2 Physical Methods and Techniques Part (iv)Infrared and Raman Spectroscopy By G.A. NEWMAN Research Division Kodak Ltd. Harrow Middx. HA I 4 TY 1 htroduction It is surprising that this topic has never been reviewed in Anlsual Reports (B). This Report is concerned with highlights in infrared and Raman spectroscopy and their application to organic chemistry with the exclusion of metal-organic compounds over the years 1971-5. It is divided into separate sections on infrared spectroscopy and Ramar; spectroscopy the latter covering the Raman and resonance Raman effects only. It is not intended to be comprehensive. Where appropriate the relevant review papers are cited but workers should also consult the SpeciaIist Periodical Reports.' The references cited are where possible the most recent provided they contain therein key referencesto provide the readerwith retrospective coverage.2 Infrared Spectroscopy Remarkably infrared (i.r.) spectroscopy shows no sign of reaching a senescent phase. At a time when other spectroscopic techniquesare advancing,particularlyin areas of structural determination and analysis previously dominated by ir* the technique shows no sign of decline. The number of publications increased by nearly 20%in 1972-3. Excellent comprehensivereviewshave been published bi-annuaHy by McDonald;' the latest covering 1972-3 contained 856 references. Twogrowth areas are those applicationsmade possible when spectrometers are interfaced with computers and also the field ofenvironmental protection and control.To appreciate these recent developments advances in instrumentation are described. Instrumentation.-A clear indication of the state of health of i,r. spectroscopy is evident from the wide range of conventiona1 (dispersive) spectrophotometers available. These have been listed together with recent trends in de~ign.~ There is a tendency towards more automation and therefore simplicity of operation and the widespread provision of ordinate scale expansion(%T)of up to x 20 enables results to be obtained more easily in energy-limited situations such as microsampIing. 'MolecularSpectroscopy',ed. R. F.Barrow D. A. Long and D. J Millen (SpecialistPeriodical Reports), The ChemicaI Society London,1975 VoI. 3. R. S.McDonald Analyr. Chern. 1974,46,52lR.3 1. A. Degen and G. A. Newman Lab. *actice 1974,23,583,and 1976 in the press. 29 30 G.A.Newman In Fourier4 and Hadamard5,6 transform methods major advances have been made in instrumentation. In dispersive instruments a great loss in energy occurs when the sample and reference beams pass through entrance and exit slits. Both of the transform methods avoid or modify this approach. Furthermore since all wavelengths are detected simultaneously interferograms of the sample are collected rapidly. These two advantages are utilized in applications. A computer is essential to perform a Fourier transform (FT) on the interferogram; consequently many applications have appeared from users of FT instrument^.^ Griffiths has written a book on chemical i.r..FT spectroscopy.’ Although an interferometer is not essential many applications are difficult if not impossible without a computer.In micro~ampling~ the energy advantage is used together with the ability of the computer to store a spectrum and rapidly to multiscan the spectrum in the signal- averaging mode (CATting) so common in n.m.r. spectroscopy. It enables i.r. spectroscopy to break the 1pg barrier spectra having been obtained from samples as small as tens of nanograms! Work has also been reported on microsampling with dispersive Here the limit is about 10 pg and use is made of beam condensers and ordinate scale expansion. Methods of identification of microquan-tities have been reviewed,12 although the coverage of references is not as up-to-date as one would like.These advances find application in the identification of products of small-scale syntheses such as electrochemical or photochemical. Microsampling methods are applicable to environmental problems. The detection of small amounts of mineral oil in both sea-water and fresh water has been widely Even with the use of dispersive spectrometers the i.r. method is very good for this (sub-p.p.m.). Hydrocarbons in water are extracted with a halogenated solvent; a freon is preferred to. the more toxic carbon tetra~hloride.’