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

with the aid of an IKA Vibro Fix instrument (Janke and Kunkel, IKA-WERK, Breisgau, Germany). 1.2.3.4. Separation and quantitation of the enantiomers Separation of the enantiomers was made according to the method of EINARSON ET AL. (1987b), using a reversed-phase analytical column packed with Kromasil octyl C8 (250 x 5.6 mm internal diameter; 5 urn particle size, EKA Nobel AB, Bohus, Sweden). To increase the lifetime of the column, a safety column was fitted between the sample injector and the analytical column (RP8, Newguard, 25 x 3.2 mm internal diameter, 7 urn particle size, EKA Nobel AB, Bohus, Sweden), and a cleaning column (CI 8, 36 x 4.5 mm internal diameter, 20 шп particle size, Rsil, EKA Nobel AB, Bohus, Sweden) was installed between the pump and the sample injector. In order to separate the enantiomers, the two component gradient system had the following composition: A = 40% methanol in phosphate buffer (9.5 mM, pH = 7.05) and В = acetonitrile. The flow rate was 1 ml/min, and the elution of the gradient as a function of time is shown below. Time (min)) A

1 B

2 Time (min)) (%) 0 95 5 10 95 5 35 83 17 55 72 28 56 67 33 74 67 33 75 62 38 '40% methanol in phosphate buffer (9.5 mM, pH = 7.05). 2

Acetonitrile 1.3. Results and discussion 1.3.1. D-amino acid composition of bovine ribonuclease as related to time and temperature Bovine ribonuclease was hydrolysed by 6M HCl at 110 °C for 24 h and at 160, 170 and 180 С for 15, 30, 45 and 60 minutes. The D-amino acid compositions of ribonuclease after hydrolysis at 110 °C for 24 h and at elevated temperatures for shorter times are in Tables 1, 2 and 3. The data in Table 1 showed that both traditional hydrolysis (6M HCl, 24 h, 110 °C) and high temperature - short duration hydrolysis, tryptophan almost completely decomposed. As a result, we shall not report this amino acid in the following tables. It is clear that, among the examined amino acids, the highest degree of racemization [D/(D+L)xl00] is recorded for aspartic acid, in both traditional and short duration hydrolysis. This is followed in decreasing order by glutamic acid, threonine, phenylalanine, alanine, valine and histidine. At 160 racemization degree increases as hydrolysis time increases. In the case of every amino acid tested, the lowest racemization was recorded at 15 minutes hydrolysis times. Increasing the hydrolysis time from 15 to 60 minutes, racemization increased from 1.73% to 3.34%» in the case of aspartic acid, 1.58% to 2.84% for glutamic acid, 1.47% to 2.12% for threonine, 1.41% to 1.73% for alanine, 1.22% to 1.54%) for valine, 2.13% to 3.21% for phenyl-alanine and 0.92% to 1.64% for histidine. 25