BIOPHYSICALCHEMISTRYPRINCIPLES AND TECHNIQUESBIOPHYSICALCHE vIISTRYPRINCIPLES AND TECHNIQUESAVINASH UPADHYAY, M.Sc., Ph.D.Department of Biochemistry,Hislop College,Nagpur (M.S.)KAKOLI UPADHYAY, M.Sc., Ph.D.Department of Biochemistry,Lady Amritabal Dga College, Shankarnagar,Nagpur (M.S.)"NIRMALENDU NATH, M.Sc., Ph.D.Retired Professor,Department of Blchemistr,Nagpur University, LIT Premises,Nagpur (M.S.)%Iimalaya PublishingHouseMUMBAI DELHI 0 NAGPUR BANGALORE HYDERABADNag u rBangalore AUTHORSNo part ofthis book shall be reproduced, reprinted or translated for any purpose whatsoever withouprior permission of the publisher in writing.First Edition: 1993Second Revised Edit/on: 1997Reprint:lkprkt:2000Rprlnt:00.eprint:-Third Revised Edition:2002 (May)Repr/nt:2003Published by:Mrs. Meena Pandcyfor HIMALAYA PUBLISHING HOUi,"Ramdoot, Dr.Bhalerao Marg, Girgaon,Mumbai - 400 004.Phones : 2386 01 70/2386 38 63 Fax : 022 - 2387 71 78F.-mail : himpub @ vsl.comWebsite: www.h/mpub,omBranch Offices:Delhi:Hyderabad"Pooja Apartments", 4-B, Murari Lal Street,Ansari Raod, Daryaganj,New Delhi - 110 002.Phone : 2327 03 92 Fax : 011 - 2325 62 86:Kundanlal Chandak Industrial Estate,Ghat Road, Nagpur - 440 018.Phones : 272 12 15 / 272 12 16:No.16/1, (Old 12/1)First Floor, Next to Hotel Highland, Madhava Nagar, -Race course Road, Bangalore - 560 001.Phones : 228 15 41,238 54 61, Fax : 080-228 66 11.: No.2-2-I 167/2H, 1st Floor,Near Railway Bridge, Tilak Nagar,- -Main Road, Hyderabad - 500 044.Phone : 5550 17 45, Fax: 040-2756 00 41: Geetanjali Press Pvt. Ltd.,Kundanlal Chandak Industrial Estate,Ghat Road, Nagpur - 440 018.PROF. CHANDRA NTHPIONEER OF IDIAN BIOCHEM/STRYPREFACE TO THE THIRD REVISED EDITIONOne of the problems facing the writers of textbooks in fast moving subjects is theobsolescence of old information and sprouting of new knowledge. Luckily, for us, evenwith tremendous growth in biochemistry, the basic techniques of study have remainedunaltered. Yet, variations and improvi.ations of the old techniques have been perfectedsince the book was last revised. The present resion is an attempt to take into accountthese changes.The Achilles' heel of the previous edition was the chapter on centrifugation. Thiswas brought to our notice time and again by students and teachers of the subject fromdiverse corners of the nation. We are happy to state that the chapter has been thoroughlyrevised, almost rewritten, and the scope of the discussion has been greatly expanded.Another aspect that has been completely revised is infrared spectrometry. Again itwas brought to our eyes by people who have taken critical notice of the book. The currentdiscussion of the aspect is again much wider in its scope and care has been taken togive several examples of its use in biochemistry and allied sciences.New techniques such as fluorescence energy transfer, fluorescence polarization,non-radioactive labeling, etc. have been added. Also, radioimmunoassay has beendiscussed more extensively along with a good discussion of its variant,radioimmunometry.A sore point with the previous editions was the lack of anindex. This shortcom, ghas been addressed and the book now has a detailed index.The above are the major additions. Numerous small changes in virtually all thewill become visible to the teachers of the subject who have seen the first twoeditions. New problems have also been added at the end of the chapters.Late in the day, when we had already submitted the revised draft for publication,we received the UGC syllabi for Biochemistry, Microbiology, Botany, Zoology,Biotec.hnology, and otherallied life science subjects We were pleasantly surprised todraftnot only covered all major points but had more to offer leavingin the syllabi. The. current edition will be of good use to studentsany life science subject.In its revised form, we feel confident that the book win be much more useful to allconcerned.We would like to thank the great number of teachers and students who have praisedour book; they provided us the support that eyery author nxis so much. We are equallyto those who criticized the book for providing the motivation to revise it.ACKNOWLEDGEMENTSA book of this expanse does not bcome possible without contribution ofseveral willing souls. We have been lucky that several colleagues and studentshelped us in our endeavour in whichever way they could, sometimes evengoing out of the way to do it.We would like to thank Prof. H. F. Daginawala, former Head of theDepartment of Biochemistry, Nagpur University, for his constantencouragement and help. We are also highly indebted to Dr. N. V. Shastri,Head of the Department of Biochemistry, Nagpur University, for hissuggestions regarding the changes to be made in the revised edition. Thefact that he taught this very subject to two of the authors (AU & KU) has!.everything to do with the writing of the textbook.. Heartfelt thanks are due to Ms. Ragi Radhakrishnan, one of our,cherished students, for designing the cover for the 3rd edition and forgoing through much of the 2rid edition in search of mistakes, typographicalotherwise.Dr. Rajnish Kaushik and Dr. Shibani Mitra Kaushik, St. Louis, Missouri,out of the way to provide us as much current literature as they couldof the fact that both of them were laden with their own research work.cannot really express our feeling of gratitude towards them.We are indebted to Dr. Saraswati Sukumar (Johns Hop.kins), and Dr.Runnebaum, The Salk Institute, La Jolla, USA, who provided usof much of the recent literature.Thanks are also due to several of our colleagues, friends and studentsDr. Irfan Rahman, Dr. Ashish Bhelwa, Dr. Saibal Biswas, Dr. S.N.Ms.Sadhana Naidu, Ms, Ramfla Bhojwani, Dr. Raymond Andrew, Dr.Deshpande, Mr. Amol Amin, Mr. S. Wankhede, Dr. Shyam Biswal,Ms. Anjali Gadkari.We record our appreciation of Mrs. Harsha Dave who was involved in,g the Ist edition.Finally, we would like to thank Shri Gokul Pandey and Shri D. P.of Himalaya Publishing House for their constant help and valuable1 - 6566- 7475 - 99CONTENTSACIDS AND BASESElectrolytic Dissociation and Electrolytes -- Ionization: Basis ofAcidity and Basicity w Bronsted-Lowry Theory: Acid is a ProtonDonor, Base is a Proton Acceptor -- Strength of Acids and Bases --Acid-Base Equilibria in Water -- Function and Structure ofBiomolecules is pH Dependent w Measurement of pH: Use ofIndicators m Electrometric Determination of pH -- Buffers: Systemswhich Resist Changes in pH -- Titrations: The Interaction of anAcid with a Base.ION SPECIFIC ELECTRODESIon Selective Electrodes Measure the Activity of Metal Long m GlassMembrane Electrodes m Solid-State Ion Exchanger Electrodes Solid-State Crystal Electrodes Liquid-Membrane Electrodes w Gas-Sensing Electrodes.3.THE COLLOIDAL PHENOMENAClassification of Colloids -- Properties of Colloids -- DonnanEquilibrium. . .DIFFUSION AND OSMOSISA Molecular-Kinetic approach to Diffusion m Methods ofDetermination of Diffusion Coefficient Significance of Diffusion Coefficient w Diffusion of Electrolytes -- Diffusion of Water AcrossMembranes: Osmosis Measurement of Osmotic Pressure Van't HoiTs Laws of Osmotic Pressure --Theories of Osmotic Pressureand Semipermeability m Osmotic Behaviour of Cells m MolecularWeight Determination from Osmotic Pressure Measurements --Significance of Osmosis in Biology.VISCOSITYFactors Affecting Viscosity Measurement of Viscosity Applications of Viscornetry Significance of Viscosity in Biological Systems.I00- 121122- 144SURFACE TENSIONFactors Affecting Surface Tension w Measurement of SurfaceTension.145-156ADSORFtlONKinds of Adsorption Interactions -- Adsorption Characteristics --Molecular Orientation Adsorption Isotherms: Quantitative157- 174Relationships -- Adsorption from Solutions n The Importance ofAdsorption Phenomena.8.SPECTROPHOTOMETRYBasic Principles -- The Laws of Absorption n Significance ofExtinction Coefficient (Box) Problems (Box) -- Preparation of Standard Graph (Box) Deviations From Beer's Law -- Absorption Spectrum -- Why is Absorption Spectrum Specific For A Substance?-- The Chromophore Concept -- Instrumentation For UV-VisibleAnd Infrared Sprectrophotometry -- Radiant Energy Sources --Wavelength Selectors -- Detection Devices -- Amplification AndReadout Double Beam Operation -- Double wavelength Spectrophotometer Applications of UV-Visible Spectrophotometry Qualitative Aalysis -- How to Interpret Absorption Spectra of Biological Macromolecules (Box) -- Quantitative Analysis Enzyme Assay n Molecular Weight Determination -- Study of Cis-transIsomerism Other Physicochemical Studies -- Control of Purification Difference Spectrophotometry Turbidimetry and Nephelometry -- Theory and Applications of InfraredSpectrophotometry -- Calculation ofVibrational Frequencies --Modes of Vibration Infrared Spectra of Common Functional Groups -- The Carbon Skeleton -- Carbonyl Group -- HydroxyCompounds Nitrogen Compounds -- Infrared Spectrophotometer: Mode of Operation Sampling Techniques -- Applications of Infrared Spectrophotometry -- Disadvantages of InfraredSpectrophotometry -- Spectro.fluorimetry Structural Factors Whichgive Rise to Fluorescence -- Fluorescence and Phosphorescence (Box)-- Fluorometry: Theory and Instrumentation -- Applications --Fluorescence Spectra and Study of Protein Structure -- ExtrinsicFluorescence -- Fluorescence Energy Transfer -- FluorescencePolarization n Luminometry -- Flame Spectrophotometry --Instrumentation for Emission Flame Photometry Instrumentation for Atomic Absorption Spectrophotometry -- Atomic Fluorescence-- Nuclear Magnetic Resonance Spectrophotometry -- MagneticProperties of the Nucleus -- Nuclear Resonance Chemical Shifts: Position of Signals '-- Hyperfine Splitting -- Instrumentation Applications Electron Spin Resonance Spectrometry n Applications -- Spin Labeling -- Mossbauer Spectrophotometry --Applications Some Solved Problems9.OTHER OPTICAL TECHNIQUES FOR MOLECCHARACTERIZATIONCircular Dlchrolsm and Optical Rotatory Dispersion -- RotationalDiffusion -- Flow Bircfringence D E1ectrlc Birefringence DPolarization of Fluorescence -- Light Scatterlng-- X-ray Diffraction.175-270271-30012.10. CENTRIFUGATIONBasic Principles of Centrlfugatlon m Relative Centrifugal Force (RCF)-- Other Factors Affecting Sedimentation -- Instrumentation --Desktop Centrifuge -- High Speed.Centrifuge The Ultracentrifuge-- Analytical Ultracentrifuge Fixed-angle Rotors -- Vertical-tube rotors Swinging-bucket Rotors n Wall Effects Preparative Centrifugation -- Differential Centrifugation -- Density GradientCentrifugation -- Rate Zonal Centrifugation -- IsopycnicCentrifugation Gradient Materials Preparation of Density Gradients --Choice of Rotors Centrifugation in Zonal Rotors --Centrifugation Analytical Basic Principles of Centrifugation --Factors Affecting Sedimentation Velocity -- Sedimentation Coefficient-- Factors Affecting Standard Sedimentation Coefficient Measurement of Sedimentation Coefficient Concentration Distribution -- Applications Of Boundary Sedimentation -- BandSedimentation Determination of Molecular Weights301-343II. CHROMATOGRAPHY344-421Survey of Chromatographic Procedures -- Techniques of Chromatography-- i. Plane Chromatography -- A. Paper ChromatographyB. Thin-Layer Chromatography 2. Column Chromatography --Types of Chromatography -- 1. Chromatography 2.Partition Chromatography A. Liquid-Liquid Chromatography B. Gas-Liquid Chromatography (GLC} -- 3. Gel PermeationChromatography 4. Ion Exchange Chromatography 5. AffinityChromatography High Performance Liquid Chromatography Some Specialized Techniques -- Hydroxyapatite Chromatography-- An Affinity System for Base Dependent Fractionation of DNA --An Affinity System for Fractionating supercoiled and Non-Supercoiled DNA -- DNA-Cellulose Chromatography.ELECTROPHORESIS422-478Migration of an Ion in an Electric Field m Factors AffectingElectrophoretic Mobility -- Types of Electrophoresis 1. Free Electrophoresis 2. Zone Electrophoresls. General Techniques of ZoneElectrophoresis -- 1. Paper Electrophoresis 2. Cellulose AcetateElectrophoresis 3. Gel Electrophoresis. Specialized ElectrophoreticTechniques I. Discontinuous (Disc) Gel Electrophoresis 2. Gradient Electrophoresis 3. High Voltage Electrophoresis (H.V.E.)4. Isoelectric Focussing 5. Two-Dimensional Gel Electrophoresis6. Immunoelectrophoresis 7. Pulse-Field Gel Electrophoresis8. Electrophoresis on Cellular Gels. Electrophoresis in GeneticAnalysis 1. Restriction Mapping. 