Laser-based optical activity detection of amino acids and proteins

Reitsma, Barbara
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Amino acid analysis by high performance liquid chromatography (HPLC) is presently a routine procedure and the field of protein HPLC has experienced rapid growth in the past decade. The optical activity detector (OAD) for HPLC, previously developed in this research group, is a selective detector for optically active substances including amino acids and proteins. This dissertation illustrates the use of the OAD in three related areas;Section I illustrates the separation of four free amino acids using cation-exchange chromatography. Detection by coupling the OAD to a refractive index detector (RI) for proline and threonine and the OAD to an ultraviolet absorbance detector (UV) for tyrosine and phenylalanine allows the calculation of enantiomeric (D/L) ratios of these amino acids without physical separation. Specific rotations of these four amino acids are also reported;Amino acid detection by refractive index is not sensitive and UV absorbance detects only three amino acids. Derivatization of amino acids to make them detectable by UV absorbance enhances the applicability of OA/UV for the determination of enantiomeric ratios. Section II illustrates the separation of 16 dansyl-L-amino acids by RP-HPLC with detection by OA/UV. Calculation of the specific rotation of 22 dansyl-L-amino acids shows that derivatization enhances the OA detectability of some amino acids but degrades that of others. This section also illustrates the first successful coupling of the OAD to gradient elution;RP-HPLC of proteins is a rapidly developing technique. Several researchers have reported the detection of multiple peaks when a pure protein is subjected to HPLC under certain conditions. These multiple peaks have been determined to be different conformations of the same protein. Since proteins are optically active, OA is a suitable detector. Section III illustrates the RP-HPLC separation of conformers of soybean trypsin inhibitor. Detection by OA/UV provides insights from the chromatogram unavailable from UV absorbance detection alone. In addition, identification of impurities is simplified with OA/UV. Specific rotations of the separated protein fractions show no significant change accompanying change in conformation. ftn * DOE Report IS-T-1131. This work was performed under Contract W-7405-Eng-82 with the U.S. Department of Energy.