APPLICATIONS OF

INFRARED SPECTROSCOPY
IN BIOCHEMISTRY, BIOLOGY,
AND MEDICINE
Applications of

INFRARED
SPECTROSCOPY
IN BIOCHEMISTRY, BIOLOGY,
AND MEDICINE

FRANK S. PARKER
Department of Biochemistry
New York Medical College
New York, New York

9? PLENUM PRESS • NEW YORK. 1971

Library of Congress Catalog Card Number 74-157930
ISBN-13: 978-1-4684-1874-3 e-ISBN-13: 978-1-4684-1872-9
DOl: 10.1007/978-1-4684-1872-9

© 1971 Plenum Press, New York

Softcover reprint of the hardcover 1st edition 1971

A Division of Plenum Publishing Corporation

227 West 17th Street, New York, N.Y. 10011

All rights reserved

No part of this publication may be reproduced in any form

without written permission from the publisher
 To my wife, Gladys,
whose love and devotion helped to write this book,
and to my children, Judith and George
PREFACE

This book is not intended to be a basic text in infrared spectroscopy. Many such books
exist and I have referred to them in the text. Rather, I have tried to find applications
that would be interesting to a variety of people: advanced undergraduate chemistry
students, graduate students and research workers in several disciplines, spectros-
copists, and physicians active in research or in the practice of medicine. With this
aim in mind there was no intent to have exhaustive coverage of the literature.
I should like to acknowledge my use of several books and reviews, which were
invaluable in my search for material:
G. H. Beaven, E. A. Johnson, H. A. Willis and R. G. 1. Miller, Molecular Spec-
troscopy, Heywood and Company, Ltd., London, 1961.
J. A. Schell man and Charlotte Schellman, 'The Conformation of Polypeptide
Chains in Proteins," in The Proteins, Vol. II, 2nd Ed. (H. Neurath, ed.), Academic
Press, New York, 1964.
R. T. O'Connor, "Application of Infrared Spectrophotometry to Fatty Acid
Derivatives," J. Am. Oil Chemists' Soc. 33, 1 (1956).
F. L. Kauffman, "Infrared Spectroscopy of Fats and Oils," J. Am. Oil Chemists'
Soc. 41,4 (1964).
W. J. Potts, Jr., Chemical Infrared Spectroscopy, Vol. I, Techniques, Wiley,
New York, 1963.
R. S. Tipson, Infrared Spectroscopy of Carbohydrates, National Bureau of
Standards Monograph llO, Washington, D.C., 1968.
C. N. R. Rao, Chemical Applications of Infrared Spectroscopy, Academic Press,
New York, 1963.
H. R. Mahler and E. H. Cordes, Biological Chemistry, Harper and Row, New
York, 1966.
T. R. Kasturi, "Steroids," in C. N. R. Rao, Chemical Applications of Infrared
Spectroscopy, Academic Press, New York, 1963, p. 403.
R. I. Dorfman and F. Ungar, Metabolism of Steroid Hormones, Academic Press,
New York, 1965.
R. N. Jones and C. Sandorfy, "The Application of Infrared and Raman Spectro-
metry to the Elucidation of Molecular Structure," in Chemical Applications of
Spectroscopy (W. West, ed.), Interscience Publishers, New York, 1956, p. 247.
S. Schwartz, M. H. Berg, I. Bossenmaier and H. Dinsmore, "Determination of
Porphyrins in Biological Materials," Methods Biochem. Anal. 8, 221 (1960).
vii
viii Preface