2. Southern Transfer. 3. Gel Retardation or Band Shift Assay. 4. DNA Sequencing. 5. DNA FootprintLng. -14.13.ISOTOPES IN BIOLOGY,Radioactive Decay =- Production of Isotopes -- Synthesis of LabeledCompounds -- Interaction of Radioactivity with Matter mMeasurement of Radioactivity -- 1. Methods Based Upon GasIonization --A. Ionization Chambers B. Proportional CountersC. Fundamentals of Geiger Counters 2. Photographic Methods3. Methods Based Upon Excitation w A. Liquid ScintillationCounting Use of Stable Isotopes in Biology -- The TracerTechnique-- Use of Isotopes as Tracers in Biological Sciences -- SomeInformation About Commonly Used Isotopes -- Safety Aspects --Dosimetry.CERTAIN PHYSICOCHEMICAL TECHNIQUES USEFULIN BIOCHEMISTRYPolymerase Chain Reaction -- Enzyme-Linked ImmunosorbentAssay (ELISA) -- Flow Cytometry.479 - 54,546 - 56!-- APPENDICES-- INDEX567-593 - 60:1ACIDS AND BASESA history of the quest to understand the molecular basis of acid - base properties ,m.akesfor a very amusing reading. For instance, in 1773 Doctor Samuel Jhonson averred that acidsiare composed of pointed particles which affect the taste in a sharp and piercing manner".iAnother attempt to explain the nature of acids was made by Lavoisier when he proposed thatithe characteristic behaviour of acids was due to the presence of oxygen. Stimulated by thisobservation, Sir Humphrey Davy went to great lengths to show that hydrochloric acid alsocontalns oxygen. He, of course, failed in his attempt thereby disproving the theory of Lavoisier.Even the later history of acid - base research is not without its share of amusement, albeit in:a manner different to the above described instances. In 1884 Svante August Arrhenius in hisdissertation proposed the theory of electrolytic dissociation and ionization on whichcurrent understanding of acid - base character is based. The doctoral dissertation was,greeted by the lowest possible pass-mark by the University of Uppsala, Sweden. For, Arrhenius was awarded Nobel Prize in Chemistry in 1903.ELECTROLYTIC DISSOCIATION AND ELECTROLYTESLet us consider a simple experiment. A pair1.1.F.Jcperlmental system for determiningelectrical conductivity of a solutWn. Thebulb does not light when there is a non-bulb l@hts when the beaker cohtalnseteces nof electrodes is connected in series to a light bulband to a source of electricity (Figure 1.1). As longas the electrodes hang separated in the air, noelectric current flows through the circuit, and thebulb does not light. If however, the two electrodesare touched to each other, the circuit is completedand the bulb lights. If the electrodes are dippedinto a beaker containing water purified byrepeated distillations, the bulb does not light. Thistella us that water is not a good conductor ofelectricity and is not capable of completing thecircuit. If we dissolve an acid, a base, or a salt inwater in which the electrodes are dipped, the bulblights up. Obviously, these substances are ableto carry the current and thereby complete thecircuit. Substances produc/ng solutions capableof conductlng electric'.tty are called electrolytes. Onthe other hand, substances producin9 solutionsincapable of conducting electricity are known asnon-electrolytes. Table 1.1 provides a fewexamples of electrolytes and non-electrolytes.2Bophs. ChemisWhat is the mechanism by which, electrolytes conduct electricity? Arrhenius' theory provingan answer. The theory proposes that acids, bases, and salts undergo dissociation in watervarying degrees, each molecule giving rise to oppositely charged long. For example, if gasehydrogen chloride is bubbled into water, virtually all the hydrogen chloride molecules re.with .water (Figure1.2) giving rise to a hydronium ion (positively charged) and a chloride i{negatively charged}. These long can now be carried to the cathode and the anode respectivthereby completing the circuit. This theory of Arrhenius is known as the theory of electrol3dissociation.WaterHydrogenChlorideCollision 'Complex'Hydronium IonChloride Io:Figure 1.2. When gaseous hydrogen chloride is bubbled in water, HCI mo/ecu/es collide with water molecuCollisions of sufficient energy and proper orientation produce hydronlum long and chloride long.Going back to the experiment we discussed, a diligent observer would note that certasubstances cause the bulb to be brightly lit, whereas other substances cause the bulb toonly dimly lit. This experimental observation permits us to subdivide the electrolytes into groups. Substances that dissociate almost completely and produce solutions that are very goconductors of electricity are known as strong electrolytes; substances which dissociate only partand produce solutions which are poor conductors of electricity are known as weak electrolyThe difference between strong and weak electrolytes was attributed by him to a difference in tdegree of ionization.IONIZATION : BASIS OF ACIDITY AND BASIClTYArrhenius Theory : H Ion is the Acid, OH- Ion is the baeFom the experiment that we have discussed above, one can safely conclude that acibase reactions are a function of ionization p-nciple. Thus, based on ionization principnhenius defined acids and bases. These definitions are elaborated below.Acls : Acids were described by Arrhenlus as compounds containing hydrogen willupon addition to water become ionized to yield 14+ long. Nitric acid (14N03), which is a solutstrong electrolyte or srong ac [Le,, it dissociates completely in water to produce t4+ long), mbe cited as an example.HNO3 H + NONitrous acid (HNO2) , a weak electrolyte {Le., dissociates only partially to produce H+ iontmay be cited as an example of a weak acid.HNO2 # H+ +NO.(A single arrow ----> denotes reactions that go completely tothe right; a double arrow x--- denotre.actions that go only partially to the right).3Acids and BasesTable 1.1 Examples of Electrolytes and NonelectrolytesStrong ElectrolytesHydrochloric acid, HCI [H+ + Cl-] .Nitric acid, HNOa [H+ + NO]Sulfuric acid, H2SO4 [H+ + HSO]Sodium hydroxide, NaOH [Na+ + OH-]Potassium chloride, KCI [K+ + CI-]Silver nitrate, AgNO3, [Ag+ + NO ]Sodium chloride, NaCI [Na + CI-]Copper fib sulphate, CuSO4 [Cu2+ + SO-]Weak ElectrolytesNonelectrolytesAcetic acid CH3COOH [CHaCOOH]Lactic acid, CHaCHOHCOOH [CHaCHOHCOOH]Ammonia, NH3 [NHa]Hydrogen sulphide, H2S [H2S]Mercury (II} chloride, HgC12 [HgCI2 ]Glucose C6H1206 [C6H1206 ]Sucrose C12H22011 [C12H22011 ]Ethyl alcohol, C2HsOH [C2H5OH ]Methyl alcohol, CH3OH [CH3OH ]Acetone CH3COCH3 [CHaCOCHa ]Species in parentheses are predominant in solution. The difference between weak andnonelectrolyte is that weak electrolytes dissociate very lltfle (not shown in the table) whereas thenonelectrolytes do not dissociate at all.Bases : According to the Arrhenius definition, bases are compounds which upon ionizationin water yield OH- (hydroxide) long. Sodium hydroxide, which dissociates completely to produceOH- long, may be cited as an example.NaOH Na+ + OH-The Arrhenius concept is important in that it has provided us with the first mechanisticapproach to acid - base behaviour and has been instrumental for the development of moresophisticated theories. There are, however, two major shortcomings in the Arrhenius model."- (0 In the AIThenius model the acid-base reactions are limited to aqueous solutions (thisis not a problem as far as biological systems are concerned since all reactions musttake place in aqueous solutions).(//)The theory limits bases to hydroxide compounds. This is very unsatisfactory becauseit is well known that many organic compounds which are not hydroxides, for exampleammonia, show basic properties in their chemistry.In the year 1923, two more theories defining acid-base character were proposed. The firsttheory, Bronsted and Lowry theory, is very satisfactory for understanding physiological processesand will therefore form the basis of all further discussions. The second theory, proposed byG. N. Lewis is much more general than the Bronsted - Lowry concept. A brief discussion of thistheory is given in Box 1.1.4Biophysical ChemistrBronsted - Lowry Theory :' Acid is a Proton Donor, Base is a Proton AcceptorThis theory defines an acid as any compound that yields protons (H+ long) and a baseas any compound that combines with a proton. In other words, acids are proton donors andbases are proton acceptors. It should be noted that as-far as acids arc concerned, Arrheniusand Bronsted - Lowry theories are similar ; in both cases acids give off H+ long. However, theconcept of a base is much broader in the Bronsted theory, hydroxyl ion being just one of thepossible bases. Cited below are a few examples which will illustrate the point much better.general equationH2SO4H+ HSOHC1H+ "+C1-HsPO4H+ [+HuPOCHaCOOHH+[+CHaCOO-HCOaH+ [+HCOHCO H+ I+CO -HsO+H+{+H.OHAH+ I+A-Concept of conjugate ac/d and conjugate base : Each of the compounds listed above as acid,pon ionization, produces. H+ long. Their ionization also produces long or molecules which canombine with a proton (HSO , Cl-, H2PO , CHsCOO-, etc). According to the definition, thesewhich can combine with a proton are bases: Thus, we can say that every acid dissociates to a proton and a base (if the reaction is reversed, a base can combine with a proton toan acid}. The Bronsted -Lowry theory thus conceives of an acid -base 'pair'. An acidits corresponding base are said to be 'conjugate', i.e., 'joined in a pair'. Thus, CI- is theof HCI, likewise H20 is the conjugate base of H30+.An acid is a proton donor. Its strength would depend upon the ease with which it cana proton. An acid will yield a proton with comparative ease if its conjugate base is weak.HCI as an example. Its conjugate base, CI-, is a weak base; it is not a very goodIn solutions, therefore, HCI is completely ionized to produce H and CI-. HCI ISi strong acid because/ts conjugate base/s wea/ Let us consider another example, that ofIts conjugate base CH COO- is stronger base compared to CI-. The acetate ion,binds the proton much more tenaciously with the result that in solution acetic acid is' ionized. CHCOOH Is a weak ac/d because/ts conjugate base Is strong. Similar conceptse drawn for bases also and their strength would epend upon the strength of their conjugateThe Bronsted -Lowry theory gives us the following reciprocal relations :-- ff an acid is strong, its conjugate base is weak:-- if an acid is weak, its conjugate base is strong.