~*~~ Long-path-length liquid cells are used in the determination. The results of g.c. and i.r. methods have been compared.16 Brown and co-workers” have used i.r. spectra as a fingerprint for the identification of the sources of oil slicks.Pierre” has pursued an on-site method for this. Atmospheric pollution has been studied with FT i.r. in~trument~’~~~~ or a fast-scanning dispersive instrument.21 Hanst Lefohn and Gay” detected 18 gases in the atmosphere at the one part per American billion level. They developed multiple-pass gas cells operated at typical path lengths of 200 4 P. R. Griffiths Analyt. Chem. l974,46,645A. 5 J. A. Decker jun. Analyt. Chem. 1972,44 127A. 6 A. G. Marshall and M. B. Comisarow Analyt. Chem. 1975,47 491A. J. L. Koenig Appl. Spectroscopy 1975,29,293. * P. R. Griffiths ‘Chemical Infrared Fourier Transform Spectroscopy’ Wiley-Interscience Chichester Sussex 1975. 9 P. R. Grif€iths and F. Block Appl. Spectroscopy 1973,27,431. 10 M. Yu. Nefedova Zhur.priklad. Spektroskopii 1974,20 664. 11 R. C. Blinn Adv. Chem.Ser. 1971 No. 104 p. 81. 12 G. M. Ayling ‘Spectroscopic Methods of Identification of Microquantities of Organic Materials’ in ‘Applied Spectroscopy Reviews’ ed. E. G. Brame jun. Dekker New York 1974,Vol. 8,Part A,p. 63. 13 D. R. Hughes R. S. Belcher and E. J. OBrien Bull. Environ. Contam. Toxicoi. 1973,10 170. l4 M. Gruenfeld Environ. Sci. Technof. 1973,7,636. H. B. Mark jun. T.-C. Yu,J. S. Mattson and R. L. Kolpack Environ. Sci. Technol. 1972,6 833. l6 R. Jeltes and W. A. M. Den Tonkelaar Water Res. 1972,6 271. P. F. Lynch S.-Y.Tang and C. W. Brown Analyt. Chem.,1975,47 1696. L. J. Pierre jun. Appl. Optics 1973,12,2035. l9 P. L. Hanst A. S. Lefohn and B.W. Gay jun. Appl. Specfroscopy 1973,27 188.2o S. H. Chan D. Nelson M. J. D. Low,and H. Mark J. Quant. Spectroscopy Radiative Transfer 1974,14 287. 2’ J. R. Combertiati Analyt. Chem. 1971,43 1497. Part (iv) Infrared and Raman Spectroscopy 31 and 700 m and used spectral subtraction of a reference air background. A portable single-beam i.r. gas analyser is used for field work this apparatus having proved useful in the determination of vinyl chloride at the p.p.m. There has been considerable activity in the use of combined techniques such as g.c.-i.r. An FT instrument is advantageou~~~ because the rapid rate of accumulation of data from a multiplex instrument enables the g.c.-separated components to be measured directly (known as ‘on-the-fly’!). A gas chromatograph has been linked to a fast-scanning dispersive spectrometer.Other workers favour trapping of the components in a vapour-phase i.r. cell.24 Ths use of i.r. spectroscopy in identifying fractions separated by means of t.l.~.~’ and h.p.l.c.26 has wide interest. Polymers.-The application of i.r. spectroscopy to polymer chemistry continues its popularity. Numerous papers are concerned with molecular orientation and confor- mation and in copolymers the stereoregularity of units and determination of composition. Two results are highly significant. Koenig and co-workers have used an FT instrument to compute the i.r. spectrum of 100°/~crystalline truns-1,4-poly~hloroprene.~~ Samples of 100%amorphous material exist in the molten state and a mixture of amorphous and crystalline material exists at room temperature and the spectrum was recorded in both states.By means of computer subtraction of one spectrum from the other the spectrum of 100%crystalline polychloroprene was simulated although a sample was not available. Koenig7 has written an excellent feature article on the applications of data-manipulated i.r. spectroscopy to chemical systems. Numerous interesting polymer applications were indicated and the differ- ence spectroscopy of plasticized PVCappears to be a method of direct determination of percentage plasticizer which has great application in industry. Miscellaneous Applications and Interpretation.