Direct Specific Rotation Measurements of Amino Acids, Dipeptides, and Tripeptides by Laser-Based Polarimetry KARNO NG, 1 THOMAS J. EDKINS, 2,1 * AND DONALD R. BOBBITT 1 1 Department of Chemistry and Biochemistry, University of Arkansas, Fayetteville, Arkansas 2 Analytical Development, The R.W. Johnson Pharmaceutical Research Institute, Spring House, Pennsylvania ABSTRACT This paper addresses direct HPLC detection of amino acids, di and tripeptides via laser-based polarimetry. Mass detection limits typically range from 0.5 to 50 μg injected on-column, corresponding to concentration levels from 0.1 to 1 mg/ml. These levels are 1 to 2 orders of magnitude lower than measurements with conventional polarimetry, implying that concentration-dependent phenomena (e.g., solute dimeriza- tion) are minimized. This is the first published report of the specific rotation of the di and tripeptides of Ala and Phe. The relationship between pH and specific rotation of selected amino acids, peptides, and lysozyme is described. Chirality 11:187–194, 1999. © 1999 Wiley-Liss, Inc. KEY WORDS: amino acid/peptide detection; HPLC; laser-based polarimetry State-of-the-art amino acid analysis is currently achieved through UV absorption spectrometry, 1–3 fluorometry, 4–6 di- rect or indirect chemiluminescence, 7–10 electrochemical means, 11–13 or gas chromatography (GC). 14–16 Most of these detection techniques require chemical derivatization of the analyte, which can enhance detection by orders of magnitude as compared to direct detection. Typical limita- tions of this technique are that it is often time consuming, it can require complicated instrumentation, and the deriva- tization products need to be isolated from reactants and excess reagents. Amino acids can be detected directly without derivatiza- tion by polarimetry. 17 Since the substituent groups of amino acids can take only one of two possible spatial con- figurations around the -carbon, all of the naturally occur- ring amino acids are optically active, with the exception of glycine. Polarimetric detection is able to provide specific rotation information on amino acids and peptides which is not available with most other detection schemes. Since specific rotation is sensitive to the arrangement of atoms at or near the stereogenic center, subtle structural differ- ences can be detected. Conventional polarimeters are not sensitive enough for trace determination. On the other hand, laser-based polar- imetry is able to provide specific rotation information on materials present at the nanogram level. 17 Recent ex- amples that illustrate the detectability of laser-based polar- imetry are the application of enantiomeric quantitation without separation to a small-molecule pharmaceutical 18 and evaluation of a flow injection analysis system for sev- eral organic molecules. 19 In this study, the application of specific rotation measurements of selected amino acids by laser-based polarimetry is extended to their di and tripep- tides. Furthermore, the polarimetric method is shown to be a sensitive method for the detection of amino acids without derivatization. The effect of changes in pH on the specific rotation of amino acids and the peptides is studied, and the sensitivity of specific rotation toward changes in the structure of these amino acids and peptides is evalu- ated. MATERIALS AND METHODS Chemicals Methanol, hydrochloric acid, sodium hydroxide (Fisher Scientific, Fairlawn, NJ), tris(hydroxymethyl) aminometh- ane (Aldrich Chemical, Milwaukee, WI). L-amino acids, di and tripeptides (Sigma Chemical, St. Louis, MO) were used as received. All specific rotation measurements were made with mixtures of methanol/2 × 10 -2 M tris(hydroxy- methyl)aminomethane (amino acids/peptides 70:30, lyso- zyme 10:90). The pH was measured from ∼2 to 9 and was adjusted by adding either 6 M HCl or 0.1 N NaOH. All reagents were used as received, except for degasification using ultrasonic agitation under vacuum prior to use. Instrumentation The laser-based polarimetric system has been described in detail elsewhere. 20 An argon-ion laser operating at  = 488 nm is used as the probing light source. The chromato- graphic system consists of a piston pump (Shimadzu Scientific Instruments, Inc., Kyoto, Japan, model LC-600), operated at 0.6 ml/min, coupled to a Rheodyne injector (Cotati, CA, model 7161) fitted with a 20 μl loop. Chromato- *Correspondence to: Thomas J. Edkins, New Product Research, The R.W. Johnson Pharmaceutical Research Institute, Welsh and McKean Roads, Spring House, PA 19477-0776. E-mail: tedkins@PRIUS.JNJ.COM Received for publication 19 January 1998; Accepted 21 July 1998 CHIRALITY 11:187–194 (1999) © 1999 Wiley-Liss, Inc.