-- if a base is strong, its conjugate acid is weak.if a base is weak, its conjugate acid is strong.Concept of an a/ka/i : In the previous pages NaOH was regarded as an Arrhenius baseionized to produce OH- long. NaOH, however, is not a Bronsted base because, as ait has little ability to accept a proton. NaOH can act as a base solely because uponit gives rise to OH- long which are very good proton acceptors. NaOH and otherhydroxides like KOH, therefore act as bases by proxy. Such compounds, under thetheory, are known as alkalies.6Biophysical ChemislAmphoteric substances : Substances which can behave both as an acid and as a basereferred to as amphoteric. Thus, under the Bronsted concept, liquid ammonia qualifies asacidNH3 NH + H+and as a base too-.NH3 + H+ NHSimilar is the case with water which behaves as an acidHOH x- H + OH-and as a baseHOH + H+ --- I-IO*Sa/ts : Under this tleory salts are thought to be compounds which are formed by replacithe ionizable hydrogen with a metal ion or with any other positively charged group. ThuCH3COONa is the sodium salt of CH-COOH formed by replacement of the proton by the Nion. KCI is a salt of HCI formed by replacement of the proton by K+ ion.CH3CO0 CH3CO0AcidIonizableSaltMetalHydrogenSTRENGTH OF ACIDS AND BASES(Throughout the dlscussiori, acids will be treated as examples. However, the discussicapplies equally well to bases, albeit, in a reverse manner).In a preceding section we have said that the strength of an acid depends upon the strengthweakness of its conjugate base. This, however, is not the only determinant of strength. Apmfrom strength of conjugate base, the strength of an acid depends upon (i) the basic strength the solvent, and (ii) the dielectric constant of the solvent. Both these factors are discussebelow.The Basic Strength of the SolventSo far we have been writing the ionization reaction of HCI asHCI .--x H+ +and the general ionization reaction of acids asHA --- H++A-It is, however, well known that H+ long do not exist in acid solutions. This is because thH+ long combine with the solvent molecules to give rise to 'lyonlum long'. Let us illustrate thcaseby considering a specific example, that of water, as a solvent. In water, the H long {formecdue to ionization of an acid) are known to combine with water molecules to give rise to H30+the. hydronlum long (also known as the oxonium or hydroxonium ionsl.H++H20 ---- H30+iAds m Boes 7Recall that Bronsted - Lowry concept states that a base is a proton acceptor, Thus waterthe above cae (and solvents in general} is acting as a base.We can now rewrite the general lonition reaction of an acid In waterHA+H20H30+A- 'The strerth of the acid, HA, now is a function of the competition between the two bases,, and H20 to accept the Ionlzable hydrogen.Coe I : A= is strorer than H20, In th-is cae A- Is a stronger base and bind to thebI hydrogen much more tenaciously than H20. A a consequence, the dissociation ofacld, HA, will be less and it not be a stror acld in water.Case 2 : A- is weaker than H-O. In this case, once the acid is dissolved in water, A= Mill 10seionlzable hydrogen to water Zhich is a stronger base. The dissociation of the acid, HA, Milland the acid may even be completely dissociated. The acid, HA, will be a strongin water.We can now generalize the above observations./f the basic strength of the solvent is lessbase, the ac will be v,,,eak in that solvent. If the basic strengthgreater tbn that of the conjugate base, the ackl will be strong in that solvent.To drive the point home, let us consider the strength of the same acid in two solvents.CaseI : Acetic acid in water. The acetate ion is a stronger base than water. Therefore,is a weak acid in water.0OCH3-C-OH+H-OH CH--C--O:+H30+ "Case2 : Acetic acid in liquid ammonia. Acetate ion is a weaker base as compared toTherefore, acetic acid which was a weak acid in water, is a strong acid in liquidOOCH3-C-O-H+NH3 CH3-C-O-+NHThe above examples show the relative nature of the designations strong and weak. Thethat an acid is strong does not convey much sense unless we know in relation todirectlon of proton transfer and its extent depend upon these relative proton - donating proton-bindlng abilities of the potential acids and the solvent. It can thus be said that theran acid s always relative to.the basic strength of the solvent used.Constant of the SolventUpon ionization the acid splits into two oppositely charged long, H+ and A-. These longattract each other and recombine. However, solvents of high dielectric constant greatlyattraction between oppositely charged partlcles dissolved in them. This action of theacid and consequently is important for the strength of acid.acid in a solvent of high dielectric constant Mill dissociate greatly and will therefore beThe same acid, in a solvent which has a low dielectric constant, will not dissociate much+ H+8B/ophysicat Chemtsand will consequently be weak.Water is a solvent which has a very high dielectric eonstan'room temperature, almost 80. On the other hand, petroleum ether has a very low dieleeconstant, just 2.2. A given acid can therefore dissociate to a much greater extent in water ttin petroleum ether. The dielectric constant is thus of great importance in determiningstre.ngth of an acld.Effectof Structure on the Strength of -cidsIt is a commonly accepted fact that carboxylic acids are stronger than other organic aciWhy is that so? The reason usually given is that the carboxylate anion (the conjugate baformed upon dissociation is stabilized by resonance (two equivalent resonance structures]such a manner that it is more stable than the original acid molecule.olR--C//u" +-R--C\oHResonance stabilized anionOn the other hand, in the alkoxide ion, TO-, the negative charge is not delocalized an(concentrated on the single oxygen atom. This anion, therefore, is not as stable as the resonarstabilized carboxylate anion. The resonance stabilization promotes dissociation in the carboxyacids making them stronger in relation to the organic acids where lack of resonance stabfllzatidecreases dissociation.If resonance stabilization were the only factor all carboxylic acids would have had tsame strength. This is not so. Carboxylic acids which contain strong electron attracting grou(halogens) on the alpha - carbon are stronger than the unsubstituted acids. On the other hacarboxylic acids bearing electron releasing groups (methyl) on the alpha - carbon atom aweaker than the unsubstituted acids. These electrostatic factors, in which electrons are eithattracted to or repelled from one atom or group of atoms with respect to another are knowninductive effects. Electron attracting groups withdraw electrons from the carboxylate grouThis weakens the oxygen - hydrogen bond thereby facilitating ionization and release of a protoMoreover, these groups also help stabilization of the conjugate base by resonance.C10CIO(I) CI-----C CH3COO- + Na + H20The strong alkali, NaOH, dissociates completely into its constituent long Na + and OH-.have increased the pH, bUt in the buffer solution they react with CH3COOH to giveto water and acetate long. The pH does not increase appreciably (it increases only into the change in the ratio of acid to salt in the solution).pH = pKa - log24Biophysical ChemlstrTo what extent can a buffer solution resist change in pH ? A simple example will be citecIf 10 ml of 0.1 N HCI is added to 990 ml of pure,water (pH 7.0), the pH of water drops 4 uniland becomes 3. Similarly, if 10 ml of 0.1 N NaOH is added to 990 ml of pure water, the p]increases by 4 points and becomes 11. However, if 10 ml f 0.1 N HCI is added to 990 ml ofbuffer consisting 0.1 N acetic acid and 0.1 M sodium acetate (pH 4.76), the drop in pH is on]0.01 points. The pH changes merely to 4.75. Similarly, addition of 10 ml of 0.1 NNaOH to 99ml of above buffer solution elicits a rise of merely 0.01 units on the pH scale. The pH become4.77. We thus see that buffer solutions resist chariges in pH to a very significant extent (we wiconsider the same example quantitatively a little later).We have seen that the conjugate base provided by salt dissociation is actually involved ithe buffering action. The metal long (like Na in sodium acetate) are not involved. We shoultherefore rewrite the definition of buffer solutions. Buffers are mixtures of weak acids and theconjugate bases.The Henderson-Hssselbslch EquationHenderson-Hasselbalch equation is important for understanding buffer action and acibase balance in the blood and tissues of the mammalian system. The equation is derived in following way. Let us denote a weak acid by the general formula HA, and its salt by the genenformula BA (B being the metal ion and A- being the conjugate base). The salt dissociatecompletely, while the weak acid dissociates only partly. We can write the equtlibrium reactiorfor the dissociation of HA and BA in the buffer solution as follows :HA -I-I + A-BA -B+ + A-We will soon find that Henderson-Hasselbalch equation is simply another way or writirthe expression for the dissociation constant of a weak acid.Solving for {H], we getTaking the negative logarithm of both sides, the equation becomesHowever, - log [H+] = pH, and - log Ka = pKa. Therefore,oralso[conjugate base]orpH = pKa + log[acid]necessary because the values of pK and activities varystrength. The value of pKa on the basis of activities can be calculaled with the help ofrelationship :and Bases25the negative sign, we invert - log [HA]/[A-] and obtainpH = pKa + logThis is Henderson-Hasselbalch equation'Now, the weak acid, HA, is only slightly dissociatedthe absence of the salt. Thus very little of the A- long come through the dissociation ofbak acid. On the other hand, we have seen that the salt BA is completely dissociated and givesEhlgh concentration of A- long. It can, therefore, be safely assumed that the concentration ofhe undissociated acid [HA] is equal to the total acid concentration. We can also assume that all." has dissociated from BA and therefore the concentration of the conjugate base, [A-] is equalthe concentration of the salt, [BA]. Taking into consideration these assumptioBs, he equation can take many differont forms.Isalt]pH = pKa + Iog [acid][proton acceptor] "'..pH = pKa + log [prot.0n donor] . .?w .As with all the equations considered so far, themore accurately when concentrations are converted topK (activity) = pK (concentration) - 1.018 fIs the ionic strength of the solution. For most calculations, however, concentrationsfairly accurate results. Now, that we have derived an equation which relates pH toconcentration (conjugate base Concentration) and the weak acid concentration,;us see the quantitative basis of buffer solutions rslsting a large change in pH. We have seenaddition of 10 ml 0.1 NHCI to 990 ml of pure water brings its pH down from 7 to 3. Let usadd