Louisiana State University LSU Digital Commons LSU Historical Dissertations and Theses Graduate School 1967 Determination and Identification of Fundamental Amino Acids by Thermometric Titration and Nuclear Magnetic Resonance Spectroscopy. William Yong-chien Wu Louisiana State University and Agricultural & Mechanical College Follow this and additional works at: https://digitalcommons.lsu.edu/gradschool_disstheses Recommended Citation Wu, William Yong-chien, "Determination and Identification of Fundamental Amino Acids by Thermometric Titration and Nuclear Magnetic Resonance Spectroscopy." (1967). LSU Historical Dissertations and Theses. 1274. https://digitalcommons.lsu.edu/gradschool_disstheses/1274 This Dissertation is brought to you for free and open access by the Graduate School at LSU Digital Commons. It has been accepted for inclusion in LSU Historical Dissertations and Theses by an authorized administrator of LSU Digital Commons. For more information, please contact [email protected]. This dissertation has been microfilmed exactly as received 67-8805 WU, William Yong-Chien, 1939- DETERMINATION AND IDENTIFICATION OF FUNDAMENTAL AMINO ACIDS BY THERMOMETRIC TITRATION AND NUCLEAR MAGNETIC RESONANCE SPECTROSCOPY. Louisiana State University and Agricultural and Mechanical College, Ph.D., 1967 Chemistry, analytical University Microfilms, Inc., Ann Arbor, Michigan Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. DETERMINATION AND IDENTIFICATION OF FUNDAMENTAL AMINO ACIDS BY THERMOMETRIC TITRATION AND NUCLEAR MAGNETIC RESONANCE SPECTROSCOPY A Dissertation Submitted to the Graduate Faculty of the Louisiana State University and Agricultural and Mechanical College in partial fulfillment of the requirements for the degree of Doctor of Philosophy in The Department of Chemistry by William Yong-Chien Wu B.A., Hardin Simmons University, I96I January, I967 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. ACKNOWLEDGMENT The author wishes to thank Dr. B. Sen for directing this work and for his patience and encouragement. He further wishes to acknowledge the faithful and loving support of his wife, Anne. A special word of sincere appreciation is due to Mr. Bill Wegner who maintained and operated the N.M.R. equipment. Grateful acknowledgment is made to the Louisiana State University, Department of Chemistry, for furnishing the Graduate Teaching Assistant- ship during the author's entire graduate training. Thanks are expressed also to the "Dr. Charles E. Coates Memorial Fund of the L.S.U. Founda­ tion Donated by Charles H. Coates" for the financial assistance for the preparation of this dissertation. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. TABLE OF CONTENTS PAGE ACKNOWLEDGMENT.................. i LIST OF TABLES........................ iv LIST OF FIGURES ............................................... v ABSTRACT........................................................ vii INTRODUCTION.................................................... ix CHATTER I. SOME CONVENTIONAL METHODS FOR THE ANALYSIS OF AMINO ACIDS A. Chemical Methods ........................ 1 B. Biological Methods ................................... 2 C. Chromatographic Methods. .. ........................ 5 D. Physico-Chemical Methods ............................ 4 E . Miscellaneous.......................................... 5 CHAPTER II. THERMOMETRIC TITRATION A. Theoretical Basis..................................... 6 B. Nomenclature......................................... 7 C . Instrumentation....................................... 9 D . Unique Features and Significance .................... 10 CHAPTER III. DETERMINATION OF AMINO ACIDS BY THERMOMETRIC TITRATIONS A. Introduction......................................... B . Selection Criteria of Experiments.................... l4 C. Apparatus............................................. 1$ D . Procedure............................................. 19 CHAPTER IV. RESULT'S AND DISCUSSIONS A. General Characterization of Thermometric Titration Curves Involving Ac id-Base Interactions............... 27 B. Analytical Study of Selected Anino Acids $0 C . Chelation Study on Monocarboxylic Acids.............. 5^ D . Thermodynamic S tudy.......................... 42 IX Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. PAGE CHAPTER V. NUCLEAR MAGNETIC RESONANCE SPECTROSCOPY STUDY OF THE FUNDAMENTAL AMINO ACIDS A. Introduction.......................................... k j B. Theoretical Aspect. .... ................. 49 C . Experimental.......................................... 52 D. Data and Discussion.................................. 