-The use of a computer evables the i.r. spectra of aqueous solutions to be obtained much more rapidly. Even 1% transmission is sufficient to accumulate and replot the data full scale.This could cause a resurgence in the i.r. spectroscopy of biochemical systems on the scale which has occurred in Raman spectroscopy. Parker2* has reviewed the current status of biochemical applications of vibrational spectroscopy and Morton’s books2’ also contain references to this. Douse and Tooke3’ have characterized over 200 amines from the spectra of their hydrochlorides and Doss3lclaims that a band in the region of 1270-1291 cm-’is characteristic of sultam rings (1). 22 ‘The Determination of Vinyl Chloride a Plant Manual’ ed. W. Thain Chemical Industries Association 1974. 23 K. L. Kizer Amer. Lab. 1973,5 No.6 p. 40. 24 R. F. Brady jun. Analyr. Ckrn. 1975,47 1425. z5 R.Kellner Mikrochim. Acta 1975 253. 26 A. A. Juhasz J. 0.Doali and J. J. Rmhio Amer. Lab. 1974,6 No. 2,23. 27 M. M. Coleman P. C. Painter D. L. Tabb and J. L. Koenig J. PolymerSci. PartB PolymerLetters 1974 12 577. 28 F. S. Parker Appl. Spectroscopy 1975,29 129. 29 R. A. Morton ‘Biochemical Spectroscopy’ Vols. 1 and 2 Halstead Press Wiley New York 1975. 30 C. S. Douse and P.‘B. Tooke Canad. Spectroscopy 1973,18,101. 31 S. H. Doss Rev. Roumaine Chim. 1972,17 1611. G.A.Newman R’ (1) R’ R2,R3 R4 = alkyl The use of audiovisual aids for the interpretation of spectra is a useful advance there being several systems commercially available. As Bellamy has remarked there are no new interpretations to report; much of the information published is an extension or refinement of previously established work.Nevertheless it is pleasing to report that the third edition of his book to which so many of us are indebted has appeared.32 3 Raman Spectroscopy Raman spectroscopy is attempting to consolidate its position since the late 1960’s as an established structure-characterization technique. The literature is prolific and several reviews have a~peared.~~-~~ The bi-annual reports in Analytical Chemistry are the latest one covering 1972-3 (348 references). Sh~ader~~ has reviewed chemical applications (355 references) and numerous books are available.3740 Instrumentation and Sampling.-There have been no major changes in instrumentation. The triple monochromator is widely used to discriminate weak Raman scatter from stray light and coherent scattering effects.The rotating sample holder has been very This is used to study coloured solids and liquids. Such compounds tend to be destroyed by the laser beam but this is reduced by spinning the sample at about 2500r.p.m. Results have been obtained from azo- compounds coloured dyes and even black compounds. This sampling method enabled extensive studies to be made of the resonance Raman effect (RRE). The RRE has been described by Bern~tein.~~ It is obtained when a sample is exposed to laser radiation of energy almost coincidental with the energy spacing of an electronic absorption band. The corresponding spectrum may exhibit either intensity enhance- ment of some bands relative to others or overall intensity enhancement of all bands.This enables weak solutions to be studied such as those of biological importance. 32 L. J. Bellamy ‘Infrared Spectra of Complex Molecules’ Vol. 1 3rd Edn. Chapman and Hall London 1975. 33 W. L. Grossman Analyt. Chem. 1974,46,345R. 34 B. Schrader Ber. Bunsengesellschaft phys. Chem. 1974,75 1187. 35 M. Delhaye and J. C. Merlin Biochimie 1975 57 401. 36 B. Schrader Angew. Chem. Infernat. Edn. 1973,12 884. 37 ‘Advancesin Infrared and Raman Spectroscopy’ ed. R. J. H. Clark and R. E. Hester Heyden London 1975 Vol. 1. 3* R. A. Nyquist and R. 0.Kagel ‘Infrared and Raman Spectroscopy of Organic Materials’ in ‘Practical Spectroscopy’ Vol. 1,ed. E. G. Brarne and J. S. Grasselli Dekker New York 1975. 39 ‘The Raman Effect’ Vol.2 ‘Applications’ ed. A. Anderson Dekker New York 1973. 