this acid to 990 ml of 0.1 N acetic acid and 0.1 M sodium acetatelong disgociating from HC1 are neutralized by the acetate long.CH3COO- +H+CH3COOHThe addition of HCI therefore lowers the concentration of the acetate ion slightly andthe concentration of acetic acid by the same amount. If we assume that all I-I+ long haveneutralized, the drop in acetate ion concentration will be 10-3 mole/litre. The concentrationacid would rise by the same amount. -260.09990.101pH = pKa + Iog--pKa of acetic acid is 4.76. Therefore,pH = pKa + log 1.0pH = pK + 0pH = pKaThus to calculate the pK of any acid one only needs to dissolve that acid and its salt equal concentrations and thenaexperimentally determine the pH of the solution. It will be equ to the pK of the acid. Some extremely important problems about buffers which can be solw using Hederson-Hasselbalch equation are provided for in Box 1.6.Henderson-Hasselbalch equation makes it clear that the pH of a buffer solution depen{upon the pKa of the acid and upon the salt to acid concentration ratio. The lower the pK oft]acid the lower will be the pH. The buffer pH will increase with increasing salt concentratioAgain, according to Henderson-Hasselbalch relationship, the actual salt and acid concentratiorcan be varied widely without any change in pH if the ratio between the two is unity. Thus,lactate buffer containing 0.01 M lactate and 0.01 N lactic acid will have the same pH even if flbuffer is diluted 10 times or even 20 times. In actual cases, however, the pH of the dilutbuffer increases slightly. This increase is not signii2cant enough.Biophysical Chemistr[CH3COO- imolemolemole-- 0.1---0.001= 0.0999--NALlitrelitrelitremolemolemolej[CH3COOH]FINAL = 0.1 -- + 0.001 -- = 01101litrelitrelitreSubstituting the final salt and acid concentrations in the Henderson-Hasselbalch equatiowe getpH = 4.76 + log 0.09990.I01= 4.75The pH of the buffer solution after addition of I0 ml of 0.1 N HCI changes from 4.76 to 4.751drop of merely 0.01 units of pH.Henderson-Hasselbalch equation gives a very important relationship which makespossible to calculate the pK 0f-any given acid with extreme ease. The relationship is, that if ttmolecular ratio oLIt to acid is unity in a solution, the pH of that solution will be equal to ttpK of flleacl tTind " transition is of a very high order and ' short wavelengths are absorbed for these transitions. The organic compoundswhich all the valence shell electrons are involved in formation of sigma bond, therefore, dothe normal Ultraviolet region (180-400 nm). The usual spectroscopicues cannot be used below 200 nm because oxygen absorbs strongly at this range. Thus,these transitions, the entire path-length is to be evacuated. It is because of this thatbelow 200 nm is known as the vacuum ultraviolet region.Energy required for n ----> * transition is lower than that of ----> a transition. This typetransition usually takes place in saturated compounds containing one heteroatom withpair of electrons. Several molecules, including water, ether, saturated alcohols, andshow absorption attributed to n * transitions.Even lower energy is required for ----> n" transitions. These transitions take place in the centres of molecules. These transitions are shown by alkenes, alkynes, carbonylcyanides, and azo compounds etc. Since these transitions are associated with aenergy, they take place at comparatively longer wavelengths easily obtained in aspectrophotometer, n ----> n transitions usually have very high extinction coeffI-= 104 - 105 M-cm-) ff not forbidden by spin or symmetry selection rules.n ---> * requires the least amount of energy. Therefore, these transitions frequently appearshoulders on the long-wavelength side of an absorption spectrum.Allthe above orbitals with ordering of their energies relative to each other along with thetransitions are shown in Figure 8.8(B).Anti-bonding electronsAnti-bonding electronsNon-bonding electronsBonding electronsBonding electronsFigure 8.8(B) Schematic rnolecular orbital energy level diagram depicting relative energy levels and allowedBy way of generalizing, it may be said that the absorption bands of almost all organicnormally found in the near ultraviolet and visible regions are due to either n ----> "n ----> * transitions. One can distinguish between n ----> n* and n ------> n* transitions byfat the extinction co-efflcients of the peaks at Xm. The n transitions are usually symmetrical considerations and therefore have extinction coefficients of the orderust 10, whereas n ---> * transition which are rarely forbidden, have extinctiongenerally in the magnitude range of 103 - 104.The transitions just discussed have been summarized in Table 8.2 along with informationthe region of the spectrum in which they absorb.190Table 8.2Types of electronic transitionsDescriptionRegion of electronic spectraFrom a bonding orbital in the. ground stateto an antlbonding orbital of higher energy.(a) o ----> (between orbitals)(b) . g* (between orhitals)Vacuum UV (e.g., ethane at 135 nm)UV (e.g., ethylene at 180 nm, benzeneat 230 nm).From a nonbonding orbital to an antlbondingorbital of higher energy(a) n ----> *Near UV and visible (e.g., acetaldehydeat 293 nm. nltrosobutane at 665 nm).Far UV, or sometlme near UV (e.g.,methyl alcohol at 174 nm, methyliodide at 258 nm).From an orbital in the ground state to a veryhigh energy orbital (towards ionization)Vacuum UVLastly, the structural character and the position of absorption maxima depend not onlyupon the structure of the compound, but also on the nature of the solvent in which they exist.The environmental factors that can cause detectable changes in the absorption spectra are pH,the polarity of the solvent or of neighboring molecules, and the relative orientation of ne/ghboringabsorbing groups.Effects due to pH : It is the pH of the solvent which determines the ionization state of amolecule (see Box 1.8 and Tabie 1.7 for clear understanding). With a change in ionization0,9"0.8.0.70.60.5.0.40.30.20.I0/ \ A240 260 280 300Wavelength (rim)Figure 8.9. travlolet absorption spectra of 3'-CMP. The nucleotlde was obtained by enzymatlc degradation oftRNA. See the difference between the absorption at neutral and acidic pH. Also note the ralchange In the absorption spectra at reglons other than the absorption maxOn25OSpectrophotometnj191status, the absorption spectra may also change. Given below are the absorption spectra (Figure8.9) of the pyrimidine nucle.otide CMP at neutral and acidic pH. Mark the difference in theabsorption spectra of the same compound in the two states of ionization.Polarity effects : In general, polar solvents shift the n =" transitions to shorter wavelengths and ----> transitions to longer wavelengths as compared to the gas phase spectra (Figure 8.10). This is true for polar chromophores. For this reason polar solvents havebeen used to distinguish between n n" and ---> =" transitions. Non-polar solvents, on the other hand, produce no such change.260 270 280 290 300 310Wavelength (nm)Fure 8.10. Effect of solvent polarlty on the absorption spectrum of tyroslne. Solid line represents absorption spectrumin water while the dashed llne corresponds to 20% ethylene glycol The transition here is n -- andtherefore the spectrum is shifted towards shorter wavelengths when water, which is more polar, is useds a solvenLOrientation effects: The best example to understand orientation effect is theDNA. Absorptioncoefficient of a single nucleotide is greater than when the nucleotides are arranged in a single-stranded polynucleotide. The effect occurs because in a polynucleotide, the bases are in closeproximity. The absorption coefficient decreases even further with a double-strandedpolynucleotide because in this structure the bases are arranged in an even more ordered manner.THE CHROMOPHORE CONCEPTThe old definition of chromophore regards it as any system which is responsible for impartingcolor to the compound. Most of the nitro compounds are yellow in color. Clearly, nitro group isa chromophore which imparts yellow color. The modem day definition of chromophore, however,akes use of the term in a broader scope. The chromophore is dejlned as any isolated covalentlybonded group that shows a characteristic absorption in the ultraviolet or the vls.lble region. Some ofthe important chromophores are carbonyls, acids, esters, nitrile group of ethylenic or acetylenicgroups. Chromophores are known to be of twotypes.(1) Chromophores containlng electrons and involved in ---> " transitions. For example, acetylenes and ethylenes (Table 8.3).(//) Chromophores containing both and n electrons and involved in ---> " and n---> n" transitions. For example, carbonyls, nitrfles and azo compounds (Table 8.3).192Btophystca/ChemfstnjAuxochromeAwcochromes are groups which by themselves do not act as chromophores but whose presencebrings about a shift of the absorption band towards the red end of the spectrum (longer wavelength).Auxochrome is thus also known as a color enhancer. Important examples are -OH, -OR,-NH2, -NHR, -NR2, -SH etc. Auxochrome exerts its effects by virtue of its ability to extend theconjugation of a chromophore by sharing of the non-bonding electrons. This results in a newchromophore which has a different absorption maximum and probably an enhancedextinctioncoefficient.In many instances the absorption and absorbance change either due to interaction withan auxochrome or due to change of the solvent. Four uch absorption and intensity shifts areknown and are detailed below.Bathochrom/c sh/Jt : This shift is due to the presence of an auxochr0me by virtue of whichthe absorption maximum shifts towards higher wavelengths. Figure 8. I .YSuch an absorptionshift is known as the red shift, or the bathochromic shift. Sometimes decreasing polarity of thesolvent may also cause bathochromic shiftHyperchromicshiftHypsochromic Bathochrom/cWavelength {rim)Figure 8.11 Different types of absorption and intensity shIjsHypsochromic shift :This is opposite of the bathochromic shift. This shift is due to removalof conjugation and a change in the polarity of the solvent due to which the absorption maximumis shifted towards shorter wavelengths (b/ue shift). See Figure 8.11.Hyperchromic effect : This effect signifies an increase in the intensity of the. absorptionmaximum, or a change in the extinction coefficient to a higher value at the same absorptionmaximum. This effect is mostly due to the presence of an auxochrome {Figure 8.1 I}.Hypochromic effect : This is the opposite of hyperchromic effect and is caused due tointroduction of groups which cause distortion in the geometry of absorbing molecules. Thiseffect signifies that the intensity of the absorption maximum is lowered {Figure 8.1 I}.INSTRUMENTATION FOR UV-VISmLE AND INFRAREDSPECTROPHOTOMETRYIn order to obtain an absorption spectrum it is necessary to measure the absorbance of asubstance at a known series of wavelengths. The instruments that are used to study theabsdrption or emission of electromagnetic radiation as a function of wavelength are calledSpectrophotometry193or spectrophotometers (colorimeters, if the instrument applies wavelengths onlyvisible range). More or less similar optical principles are