53 SELECTED BIBLIOGRAPHY. .................................... J2 APPENDIX ............ T6 VITA .......................................................... 78 iii Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. LIST OF TABLES TABLE PAGE I. Abcissa Calibration Chart............................. 21 II. Analytical Results for Neutral Amino Acids .......... 51 III. Analytical Results for Acidic Amino Acids............ 52 IV. Analytical Results for Basic Amino Acids . ........ 53 V. Analytical Results for Mixtures of Amino Acids .... 58 VI. Analytical Results for Mixtures of Amino Acids .... 59 VII. Thermodynamic Data of Neutral Amino Acids............. 45 VIII. Comparison of Data Based on Thermodynamic Titrations and on Literature Values................... 44 IX. Copper (1 1 ) Chelation Data with Selected Amino Acids . 45 X. Characteristic NMR Spectral Positions for Protons in the Series of Amino A c i d s ......................... 60 IV Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. LIST OF FIGURES FIGURE PAGE 1. (a) Theoretical Exothermic Titration Curve............ 8 (b) Experimental Exothermic Titration Curve .......... 8 2 . Potentiometrie Titration Curves of 0.01 M Acids with Sodium Hydroxide.................................... 11 3 . Thermometric Titration Curves of 0.01 M Acids with Sodium Hydroxide................................. 15 4. Block Diagram of Thermometric Titration Apparatus . 16 5. Thermistor Bridge Circuit ............................. 18 6. Typical Calibration Curve ............................. 25 7. Thermometric Titration Curve of a Mixture of Aspartic Acid and A l a n i n e ............................. 25 8. Typical Thermometric Titration Curves for Monoprotic, and Diprotic Amino Acids................... 28 9 . Thermometric Titration Curve for Glutamic Acid........ 29 10. Plot of Percent Error vs. Concentration.............. 55 11. Thermometric Titration Curves for Histidine Monohydrochloride ...................................... 57 12. NMR Spectrum of Alanine in Water....................... 5^ 1 5. NMR Spectrum of Valine in W a t e r ....................... 56 14. NMR Spectrum of Valine in 0.25 M NagCOs and NaHCOg. 58 1 5. NMR Spectrum of Isoleucine and Leucine................. 65 1 6. NMR Spectrum of (l) Lysine, and (ll) Mixture of Lysine and Histidine.................................... 66 1 7. NMR Spectrum of (l) Aspartic Acid, and (ll) Mixture of Aspartic and Histidine.................................. 67 1 8. NMR Spectrum of (l) Pure Methionine, and (ll) Mixture of Methionine and Histidine............ 68 1 9. NMR Spectrum of a Mixture of Aspartic Acid and Phenylalanine ...... ............................. 69 V Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. FIGURE PAGE 20. NMR Spectrum of (l) Serine, and (il) Mixture of Serine and Phenylalanine 70 21. NMR Spectrum of (l) Glycine, and (il) Mixture of Glycine and Alanine....................................... 71 vx Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. ABSTRACT The essential constituents of protein molecules are the amino acids, cy-Amino acids have in common an amino group in the cy-position to the carboxyl group, and have the general structure of R—^— COOH. NH2 The carboxyl groups of the amino acids have extremely low ioniza­ tion constants, of the order of 10“^°. Thermometric titration, whose only detection criterion is the enthalpy change, has been adapted to the determination of twenty fundamental, and biologically important amino acids in the millimolar and centimolar range. Methods for quan­ titative determination of amino acids utilizing the acid-base neutrali­ zation reaction, and the metal ion (Cu"^^) chelation have been developed, Individual and binary mixtures have been investigated. Potentialities and limitations of the methods have been explored in terms of analytical precision, accuracy and range of application. In general, the thermometric titration technique proves to be a sensitive, rapid, simple and reliable method for the quantitative deter­ mination in the 10”^ to 10"'^ range, and sample size of 25 ml. The method is of particular value for routine analysis. Referring back to the general structure, it is obvious that R is the only distinguishing functional group among all the acids. Proton nuclear magnetic resonance spectroscopy has been chosen as an ideal tool for qualitative identification. In an alkaline (0.25 M NagCOg and NaHCOs) aqueous solution the protons of the carboxyl group and the amino group exchange rapidly with the solvent, therefore only absorption peaks