M. Tsuboi, "Application of Infrared Spectroscopy to Structure Studies of

Nucleic Acids," Appl. Spectrosc. Rev. 3, 45 (1969).
M. Tichy, "The Determination ofIntramolecular Hydrogen Bonding by Infrared
Spectroscopy and Its Applications in Stereochemistry," in Advances in Organic
Chemistry, Methods and Results, Vol. 5 (R. A. Raphael, E. C. Taylor and H. Wynberg,
eds.), Interscience Publishers, New York, 1965, p. 115.
R. D. Stewart and D. S. Erley, "Detection of Volatile Organic Compounds and
Toxic Gases in Humans by Rapid Infrared Techniques," in Progress in Chemical
Toxicology, Vol. 2 (A. Stolman, ed.), Academic Press, New York, 1965, p. 183.
J. J. Katz, R. C. Dougherty, and L. J. Boucher, "Infrared and Nuclear Magnetic
Resonance Spectroscopy of Chlorophyll," in The Chlorophylls (L. P. Vernon and
G. Seely, eds.), Academic Press, New York, 1966, p. 185.
W. P. Jencks, C. Moore, F. Perini, and J. Roberts, "Infrared Spectra of Activated
Acyl Groups in Deuterium Oxide Solution," Arch. Biochem. Biophys. 88,193 (1960).
M. Jacobson, Insect Sex Attractants, Interscience Publishers, New York, 1965.
R. A. Kekwick, ed., 'The Separation of Biological Materials," British Medical
Bulletin 22, 103 (1966).
Besides the titles listed above are all the papers, brochures, books, etc., which I
have referred to in the various chapters. It is a pleasure to thank all those authors who
kindly sent me reprints of their papers.
F.S.P.
ACKNOWLEDGMENTS

I appreciate very much the help given to me by many friends and associates during the
preparation of this book. Dr. K. R. Bhaskar, Dr. Irving Rappaport, and Martin
Stryker have read various chapters critically and have made valuable suggestions.
My colleagues on the staff of the Department of Biochemistry have been very co-
operative. Marji Gold, Randy Mound, George Parker, and Nancy Rodriguez have
assisted in various ways. I thank Juanita O'Brien and Ruth Prunty for their readiness
to help at all times, and want especially to express my appreciation to Anne Zver
for her willing cooperation and exceptional typing throughout all phases of the
writing of the manuscript. Also, I want to thank the staff ofthe Lillian Morgan Hetrick
Library of New York Medical College for their help.
To two other persons whose help in the past years has been very meaningful to
me, I give my sincere thanks: Dr. J. Logan Irvin, for introducing me in his kind and
thorough way to spectroscopy; and Dr. Murray Lieberman, for his assistance in
many ways, including the writing of this book.
It is a pleasure to acknowledge here the various research grants I have received
from the United States Public Health Service, National Institutes of Health. I ap-
preciate also the support of my research and a Career Scientist Award from the
Health Research Council of the City of New York.
F.S.P.

ix
CONTENTS

1. Introduction and Brief Theory ..................................... .

Theory (1), Wave Number and Wavelength Units (2), The Double-Beam
Recording Spectrophotometer (2), The Infrared Spectrum (4), Calibration
of Wave Number and Wavelength (5), Examples of the Interpretation of
Spectra (7), Spurious bands (20), References (24)

2. Near-Infrared Spectroscopy. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 25
Introduction (25), Applications (26), References (39)

3. Sampling Methods. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 41

Solids (41), Solution Techniques (46), Calibration of Infrared Absorption

Cells (56), Cells for Gases (57), Micromethods (58), Low-Temperature Work
(61), Attenuated Total Reflection (70), The Use of Polarized Infrared
Radiation and the Measurement of Dichroism (73), References, (76)

4. Quantitative Analysis. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 80
References (83)

5. Hydrogen Bonding. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 84
Introduction (84), Intramolecular Hydrogen Bonding (86), Intermolecular
Hydrogen Bonding (91), References (98)