employed in these instruments.are, however, some important differences in the specific components used in the variousspectrum. The essential components of a spectrophotometer include: (/) a stable and cheap radiantsource, (tO a monochromator to break the polychromatic radiation into componentor "bands" of wavelengths, (///) transparent vessels {cuvettes) to' hold the sample,(/v) a photosensitive detector and an associated readout system (meter or recorder).available commercially involve quite a bit of complex arrangements, but allrepresent variations of the block diagram in Figure 8.12.Sample --- Detectr -Amplifier &1Holder " [ RecorderFigure 8.12 Block diagram of a spectrometer ,adiant Energy SourcesMaterials whlcL can be excited to h/gh energy states by a gh voltage electric discharge or,by electric heating serve as excellent radiant energy sources. As the electrons of these materialsreturn to the/r ground state, they em/t radiation of characteristic energies corresponding to AE,the energy difference between the excited and the ground energy levels. Some materials havenumerous energy levels very close to each other. Consequently the wavelengths of radiationera/tied by these substances take the form of a continuum of radiation over a very broad region.These materials would constitute an ideal source for absorption measurements if the intensityof all the wavelengths is alike. However, inpractice, this is not so and so we have differentsources for different regions of the spectrum.Sources of ultraviolet radton : Most commonly used sources of ultraviolet radiation arethe hydrogen lamp and the deuterium lamp. Both the systems. :consist of a pair of electrodesenclosed in a glass tube provided with a qumz window, Te glass tube is tflled with hydrogenor deuterium gas at low pressure. When a stabilized high voltage is applied they ernit radiationwhich is continuous in the region roughly between 180 and 350 nm. Xenon lamp may also beused for ultraviolet radiation, but the radiation produced is not as stable as the hydrogen ]amp.Sources of visible radon :'lngsten filament Imp is the most commonly used source forvisible radiation. It is inexpensive and emits continuous radiation in the region betweenand 2500 nm. Carbon. arc, which prov/des more intense v/sible radiation is used in a smallnumber of commercially available instruments.Sources ofnfrared radton : Nemst Glower and Globar are the most satisfactory sourceso infrared radiation. The Globar consists o a s/I/con carbide rod which when heated toapproxLrnate]y 1200C, emits radiation in the 1-40 region. emst Glower employs a hollow rod of zh-'con/um and yttrium. It requires to be heated up to 1500C before it emits radiation inthe range of 0.4-20 . Globar is more stable than the Nemst Glower.Wavelength SelectorsAll the sources discussed so far em/t continuous radiation over w/de range of wavelengths.However, as pe/nted out earl/er in the chapter, the laws of absorption in the strictest senseapply only to monochromatic radiation. Thus, absorption of nrrow band width will show greateradherence to Beer's law. Moreover, narrow band radiation will allow the resolution of absorptionbands which are quite close to each other. Therefore, a narrow band width will go a long way inincreasing the sensitivity of absorbance measurement. Narrow band widths are made possibleby using wavelength selector(s).SourceSampleholder194Biophysical Chemistry'fffYgl'tlg'ffi sel'ecfors are of'two types, filters and mon0chromators.lailters : Filters operate by absorbing light in all other regions except for one, whlch theyreflect. Gelatin filters are made of a layer of gelatin, colored with organic dyes and sealedbetween glass plates. Most modern filter instruments, however, use tinted-glass filters. Filtersresolve polychromatic light into a relatively wide bandwidth of about 40 nm and are used onlyin coldrimeters. One disadvantage of glass filters is their low transmittance (5-20%).Monohromators : As the name suggests, a monochromator resolves polychromatic radiationinto Its individual wavelengths and isolates these wavelengths into very narrow bands. Theessential components of a monochromator are: (i) an entrance slit whlch admits polychromaticlight from the source, (tO a collimating device such as a lens or a mirror whlch collimates thepolychromattc light on to the dispersion device, (No a wavelength resolving device like a prism ora grating which breaks the radiation into component wavelengths, (iv) a focussing lens or amirror, and (v) an exit slit which allows the monochromatic beam to escape. The entire assemblyis mounted in a light-tight box. For obvious reasons, all the components in a monochromatorassembly must not absorb in the range of wavelengths which are to be studied. A monochromatoremploying a prism for dispersion is shown schematically in Figure 8.13. The effective bandwidth of the light emerging from the monochromator depends mostly upon the dispersing element(prism or diffraction grating) and the slit widths of both the entrance and the exit slits. Narrowslit widths isolate narrow bands. However, the slit width also limits the radiant power whichreaches the detector. Since the effectiveness of the resolving element is of primary importance,the two kinds most widely used, namely, prlsms and diffraction gratings are considered below..... lenslens"Entrance slitExit slitMONOCHROMATORl%3ure 8.13 Pasta monochromatorPrisrr" A prism disperses polychromatic light from the source into its constituent wavelengthsby virtue of its ability to refract different wavelengths to a different extent;, the shorter wavelengthsare diffracted most. The degree of dispersion by the prism depends upon (/) the apical angle ofthe prisln (usually 60), and (it) the material of which It is made. Since it disperses the shortwavelengths more and long wavelengths less, the wavelengths at the red end of the spectrumare not fully resolved; they are said to be crowded. This is a major disadvantage of a prism.Two types of prisms, namely 60 Cornu quartz prism and 30 Littrow prism are usuallyemployed in commercial instruments. The latter is preferred, Simple glass prisms are used forvisible range. For ultraviolet region silica, fused silica or quartz prism are used. F1ourlte is usedin vacuum ultraviolet range. Ionic crystalline materials are used in the infrared region. Someexamples are NaCI, KBr, CsBr, and the mixed crystalline material commonly called KRS-5,SpectrophotometryFigure 8.14 Diffraction grating and the dispersion of polychromatic radiationGratings : Gratings (Figure 8.14) are often used in the monochromators ofspectrophotometers operating in ultraviolet, visible, and infrared regions. The grating possessesa highly aluminized surface etched with a large number of parallel grooves which are equallyspaced. These grooves are also known as lines. A grating may have anywhere between 600 to2000 lines per mm on the surface depending on the region of the spectrum in which it isintended to operate it. The principle behind dispersion of radiation by a grating is that it resolveslight into its component wavelength by virtue of constructive reinforcement and destructiveinterference of radiation reflected.Very often, the monochromator consists of both, a prism and a grating. The prism, placedbefore the grating is known as the foreprism. It preselects a portion of the spectrum which isthen allowed to be diffracted by the grating. This arrangement allows resolution of a singlewavelength. The major advantage of diffraction grating monochromators is that their resolvingpower is far superior to that of prisms. In addition they.yield a linear resolution of spectrumwhich is not possible when prisms are used.Sample ContainersSamples to be studied in the ultraviolet or visible regionare usually gases or solutions andare put in cells known as cuvettes. Spectra of gases are taken using enclosed cells, with anevacuated cell as a reference. Standard path-length of gas cells is usually 1 mm but cells withpath-length of 0.1 to 100 mm are available for special cases. Sometimes spectra of solids maybe taken directly. For this purpose, the solids are generally in the form of pellets. The pellets arekept in pellet holders for absorption measurements.Most of the spectrophotometric studies are made in solution. The solutions are dispensedin cells known as cuvettes. Cuvettes meant for the visible region are made up of either ordinaryglass or sometimes quartz. Since glass absorbs in the ultraviolet region, quartz or fused silicacells are used in this region. Standard path length of these cuvettes is usually 1 cm. However,cuvettes of path-length of I mm to 10 cm are available for special purposes. The surface of thecuvettes must be kept scrupulously clean; fingerprint smudges and traces ofprevious samples,by causing interference in the optical path, might cause serious errors in quantitative measurements. Rinsing with water should normally clean quartz or glass cuvettes. If, however,the dirt is abnormally tenacious, sulfonic detergents or nitric acid may be used. The use ofrectangular cuvettes in spectrophotometers effectively curtails the chances of dirt beingtransferred during handling. The two sides of such ceils through which the light passes areprecision ground and polished to be optically flat The other two sides are rough ground glassand the cell may be handled by these.Since most of the spectrophotometric studies are made in solutions, the solvents assumeprime importance. The most important factor in choosing the solvent is that the solvent shouldnot absorb (opt/ca//y transparent) in the same region as the solute. However, as we have discussedearlier, one should also take into consideration the effect that the solvent may have on the196Bophysical Chemistryabsorption spectrum of the solute. The solvents which can be used in the UV and visible regionare water, methyl-, ethyl-, isopropyl-alcohols, chloroform, hexane etc. Some often used solventswith their upper wavelength limit of absorption are given in Table 8.4.Table 8.4 Upper wavelength absorption limits of some solventsSolventUpper wavelength limit (nm)WaterEthanolButanoll-PropanolEthyl-etherIso-octaneHexaneCyclohexaneAcetonitrileMethanolDichloromethaneChloroformCarbon tetrachlorideBenzenePyridineAcetone20521021021021021021021021021523524526528O3O5330Infrared gas cells are made up of glass. These glass tubes possess NaCl, KBr, or CaF2windows for the passage of infrared radiation. The cells have varying path length; from a fewcentimeters to several meters, The latter is achieved by multiple reflections within the cell.Liquids are studied as thin films or solutions between NaCI, KBr, or CaF2 plates. The distancebetween the plates (path length) is usually 0.005 to 1 mm. Solids are examined in the infraredas pressed KBr discs or as suspensions in high molecular weight liquids ("mulls").Detection DevicesMost detectors depend on the photoelectric effect, where incident light (photons) liberateselectrons from a metal or other material surface. Some sort of external circuitry collects theseelectrons and measures their number as current. The current is then proportional to the lightintensity and therefore a measure of it. Important requirements for a detector include (i highsensitivity to allow the detection of low levels of radiant energy, (ii) short response time, (iii) longterm stability, and (/v) an electronic signal which is easily amplified for typical readout apparatus.Change in thermal energy, however, is the basis of detection for infrared radiation,Consequently detection devices used for this region are different than those operating in theultraviolet and visible regions.Ultraviolet and visible radiation detectors : There are three basic kinds of detectors in thisregion. Photocells, phototubes, and photomultiplier tubes.