40 W. Kemp ‘Organic Spectroscopy’ Macmillan London 1975. 41 H. J. Sloane and R. B. Cook,Appl. Spectroscopy 1972,26 589. 42 H. J. Bernstein in ‘Advances in Raman Spectroscopy’ ed. J. P. Mathieu Heyden London 1973 Vol. 1 p. 305. Part (iv) Infrared and Raman Spectroscopy 33 The RR spectra of ferrocytochrome c and oxyhaemoglobin solutions have been at solute concentrations approximately three orders of magnitude lower than those required normally. Kiefer44 has reviewed the RRE results on small molecules. A great obstacle to widespread use of Raman spectroscopy is its inability to combat luminescence observed as a broad intense band which often obscures Raman shifts. Two new approaches to reduce or eliminate this effect have been reported.Shriver and co-w~rkers~~ use a mode-locked tuneable pulsed laser and a triggered gate mechanism to reject much of the luminescent emission. This depends on the difference in time-scale between the Raman scatter which is essentially instantaneous and the luminescence which requires a longer time to build up. The other method is coherent anti-Stokes Raman Spectroscopy (CARS).46 A pulsed laser is frequency-doubled in order to pump a dye laser. The sample is pumped to an excited state by the dye laser and irradiated by the pulsed laser as it might be in conventional Raman spectroscopy. By populating the higher-energy levels the anti-Stokes Raman spectrum i.e. bands at shorter wavelength than the exciting line may be recorded and luminescence is less troublesome.Applications.-Raman activity tends to be a function of the covalent character of bonds; the vibrations of non-polar groups and molecular skeletons (backbone structure). Totally symmetric vibrations are strong since they modulate strongly the polarizability of the molecule. Generally vibrations which are strongly i.r.-active are weakly Raman-active and vice versa Very small amounts of sample may be examined for example single beads of polystyrene 40pm in diameter47 and ‘nibs’ (microscopic defects) in plastic films. The identification of trapped g.c. fractions is a routine method. Since the intensities of the Raman shifts are proportional to concentration the technique is directly quantitative.The level of pollutants in water has been deter- mined from the intensity of the C-H stretching vibrations of the total organic content.48 Interpretation.-The appearance of two books which aid interpretation has been welcomed. Freeman’s book49 contains applications of Raman spectroscopy as well as group frequency assignments. The one by Dollish Fateley and Bentley” is already a standard text on characteristic frequencies of organic compounds. The number of methyl groups bound to ethylenic groups in di- and tri-substituted aliphatic and alicyclic molecules may be deterrnir~ed.~’ A characteristic band for the thiomethyl group5* has been found near 690 cm-’. A group-frequency correlation 43 G. J. Thomas jun. in ‘Vibrational Spectra and Structure A Series of Advances’ ed.J. R. Durig Vol. 3 Dekker New York,1975. 44 W. Kiefer Appl. Spectroscopy 1974,211 115. 45 R. P. Van Duyne D. L. Jeanmaire and D. F. Shriver Analyt. Chern. 1974,46,213. 46 R. F. Begley A. B. Harvey R. L. Beyer and B. S. Hudson Appl. Phys. Letters 1974,25 387. 47 G. J. Rosasco E. S. Etz and W. A. Cassatt Appl. Spectroscopy 1975,29 396. 48 G. Breunlich G. Games and M. S. Petty Water Res. 1973,7 1643. 49 S. K. Freeman ‘Applications of Laser Raman Spectroscopy’ Wiley-Interscience Chichester Sussex 1974. F. R. Dollish W. G. Fateley and F. F. Bentley ‘Characteristic Raman Frequencies of Organic Compounds,’ Wiley-Interscience Chichester Sussex 1974. 51 S. K. Freeman and D. W. Mayo Appl. Spectroscopy 1972,26 543.52 S. K. Freeman and D. W. Mayo Appl. Spectroscopy 1973,27 286. 