6. Carbohydrates .................................................... 100

Comparison of Samples (100), Interpretation of Spectra (100), Group
Frequencies in Carbohydrates and Their Derivatives (101), C-H Bands:
Stretching and Deformation Frequencies (106), N -H Bands: Stretching
and Deformation Frequencies (107), O-H Bands (108), C-C and C=C
Bands (110), C N, C=N, and C-N Bands (110), C=O Bands (111),
C-O Bands (113), N N, N=N, and N0 2 Bands (113), S=O, -S02-'
and C=S Bands (113), Correlations for the Fingerprint Region and Beyond
(l14), Conformational Studies (119), Additional Examples of Applications
xi
xii Contents

of Infrared Spectra to Carbohydrates (125), Specialized Techniques (125),

References (138)

7. Lipids ........................................................... 142

Introduction (142), Fatty Acids and Esters (149), Fatty Amides (152),
Ethers, Esters, and Ether Esters of Glycerol and Various Diols (152),
Glycerides and Cholesterol Esters (152), Lecithins, Cephalins, and Phospho-
lipids (154), Phosphonic Acids, Phospho nates, and Phosphonolipids (157),
Plasmalogens (158), Prostglandins (158), Erythrocyte Composition (159),
Tissue Analysis (160), References (163)

8. Amides.......................................................... 165
N -H Stretching Modes (166), The Amide I Band: C=O Stretching (167),
The Amide II Band (169), Other Correlations (169), Amines (171), Refer-
ences (171)

9. Amino Acids, Related Compounds, and Peptides. . . . . . . . . . . . . . . . . . . . .. 173

Introduction (173), Spectra-Structure Correlations and Other Structural
Considerations (174), Chelate Compounds (178), Determination of Ioniza-
tion Constants (181), Amino Acid Derivatives (181), Simple Peptides (183),
Aqueous Solutions of Dipeptides (183), Low-Temperature Studies with
Amino Acids and Simple Polypeptides (184), References (185)

10. Proteins and Polypeptides .......................................... 188

Conformational Studies of Proteins and Polypeptides (188), Other Studies
on Polypeptides (206), Globular Proteins (216), Other Studies on Proteins
and Related Substances (221), References (229)

11. Hydrogen-Deuterium Exchange. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 232

Methods (233), Theory (238), Factors Affecting Hydrogen-Deuterium
Exchange of Biological Molecules (240), Applications (248), References
(268)

12. Nucleic Acids and Related Compounds .............................. 271

Absorption Band Assignments (274), Studies of DNA (278), Studies of
Deoxyribonucleoproteins (282), Double-Helical RNA (283), DNA-RNA
Hybrid (283), Aqueous Solutions of Nucleosides, Nucleotides, Poly-
nucleotides, and DNA (284), Pairing of Bases in Various Nucleic Acids
(298), Hydrogen Bonding (302), Complexes of Nucleosides, Nucleotides,
DNA, and Related Compounds with Metal Ions (304), Raman Spectra
Investigations (306), The Conformation of Nucleosides in Solution (309),
Contents xiii

Photochemistry of Nucleic Acids and Related Compounds (311), Refer-

ences (312)

13. Steroids ......................................................... 315

Steroid Nomenclature (315), Conformation of Steroids (320), Polymor-
phism (320), Correlations of Spectra with Structures (321), Metabolism in
Men and Animals (341), Metabolism in Plants (345), Metabolism in Micro-
organisms (346), References (347)

14. Porphyrins and Related Compounds ................................. 350

Sample Preparation (350), Characteristic Group Frequencies (350), Por-
phyrins and Metalloporphyrins (350), Porphyrin-Containing Proteins (354),
The Binding of Carbon Monoxide (355), Chlorophyll and Related Com-
pounds (357), References (363)

15. Enzymology ...................................................... 364

Mechanism of Enzyme Action (364), Stereospecificity (369), Enzyme
Inhibition (371), Enzyme Kinetics (372), Properties of Nitrilase and Its
Substrates (383), Characterization of Reaction Products (383), Identification
of Organisms by Their Enzymic Action (385), References (389)