(i) Photovoltaic or barrier layer cells : It employs semiconductor materials.Semiconductors are crystalline, and the bonding electrons between the crystals of somesemiconductors can be knocked out of their positions by incident radiation. Although a numberof materials are used in photocells (cadmium sulphide, silicon, selenium) selenium basedphotocells are most common. A typical photocell consists of a thin coating of selenium over aSeleniumAnodeCathodeRecorderSpectrophotometry197thin transparent silver film on a steel base. This arrangement ensures that electrons passeasily from selenium to silver but not in the reverse direction. Due to the inability of electro ,ns tomove away from the silver film, the silver acts as the collecting electrode for electrons liberatedfrom selenium by the incident radiation. The steel plate functions as the other electrode. Thecurrent flowing between the two electrodes is then measured by a microammeter. A representativediagram of the cell is given in Figure 8.15.SilverSteel back-plateFigure 8.15 Selenium based photovoltalc cellPhotocells have a long life and are inexpensive and reliable. They are widely used incolorimeters but their use in spectrophotometers is becoming limited.(ll) Phototubes or photoemiasive tubes : The components Of a phototube include(A} anevacuated glass envelope (with a quartz window), (B) a semi-eylindrical cathode whose innersurface is coated with alkali or alkaline earth oxide, and (C) a centrally located metal wiremode. A potential difference of approximately 90 volts is applied across the electrodes. Thequartz window allows the passage of radiation which strikes the photoemissive surface of thecathode. The energy of the photon is transferred to the loosely bound electrons of the cathodesurface. The electrons become excited and finally leave the surface and travel towards theanode causing current to flow in the circuit, If the electron collection is 100% efficient, thephototube current should be proportional to the light intensity. A schematic diagram of aphototube and the associated circuitry is provided in Figure 8.16.Evacuated glassenvelopeI AnodeQuartzCathodewindowlgure 8.16 Dlram of a photoemislve the. R stands for res/stancePhototube currents are quite small and require amplification.This is usually accomplishedby placing a high resistance (R in Figure 8.16) in the phototube circuit.(|iI) Pht.multl[r : These detectors are designed to amplify the initial photoelectriceffect and are suitable for use at very low light intensities. A photomultiplier consists of (A) anevacuated glass tube into whlch are sealed the cathode and the anode, and (B) additionalIncidentradiation198Biophysical Chemtstrintervening electrodes known as dynodes. The arrangement is shown in Figure 8,17. The externalcircuitry is arranged so that a high voltage (1000 volts) exists between the anode and thecathode. As the radiation strikes the photocathode, electrons are liberated and the appliedpotential difference accelerates the electrons towards the first dynode. Each successive dynodeis at a higher electrical potential and thus acts as an amplification stage for the original photon.The applied voltage causes sufficient electron acceleration to knock out other electrons fromeach dynode surface. The liberated electrons are dragged onto the next dynode where moreelectrons are released and this process goes on as a cascade till the last dynode. By the time theelectrons arrive at the collecting anode, the initial photoelectric current is amplified by a factorof approximately 106. In practice, photomultiplier tubes are used only for low light intensities.At higher light intensities, due to their great amplification power, photomultipliers exhibit greatinstability. Inspire of this tendency to be unstable, photomultipliers are the detectors of choicein all modern spectrophotqmeters.PhotocathodeAnode-High voltageFigure. 8.17 A photomultiplier tube(iVl Phottditdes : Photodlodes are semiconductors that change their charged voltage(usually 5 VI upon being struck by Light. The voltage change is converted to current and ismeasured. A photodiode array is a two-dimensional matrix composed of hundreds of thinsemiconductors spaced very closely together. Light from the instrument is dispersed by eithera grating or a prism onto the photodiode array. Each position of diode on the array is calibratedto correspond to a specific wavelength. Each diode is scanned, and the resultant electronicchange is calculated to be proportional to absorption. The entire spectrum is essentially recordedwithin milliseconds.Near infrared detectors : These are usually photoconductive ceLls which detect infraredradiation in the range 0.8- 3.0 . The sensing element is a semiconductor (germanium,lead sulphide, or lead teLlurlde). Upon illumination with radiation of appropriate wavelength,the electrons of the semiconductor are raised to conduction bands. This causes a drop inelectrical resistance. Consequently, if a small voltage is applied, a large increase in current canbe noted. The resistance of the system is such that the current may be amplified and finallyindicated on a meter is recorded.Middle and far infrared detectors : When middle and far infrared photons are absorbed,their energies are convened to thermal energy leading to a rise in temperature. Obviously then,rapid response thermometers such as thermocouples, resistance thermometers (bolometers),and gas thermometers (pneumatic or Golay ceils) are used as detectors in this region.Thermocouples used in the infrared receivers typically consist of a blackened gold lead-teLluriummetal pin junction which develops a voltage that is temperature dependent. To avoid heat loss,the the.rmocoupl is usually enclosed in a shielded evacuated housing. This prevents errorcausing temperature fluctuations.VisibleGlass.DetectorWave number (cm-1)Source of radiationSpectrophotometry199Amplification and ReadoutRadiation detectors generate electronic signals which are proportional to the transmittedlight. These signals need to be translated into a form that is easy to interpret. This is accomplishedby using amplifiers, ammeters, potentiometers, and potentiometric recorders.Components of UV-visible and i.nfrared spectrophoometers have been summarized inTables 8.5 and 8.6 respectively.Table 8.5 A summary of components of spectrophotometers and colorimetersRegion of electromagnetic spectrumUltravioletRadiation sourceOptical systemMaterial used in theOptical systemHydrogen or deuterium lampPrism or diffraction grating, ora foreprism grating doublemonochromator.Quartz or fused silica,Tungsten filament lamp,carbon arc (less used)Tinted glass filters or interferencefiltersSample holdersQuartz or fused silica, rectangularcells.Round glass cells.Photovoltaic cell.DetectorPhotomultiplierTable 8.6 A summary of the components of infrared spectrophotometersRegion of electromagnetic spectrumNear-InfraredMid-InfraredFar-Infrared12,500,4000 200 , -10Tungsten filamentlampCoil of Nichromewire, or NernstGlower, or Globar.High pressuremercury arc lamp.Optical systemQuartz prisms or prismgrating double mono-chromatorDiffraction gratingswith a fore-prismmonochromator.diffraction gratings.Optical elements are made up of ionic crystalline materials like NaCI,KBr, CsBr, or KRS-5.Lead sulphide, orlead telluride photoconductivecells.Thermocouple, ther- Pneumatic or Golaymistor, or pyroele- cells, pr bolometers.ctric.lflecUngsector200Bphysol ChemtryDouble Beam OperationVoltage fluctuations inducing fluctuations in the source intensity can cause large scaleerrors in spectrophotometer operation. To obviate this situation, double beam spectro-photometers have been designed (Figure 8.18}. Double beam instruments employ some type ofbeam splitter prior to the sample containers. One of the split beams passes through the "blank"or reference cell while the other passes through the sample cell. The two transmitted beams arethen compared either continuously or alternately several times in a second. The double beamdevice, therefore, compensates for fluctuations in the source intensity, the detector signal, andamplifier gain by observing the differences in signal between reference and sample at virtuallythe same time. The modifications described above make the double beam devices moresophisticated mechanically and electronicaliy as compared to the single beam devices. Obviously,the double beam devices are expensive.Mirror ., ,,. MirrorBlankIIMiulflplieonoChopper pleOpen sectorRotating -sectorchopperFigure 8,18 Optical arrangement of a double-beam instrument. A rotaW sector chopper s also shown.The beam splitting usually occurs after the monochromator. Rotatingsector mirrors arecommonly used for splitting or *chopping" the beam (Figure 8,18). The chopped beams reachsample and reference and subsequently to the detector at intervals which depend upon therotational frequency of the chopper. The device then records the ratio of the reference andsample signals.Dual Wavelength SpectrophotometerSome metal chelators absorb at One wavelength before chelating the metal ion and absorbat a completely different wavelength after the chelation has taken place. An example is that ofarsenazo III, a calcium chelator. This chelator absorbs at 675 nm before binding to calcium andat 685 nm after the binding has taken place. If this chelator is incubated with a biologicalsystem and the absorbance of the chelator is measured simultaneously at the wavelength pair675 nm. 685 nm, the ratio of the two absorbances can provide an idea of the calciumconcentration in the given biological system. Similarly, in many reaction kinetic studies it isnecessary to monitor the absorbance changes of two chemical species simultaneously. In manyexperiments it is necessary to measure the relative absorbances of proteins {280 nm} andnucleic acids (254 nm} simultaneously. For all these experiments it is necessary to use dualwavelength spectrophotometer.Z2CuvetteSpectrophotometry201Dual wavelength spectrophotometry refers to the photometric measurement of a materialby passing radiation of two different wavelengths through the same sample before reaching thedetector. Light from two different sources is allowed to be resolved into two different wavelengthwith the help of a pair of diffraction gratings. Subsequently the two beams of different wavelengthsare made to pass through the same sample by a complex arrangement of a large number ofmirrors (Figure 8.19). Only a single detector, which is always a photomultiplier tube, is used.Light sourceMonochromatorMonochromatorFigure 