34 G.A.Newman of pyra~ines~~ was established from a study of 32 compounds. Schrader and Meier54 have produced a scheme for characterizing some substituted benzenes that is more reliable than the corresponding i.r. method. Collections of spectra are always useful. A book of assignments for the vibrational spectra of 700 benzene derivatives has been published.ss A Sadtler collection totalling 2000 spectra has been produced together with the i.r. spectra in a continuing series.s6 An atlass7 is available of the Raman i.r. and far-i.r. spectra of 1000 compounds. The identification of unknown compounds may be achieved by means of a computer search pr~gram.’~ OrganicCompounds.-The coverage of aliphatic compounds has been fairly random except for n-alkanes which has been an active area.The interest here has been in the low-frequency vibrations of the solids particularly the determination of the lon- gitudinal acoustic (LA or accordion) modes9 of the carbon backbone (2) which enables each homologue to be identified. Similarly the chain length may be determined in some fatty acids.60 This has been used for the qualitative and quantitative analysis of mixtures of acids in the range C1z-Cz4. 2495*86 (2) LAmodeAv=-cm n n =number of carbon atoms Aromatic alicyclic and heterocyclic compounds have been extensively studied but there are no known systematic collations of such work. Polymers.-This has been an active area with the emphasis of study shifting from synthetic polymers to natural polymers particularly those of biological importance.Hendra has reviewed the field.61 Compared with other spectroscopic techniques the method of direct sampling is attractive. For certain functional groups the Raman effect is strong but it also has the ability to detect slight changes in the skeletal backbone of the polymer. These can be interpreted in terms of chain conformation. In polymers with unsaturation geometrical isomerization is of interest. Stereoregu- larity may be determined. This is important because of the greatly improved physical and chemical properties which are dependent on the regular geometrical arrange- ment of molecular structure. 53 R. P. Oertel and D. V. Myhre Analyt.Chem.,1972 44 1589. 54 B. Schrader and W. Meier 2.analyt. Chem. 1972,260,248. 55 G. Varsanyi ‘Assignments for Vibrational Spectra of Seven Hundred Benzene Derivatives’ Vols. 1 and 2 Akademiai Kiado Budapest and Adam Hilger London 1974. 56 Sadtler Standard Spectra-Raman Spectra Vols. 1-5,1974-5 Heyden London. 5’ B. Schrader and W. Meier ‘Raman/IR Atlas of Organic Compounds’ Verlag Chemie Weinheim West Germany 1974. 58 I. A. Degen L. Birmingham and G. A. Newman Analyst 1976,101,212. 59 H. Takeuchi T. Shimanouchi M. Tasumi G. Vergoten and G. Fleury Gem. Phys. Letters 1974,28 449. 60 C. H. Warren and D. L. Hooper Canad.J. Chem. 1973,51,3901. P. J. Hendra in ‘Polymer Spectroscopy’ ed. D. 0.Hummel Verlag Chemie Weinheim West Germany 1974. Part (iv)Infrared and Raman Spectroscopy 35 Pol ye thylene despite its structural simplicity has been subjectto much discussion of its vibrational assignments.These can be used to determine the state of crystallinity. The LA mode similar to that found in n-alkanes was first found in highly crystalline materials. More recently the variation in LA mode with bulk sample crystallinity and annealing has been st~died.~'.~~ The long polyethylene chain is directed roughly parallel to the lamellar surface and folds back and forth many times. The nature of the chain folds is of current intere~t.~~ Polypropylene is interesting because of its tacticity; it occurs in three isomeric forms of stereoregular-ity of which the isotactic and syndiotactic structures assume planar and helical conformations re~pectively.~~ The spectrum of the atactic form of disordered conformation remains to be unravelled.The vibrational spectrum of syndiotactic PVChasbeenreported.66 Biochemicals and Biopo1ymers.