16. Drugs, Pharmaceuticals, and Pharmacological Applications ............. 390

Spectra Collections (391), Antibiotics (399), Sulfonamides (403), Alkaloids
(406), Barbiturates and Ureides (411), Amphetamines (413), Marijuana
(413), Caffeine and Theobromine (413), Choleretics (414), Cyclamate,
Dulcin, and Saccharin (415), Isopropamine (415), Thyroid Drug Prepara-
tions (415), Analysis of Dicumarol in Blood Serum (416), Addendum (416),
References (416)

17. Microbiology ..................................................... 418

Identification of Organisms (418), Capsular and Cell-Wall Materials (424),
Viruses and Viral Infection (426), DNA of Viruses and Cells (432), Metabolic
Products (432), References (438)

18. Applications in Medicine and Related Fields. . . . . . . . . . . . . . . . . . . . . . . . .. 440

Diagnostic Use of Spectra of Blood Serum and Other Biologic Fluids (441),
Blood Clotting (444), Examination of Normal and Diseased Skin (445),
Examination of Urine (447), Analysis of Blister Serum (449), Analysis of
Lipoproteins in Serum (449), Analysis of Calculi (Gallstones) (450), Body
Water Content (451), Determination of Toxic Gases and Volatile Organic
Compounds in Human Beings (452), Analysis of Expired Breath (453),
Analysis of Body Fluids after Extraction with Solvents (456), Tissue
xiv Contents

Extract Analysis (458), Analysis of Organs and Organ Content (458), Use
of Gases in Anesthesiology (459), Other Medical Applications Using Gas
Analyzers (460), Other Examples of the Use of an Infrared Gas Analyzer
(461), Monitoring of Environments (462), Analyses of Space-Flight and
Submarine Atmospheres (465), References (465)

19. Special Topics ofInterest in Biochemistry and Related Fields ............ 468
Water Structure (468), Aqueous Solution Infrared Spectroscopy (472), Metal
Chelates and Complexes (485), Membranes (487), Mitochondria (491),
Some Early Studies on Tissues (492), Studies on Bone and Collagen (494),
Bones, Teeth, and Minerals (497), Vitamins, Hormones, and Coenzymes
(511), Quinones (515), Catechol-Related Polymers (516), Synthetic Blood
Substitutes (517), Botanical Products, Botony, and Plant Physiology (518),
Prebiology (522), Paleobiochemistry (522), Odor (523), Insect Sex Attract-
ants (526), Insect Defense (527), Pesticides (528), Metabolism of Herbicides
(528), References (529)

20. Information Sources Concerning Infrared Spectroscopy and Spectra. . . . .. 536

A Partial Listing of General References to Infrared Spectroscopy (536),
Spectra References (Including Theory in Some Cases) (537), Unmechanized
Spectra Indexes (539), Mechanized Use of Spectral Indexes (539), Computer
Techniques for Using the IBM Card File (540), "SIRCH," the ASTM-Dow
Infrared File Searching System (542), FIRST-1 (542), Eastman Infrared
Spectral Data Retrieval Service (543), Computer Programs of the National
Research Council of Canada (543), References (543)

APPENDIXES
1. Fractionation Methods Before the Use of Infrared Spectroscopy. . . . . . . .. 544
Miscellaneous Types of Chromatography (544), Combined Use of Gas-
Liquid Chromatography and Infrared Spectroscopy (548), Ion-Exchange
Chromatography (549), Countercurrent Chromatography (549), Molecular-
Sieve Chromatography (Gel Filtration) (550), Electrophoretic Techniques
(552), Differential Dialysis (553), Density-Gradient Separations in the Ultra-
centrifuge (553), Countercurrent Distribution (554), Use of the Solubility
Diagram for Determining the Presence of Impurities in Soluble Materials
(544), References (554), Additional References (558)

2. Conversion Table-Wave Numbers and Wavelength. . . . . . . . . . . . . . . . . .. 560

General, Index. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 563
Chemical Compound Index. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 591