8.19 Optical arrangement of a dual wavelength spectrophotometerFrom the above it is clear that dual wavelength spectrophotometry provides informationfrom two wavelengths per unit time. All other factors being equal, the resultant data should bemore useful than data from a double beam spectrophotometer.APPLICATIONS OF.UV-VIS SPECTROPHOTOMETRYPhotometry being a very versatile technique, has diverse applications, only a few of whichare summarized below.Qualitative AnalysisVisible and ultraviolet spectra may be used to identify classes of compounds in both thepure state and in biological preparations. This is usually done by plotting absorption spectrumcurves. Since these curves are specific for a class of compounds, a knowledge of the absorptionspectrum can help in identification of a substance in biological milieu. Table 8.3 providesinformation about the absorption ranges of the most commonly occurring functional groups ofbiomolecules.It is quite beyond the scope of this book to deal with the details of identification of anunknown compound or the assignment of structure on the basis of its absorption spectrum (fordetails the student is referred to literature cited at the end of this chapter). However, a briefdiscussion is possible. Before attempting to interpret the absorption spectrum of a givencompound, suffioient chemical information about the substance such as the elements presentshould be known. Absorption by a compound in different regions gives some hints of its structure.Thus, compounds which do not absorb in 220-280 nm region are usually aliphatic or alicyclichydrocarbons or their derivatives. Sometimes they might be simple olefinic compounds. If the202Biophysical Chemistrycompound absorbs between 220-250 nm range, it will usually contain two unsaturated linkagesin conjugation. Absorption in this range could also be due to benzene derivatives. Presence ofmore than two conjugated double bonds usually gives rise to absorption in the range 250-330nm. It is a fact that as the number of conjugated double bonds increases, the absorption rangeis shifted more and more to higher wavelengths. Thus, -carotene, a precursor of vitamin A haseleven double bonds in a conjugated system and appears yellow because the light in visibleregion (450-500 nm) is being absorbed by it. Complex systems will give rise to absorptioncurves with several maxima, each of them possessing a characteristic shape and range indicatingthe presence of the particular functional group.As an example to prove how valuable absorption data can be, one can cite the example ofvitamin K whose structure was determined by the use of its spectral data. The absorptionspectrum of vitamin K has the following absorption maxima: 249 nm, 260 nm, and 325 nm.The maxima around 250-330 nm are characteristic ofa naphthaquinone. Chemical manipulationand comparison of K spectra with several model compounds indicated that the positions of theabsorption maxima were similar to those of 2,3-dialkyl- 1, 4-naphthaquinone. The structure ofvitamin K determined later is as below:Also see Box 8.3 for further discussion.Abetter example, perhaps, is afforded by the four nucleotides (Figure 8.20). If you dissolvethe four nucleotides GMP, CMP, AMP, and UMP in aqueous solution at neutral pH, the absorptionmaximum at highest wavelength for GMP occurs at 255 nm, for CMP at 271 nm, for UMP at262 nm, and for AMP at 259 nm. With just these absorption maxima, one can tentativelyidentify GMP from CMP since their absorption maxima are quite removed from each other.However, it would be difficult to distinguish between AMP and UMP owing to their absorptionspectra being quite close. However, one can resort to changes in pH of these solutions and thedata so obtained can help us in identification. A look at Figure 8.20 and the text therein willexplain the point.'0pounds o,t,'Oc"09"0L'Og'Owv - , .7 6"0204Biophysical Chemistry205rise?behavior ofSpectrophotometrybe ,zerofthe pH is now raisedbeyondt 0.0, what do youare rosine.rise in absorption at295 nm will be seen because these tyros nes w I be sh elded, f only twotyrosines an; On the surface and two buried in the folds, riein absorpt on at 295nm w I onlybe hail of what is expected for four tyrosines. Inthe last Case if the pH is raised tosay 13.(,all tyrosines wi be available for titri0n becausethe protein is now denatured.largescale unfold ng of the proteinwere buried when the protein was in thesurface brings about, a change n theproteins: Whenever the substrate orthe competitive inhibitorregion or makes certain aminc acid residuesIn doing so, it might quite frequspectral changes areobtained with other stud es such as the so ventquite a bit of information about the structure of the active stem ght bestudies. Solventof lysozyme, one observesmaximum of tryptophan is shifted to a longer wavelength.pectral change corresponds with only one tryptophan residue.. If thesolution of the enzyme with the substrate is again studied by solvent perturbation method,only'three tryptophan residues seem to be on the surface. The conclusion is quite Obviousreally. It seems that the e.nzyme has one tryptophan residue in the active site which might beinvolved in the binding. Studies with x-ray diffraction confirm this conclusion.text, can you answer the followingquestions?(a)A protein solution is being heated and its spectral characteristics are being determined at250 nm (., for cysteine). At:45oc the extinction of the system suddenly increases. Thisincrease plateaus Out at 55oc. What conclusion should you draw?(b)A DNAsolution is being cooled after it was heated..Tle ext nction at 260 nm shows suddendrop. What happened?Wavelength206Bophystcal ChemistryQuantitative AnalysisIn developing a quantitative method for determining an unknown concentration of a givenspecies by absorption spectrometry, the first step is the choice of the absorption band at whichthe absorbance measurements are to be made. If the chemical species of interest has alreadybeen researched upon, its .ultravlolet/vlsible absorption spectrum would be available in theliterature. If this is not the case, the absorption spectrum of the chemical specie can be dctermcdexperimentally by means of a scanning double beam spectro-photometer. A suitable absorptionband is then selected from within the absorption spectrum for quantitative measurements.Absorptivity at any given wavelength is constant and is an inherent characteristic of the absorbingsubstance. Most of the organic compounds of biological interest absorb in the UV-visible rangeof the spectrum. Thus, a number oflmportant classes of biological compounds may be measuredsemi-quantitatively using UV-visible spectro-photometers. Nucleic acids at 254 run and prote/nat 280 nm provide good examples of such use. The absorbance at 280 um by proteins dependson their tyrosine and tryptophan content. All proteins will therefore have a different absorbanceat 280 nm and may be only accurately assayed if a calibration curve {see Box 8.2} is plotted forthe pure protein.What do we mean by selecting a suitable wavelength or alsorption band? There are certainrules for the choice. These may be summarized as under (Figure 8.21).1. Choose an absorption peak with the greatest possible molar absorptivity.2. Choose a relatively broad peak.3.Choose a peak that is as far as possible from the absorption peaks of commonlyinterfering chromogens. The same is true for solvents and other reagents used - theabsorption band chosen should be as far away as possible from the absorption peaksof the solvent and the reagents.Figure 8.21. Choice of the absorption band for quantitative analysts.X has a small absorption coefficient. Quantitative analysis should normally not be carriedout here. X has the highest absorption coefficient but the peak is too sharp. '3 has a sufficientlyhigh absorption coefficient (though lower than .) and the peak is broader than .. k3 thenbecomes the best choice. Of course, this must not correspond to absorption peaks of otherinterfering chromogens or the solvent.207ayThe quantitative assay of enzyme activity is carried out most quickly and convenientlythe substrate or the product is colored or absorbs light in the ultraviolet range becauseor disappearance of a light absorbing product or substrate can be followedwhich gives a continuous record of the progress of the reaction on achart recorder. Other light absorbing or light scattering substances must either be absentby appropriate blank measurements. Example can be cited of the measurementof the enzyme lactate dehydrogenase, which is engaged in the transfer of electrons lactate to NAD*. The products of the reaction are pyruvate, NADH, and a proton.Lactate + NAD - Pyruvate + NADH + HOne of products, NADH, absorbs radiation in the ultraviolet range at 340 nm while itsNAD, does not. No other component of the reaction, either substrate ortbsorbs at 340 rim. It is thus very obvious that the progress of the reaction in thellrection can be followed by measuring the increment in light absorption of the systemnm in a spectrophotometer.simplicity and convenience of optical assays prompts their use in following the timeof an enzymatic reaction in which neither the substrate(s) nor product(s) have anyabsorption maxima. Such type of enzyme reactions are coupled to some otherreaction which has an easily measured optical change. An example of the reactionhosphoenolpyruvate and ADP yielding pyruvate and ATP catalyzed by pyruvate kinasebelow.Phosphoenolpyruvate + ADP v- Pyruvate + ATPAlthough neither the substrates nor products of this reaction absorb light in the 300-400the reaction is easily measured if lactate dehydrogenase and NADH are added to thelarge excess. By manipulating the system in such a manner we obtain the followingI reactions, which have the common intermediate pyruvate:Phosphoenolpyruvate + ADP Pyruvate + ATPPyruvate + NA{)H + H -- Lactate + NAD Since we have added a large excess of NADH to the system, the system now absorbs at 340 But from the reactions written above it is clear that for each molecule of pyruvate formedfirst reaction, a molecule of NADH is oxidized to NAD in the second reaction when theconverts pyruvate to lactate. Since NAD does not absorb at 340 nm the absorbanceon decreasing with increased pyruvate generation. Such measurements are known asassays and are fairly routinely used.Weight DeterminationIf a compound forms a derivative ith a reagent which has a characteristic absorption' at a wavelength where the compound does not absorb, then the extinctionf the derivative is usually the same as that of the reagent. Although the extinctionof the absorption band remains constant in all the derivatives, the optical density,is different for compounds of different molecular weights. The molecular weight, M, of thebe readily calculated on the basis of its absorption data.M = awb/ODor M= 10a/E IL208Biophysical Chemistrywhere w is the weight of the compound in grams per litre, and b is the pathlength.Molecular weights of amine picrates, sugars, and many aldehyde and ketone compoundshave been determined by this method. The method has an accuracy of+ 2%. Molecular weights iof only small molecules may be determined by