-This is probably the most rapidly growing and exciting area of application. Molecular structure may be studied in H20or D20and over a range of pH's or pD's. New information much ofit conformational has been obtained from amino-acids lipids carbohydrates,6* steroids and nudeic acids. Some extensive general reviews have appeared.43*69,70 Much interest in amino-acids is centred around the assignment of Raman-active vibrations to certain functional group motions particulary the amide groups. The various amino-acids are recognized by the vibrations of their side chains.This knowledge has been applied to the study of proteins and polypeptides. Their structures are built up from information concerning the peptide and amino-acid side-chain residues derived from the assignment of spectral bands their intensities and polarizabilities and comparison with model compounds such as poly-a -amino-acids. In gelatin amino-acids such as poly-L-proline and poly-L-hydroxyproline and their relative composi-tions have been identified.71 In the spectra of collagen and gelatin use was made of the amide I bands (1668 cm-') and amide 111bands (1271and 1248cm-') to deduce possible arrangements of the molecules. The amide I band is due predominantIy to C=O stretching and the amide III band to C-N stretching and NH in-plane bending in the polypeptide backbone.In particular the number and positions of the amide III bands may be interpreted in terms of conformational structure of proteins (Table).72 Much workhas been reported on the diflerences in spectra between native and denatured proteins such as insulin. 62 H. G. Olf A. PeterIin and W. L. Peticolas J. Polymer Sci. Part A-2 Polymer Phys. 1974,12 359. 63 J. L. Kuenig and D.L. Tabb J. Mucromol. Sci.,1974,9 141. 454 M.3. Folkes A. Keller,J. Stejny P.L. Goggin G. V. Fraser andP.J. Hendra Kulloid-2. Polymere 1975 253 354. 65 R.T. Bailey A. J. Hyde and J. J. Kim Spectrochim. Actq 1974,30A,91. 66 W. M. Moore and S. Krimm Makromol. Chern. 1975 Supp!. I 491. 67 J. L. Koenig and B. G. Frushour in 'Advances in Infrared and Raman Spectroscopy',ed.R. J. H. Clark and R. E. Hester Heyden London 1975 Vol. 1. 6% J. J. Caei K.H. Gardner J. L. Koenig and J. BIackwelI 1Chem. Phys. 1975,62,1145. 69 'Chemical and Biochemical Applications ofLasers' Vol. 1,ed. C. 3.Moore Academic Press New York 1974. 70 W. L. Peticolas Biochimie 1975,57,417. 71 0. G. Frushour and J. L. Koenig Biopolymers 1975,14,379. 72 M. C. Chen and R. C. Lord J.Arner. am.Soc. 1974,96,4750. G.A.Newman Table Vibration wavenumbers/cm-’ and conformations of sume proteins Conformation Amide I Amide III a-Helix 1660 1265-1300 Random coil (H-bonded)Antiparallel p -structure 1665 1672 1243-1253 1229-1235 Some significant advances have been made in the study of the very complex nucleic The primary structures of the constituents are elucidated by the identifica- tion of bases such as purine pyrimidine adenine uracil guanine and cytosine and their tautorneric equilibria.Base and sugar residues and ribose phosphate diester backbones have been identified in the spectra. The secondary structural investiga-tions involve weak hydrogen-bonding between the bases (70-1 25 cm-’) base-stacking interaction and backbone conformation. All of the prominent bands in the spectrum of aqueous yeast phenylalanyl transfer RNA (tRNg::sJ have been assigned. Comparison has been made with the spectrum of tRNA~,~oii in terms of similar conformation but with the absence of a pyrimidine base in tRNAz:dp74 The RRE has been utilized in coloured systems such as plant and respiratory pigments visual pigments enzymes vitamin derivatives metal porphyrins and haemoglobin~.’~ Outstanding examples are the This work has been re~iewed.’~ spectra obtained from live carrot root and live tomato fruit which are similar to the spectra obtained from canned carrot juice and bottled tomato sauce.In turn these spectra are similar to those of @-caroteneand lycopene respectively examined in n-he~ane.~~ 73 G.J. Thomas,jun.,Conferenceon ‘ImpactofLasers in Spectroscopy’ Prm.Soc.Pholo-opfkd1nstrurnen-tation Engineers 1974,49 127. 74 M.C. Chen and G. J. Thomas jun. Biopolymers 1974,13,615. 7s T. G. Spiro Accounts Chem. Res. 1974,7 339. 76 T. G.Spiroand T. M. Loehr ref. 37 chapter on ‘Resonance Raman Spectra of Heme Proteinsand other Biological Systems’. 77 L. Rimai D. Gill and J. L. Parsons J. Amer. Chem. Soc. 1971 93 1359.