Achaeometrical Research in Hungary II., 1988

PROSPECTING and DATING - János CSAPÓ - Zsuzsanna CSAPÓ-KISS - János CSAPÓ JR.: How the amino acids and amino acid racemization can be used and with what limits for age determination of fossil materials in archaeometry

acids as follows: L-aspartic acid, L-glutamic acid, L-threonine, L-alanine, L-valine, Lphenylalanine, L-histidine and L-tryptophan. The protein content of bone samples (520%) was determined using a Kjel-Foss 16200 (Foss Electric, Denmark) rapid nitrogen analyser. The protein content was calculated from nitrogen % using a conversion factor of 6.25. Peptides and proteins were hydroIysed at varying temperature-time combinations. The free amino acids samples were subjected to the same temperature-time treatments. HPLC was used for determination of L-aspartic acid, L-glutamic acid, L-threonine, Lalanine, L-valine, L-phenylalanine, L-histidine and L-tryptophan content of the samples. The following materials were used for testing the racemization during hydrolysis: Bovine ribonuclease, lysozyme, citochrom C, fossil bone sample, and individual free amino acids as follows: L-aspartic acid, L-glutamic acid, L-threonine, L-alanine, L-valine, Lphenylalanine, L-histidine and L-tryptophan. The protein content of bone samples (520%) was determined using a Kjel-Foss 16200 (Foss Electric, Denmark) rapid nitrogen analyser. The protein content was calculated from nitrogen % using a conversion factor of 6.25. Peptides and proteins were hydrolysed at varying temperature-time combinations. The free amino acids samples were subjected to the same temperature-time treatments. HPLC was used for determination of L-aspartic acid, L-glutamic acid, L-threonine, Lalanine, L-valine, L-phenylalanine, L-histidine and L-tryptophan content of the samples. 1.2.3. High performance liquid chromatography (HPLC) for separation and determination the D- and L-amino acids 1.2.3.1. Instruments The chromatographic system was assembled from ISCO 100 DM syringe pumps (Isco Inc. Lincoln, Nebraska, USA) and a Rheodyne (Berkeley, California, USA) injector equipped with a 20-ul loop. The separation process was monitored and chromatograms stored on an ISCO Chem Research (Isco Inc. Lincoln, Nebraska, USA) system. The derivative formation and sample injection were performed manually. The excitation and observation wavelengths were 325 and 420 nm, respectively. 1.2.3.2. Reagents Acetonitrile and methanol were purchased from Rathburn Ltd (Walkeburn, England). The AA standards, the o-phthalaldehyde and the TATG were obtained from Sigma Chemical Co., Inc. (St. Louis, MO). The buffers used for elution were prepared from mono- and disodium phosphate. The pH was adjusted with 4M sodium hydroxide. 1.2.3.3. Synthesis of derivatives The reaction was carried out in a 120-ul microvial which was placed in another vial (volume, 1.8 ml) that had Teflon

R coating, internal cover plate, and a screw cap. The sample (free AA or protein hydrolysate evaporated by lyophylysation), dissolved in 90-ul borate buffer (0.4M; pH 9.5), was mixed with 15-ul of reagent (8 mg of o-phthalaldehyde (OPA) and 44 mg of 2,3,4,6-tetra-O-acetyl-l-thio-ß-D-glucopyranoside (TATG) dissolved in 1 ml of methanol). The mixture was then homogenized by bubbling through approximately 100-ul of nitrogen and left standing for 6 min. Then, 25-ul of the reaction mixture were injected into the analytical column. After injection, the system was rinsed three times with approximately 100-ul of a 70:30 acetone-water (vol/vol) solution. Synthesis of derivatives was performed manually and mixing of reagent solution was made 24