this method'.Study of Cis-Trans IsomerismSine geometrical isomers differ in spatial arrangement of groups ab.out a plane, theabsorption spectra of the isomers also differs. The trans-isomer is usually more elongated thanits cis counterpart. It is usual therefore, for the trans-isomer to have a higher wavelength ofmaximum absorption and also to have a higher ema' Absorption spectrometry can thus beutilized (indeed it has been) to study cis-trans isomerism.Other Physicochemical StudiesOver the years, spectrophotometry (UV-VIS) has been used to study such physicochemicalphenomena as heats of formation of molecular addition compounds and complexes in solution,.determination of empirical formulas, formation constants of complexes in solution, hydrationequilibria of carbonyl compounds, association constants of weak acids and bases in organicsolvents, tautomeric equilibria involving acid base systems, protein-dye interactions, chlorophyll-protein complexes, vitamin A aldehyde-protein complex, association of cyanine-dyes,determination of reaction rates, determination of labile intermediates, and dissociation constantsof acids and bases.Control of PurificationThis is one of the most important uses of UV-VIS spectrophotometry. Impurities in acompound can be detected very easily by spectrophotometric studies by experimentally verifyingwhether the given compound shows an absorption maxima not characteristic of it. Thus carbondisulphide impurity in carbon tetrac, hloride can be detected easily by measuring absorbance at318 nm where carbon disulphide absorbs. Similarly benzene impurity in commercial absolutealcohol can be detected by measuring absorbance at 280 nm where alcohol (210 nm) does notabsorb. A lot many commercial solutions are routinely tested for purity spectrophotometrically.Difference SpectroscopyDifference spectroscopy provides a sensitive method for detecting small changes in theenvironment of a chromophore. It may also be used to demonstrate ionization of a chromophoreleading to identification and quantitation of various components in a mixture. Differencespectroscopy involves comparison of absorption spectra of two samples which differ only slightlyin their physical states. The common features in the spectra cancel out and the bands whichare recorded can be interpreted in terms of known differences between the samples.Difference spectroscopy was developed by Chance and Williams in course of their researchon electron transport chain proteins in the mitochondria. The technique subsequently providedmuch needed information about the state and sequence of the electron transport proteins.Difference spectroscopy has also been utilized in toxicology laboratories for analysis of manytoxic drugs. Example can be cited of barbiturates which show characteristic changes in absorptionspectra between their keto and enol forms. Moreover, difference spectroscopy is a necess.arytool to study globular protein conformation.Turbiflimetry and NepheloraetryBacterial Or any other particulate suspension makes the liquid turbid. This is due toTyndall effect which addresses itself to light scattering by colloidal particles (see chapter 3).While. the liquid in this system might absorb at a particular wavelength, the particles scatterSpectrophotometry209the incident light. If, then, radiation of a wavelength which is not absorbed by the liquid ismade to pass through this suspension, the apparent absorption will be solely due to light scatteringby the particles. The light transmitted by the suspension will have lesser intensity than thelight which was incident. Measurement of the intensity of this transmiR, ed light will allow one tohave an idea of the number of particles in the suspension. Using this technique, known asturb/d/metry, one can arrive at a fair approximation of the number of particles in a givensuspension. This technique is routinely used to measure the number of bacteria in a givensuspension. The wavelength used for this purpose is 600 nm. The technique, however, is verytedious to standardize as the particle size is critical for accuracy (larger particles scatter morelight as compared to smaller particles; thus contamination of small particle suspension by asmall number of large particles will give a value far in excess of the number of small particlesactually present). The principle of turbidimetry is shown diagrammatically in Figure 8.22(A).Nephelometry is a term many times used synonymously with turbidimetry probably becausethe two techniques are based on a common principle. The major difference between turbidimetryand nephelometry is that while the former measures the intensity of transmitted light comingout of a suspension, the latter measure the intensity of the light scattered by the particles insuspension. The scattered intensity is usually measured at right angles to the direction ofincident light. For low concentration this method is more sensitive since zero concentration isrepresented by a dark background. On the other hand zero concentration in turbidimetry meansfull illumination. The principle of nephelometry is diagrammatically shown in Figure 8.22(B).Nephelometry is commonly used for estimating the concentration of microorganisms. It is alsoused for waste-water analysis as well as in the beverages and pharmaceutical industries toevaluate the amount of haze present in the preparations.Sample cellcontaining suspension] [ (A) f ,Photocell oLight Slit,Lenssource(B)ILens-Photocell detectorFigure 8.22 Principle of turbidimetry and nephelometry.(A) Light is scattered by particles in the suspension in sample cell. The transmitted light is therefore ofa weaker intensity. A turbidimeter measures the intensity of this transmitted light and a calibrationcw' beaueen transmitted intensity and particle concentration can be drawn.(B) Nepheiometry measures the intensity of scattered light rather than transmitted light.THEORY AND APPLICATIONS OF INFRARED SPECTROSCOPYAs evident from the electromagnetic spectrum diagram, infrared radiation is of muchhigher wavelength as compared to the ultraviolet and the visible region. Consequently,electromagnetic radiation of this region has considerably lower energy. Infrared radiation is,therefore, not associated with electronic transitions; rather, it is associated with vibrationaltransitions of molecules as we will see below.210In-plane deformationsScissor and Rock"NFSymmetrlcAntlsymmetrlcDFINOut-of-plane deformationsTwistand WagFigure 8.23 Types of vibrations : (A) Stretching, (B) BendingAll the molecules are continually vibrating. These vibrations are of two types. The bonddistances between the atoms in a molecule fluctuate to about 0.5A. It should be rememberedthat while this increase or decrease in bond length is occurring, the atoms remain in the samebond axis. These vibrations are known as stretching vibrations (Figure 8.23{A)). The other typeof molecular vibration, known as bending vibration (Figure 8.23(B)) involves changes in thepositions of the atoms with respect to the original bond axis. Such variations in bond anglesmay be about 0.5. Vibrational transitions are low energy transitions and these energy levelscorrespond to the energies of electromagnetic radiation in the infrared region of the spectrum.Consequently, a molecule can absorb infrared radiation of an appropriate frequency, accompaniedby promotion of the molecule to an excited vibrational state. The presentation of infrared spectradiffers from UV-visible spectra in that wave number is used in this region rather than wavelength;infrared spectra are typically presented as percent transmission (transmittance x 100) versuswave number.Cslculatlon of Vibrational FrequenciesThe vibrational frequency of a bond can be calculated with the help of Hooke's lawwhich correlates frequency with bond strength and atomic masses.VlOrV--,mass2n mim2/(m, +rni)where v is the frequency, k is the force constant of the bond and m and m2 are the masses ofthe two atoms involved in bond formation. The quantity mrn/(m+ m2) is often expressed as m,..the reduced mass of the system.Let's try and calculate the approximate frequency of the C--H stretching vibration fromthe data given below.k=5.0 x 10Sgs-2mass of carbon atom = 20 x 10-24.g211Specrophotorrtr5.0xlO5gs-272x22= 9.3x10as-Dividing the above value with the speed of light we get the value in wave number which is3100 cm-L When you experimentally determine the vibrational frequency for C--H stretchingyou find it ranging from 2800 to 3100 depending on the compound you choose. This simpleobservation tells us that the vibration of any given bond will be affected by the other atoms andtheir bonds that it coexists with.If you look at the above equation, it should be easy to surmise that the vibrational frequencyof a bond should increase with the strength of the bond. Also when the reduced mass of thesystem decreases, the frequency should increase.The foregoing may make us think that we can predict the frequencies of vibration for agiven bond. To an extent we can. For example, double bonds are stronger than the single. So wecan safely say that C--C should have a frequency lower than that of C---C. This we predicted onthe basis of the bond strength. On the basis of mass we may hazard a guess that C--H shouldabsorb at a higher frequency as compared to CmC since the latter has a higher reduced mass.However, we are stepping onto treacherous territory if we start predicting frequencies on thebasis of masses without knowing the force constant. One example will prove this. Given beloware a few bonds. Guess which of these bonds will absorb at the highest frequency solely on thebasis of the reduced masses.C--H ,. N--H, O--H, and F--H.Solely on the basis of masses your answer must have been that the absorption frequencyfalls along the series given. The actual observation is quite to the contrary. The frequencies risealong the series. They rise because all along the series the electronegativity rises and with itrises the force constant.Thus, although it is possible to predict the frequencies in a general manner taking thehelp of Hooke's law, some caution .must be exercised in doing so.Modes of VibrationThe theory of molecular vibrations predicts that an asymmetrical molecule which containsn atoms will have 3n - 6 modes of fundamental vibrations. This means that a molecule like CO2o should possess (3 x 3) - 6, i.e., 3 fundamental modes of vibrations. Likewise, methane shouldhave 9 and ethane 18.Figure 8.23 shows vibrational modes available for AX systems (any atom joined to twoother atoms, e.g., NH2, NO2, CH2 , etc.). Normally each vibration mode absorbs at a differentfrequency. Thus a CH group may give rise to two C--H stretch bands, symmetric and asymmetric.However, this is not always true. There will be some vibrations which may absorb at the samefrequency. Naturally their absorption bands will overlap. Such vibrations are called degenerate.Also, there are vibrations whose absorption frequency may lie outside the normal infraredregion examined.So far we have been talking about vibrations which are dubbed fundamental. There areother frequencies at which bands appear in.an infrared absorption spectrum. Some of thesebands ar