of the analyte in the solution. • 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 • . hese methods can be further classified into the following methods a) &hromatographic methods b) .

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