M. Járó - L. Költő szerk.: Archaeometrical research in Hungary (Budapest, 1988)
Dating - CSAPÓ János, KÖLTŐ László , PAP Ildikó: Archaeological age determination based on the racemization and epimerization of amino acids
4.2. Determination of isoleucine and D-alloisoleucine When determining isoleucine and D-alloisoleucine the hydrolysed material is diluted in a pH = 2.2 citrate buffer, and the 25-50 nanomole concentrate solution is fed to the LKB— 4101 type automatic amino acid analyser. The description of the analysis and the applied buffers are discussed elsewhere (Csapó et al. 1986). D-alloisoleucine appears on the chromatogram between methionine and isoleucine. It can easily be separated from the neighbouring amino acids, and the peak can easily and precisely be evaluated. 43. Determination of D- and L-amino acids by high présure liquid chromatography The most suitable method for separating D- and L-amino acids by high pressure liquid chromatography was elaborated in conjunction with the Department of Organic Chemistry of Eötvös Loránd University, Budapest. The intent was to prepare a method by which all protein forming amino acids' D- and L-enantiomer can be separated from the hydrolysed compound. By this method the losses due to manipulation could be minimized (the separation of amino acids, the determination of the separated fractions), while the information value of the result could be considerably increased. 4.4. Determination of D- and L-amino acids in chiral silical gel In silica gel thin layers the mixture of D- and L-amino acids cannot be separated, not even by the application of chiral reagent. The amino acids first have to be separated from one another. It is then possible to separate the D- and L-enantiomers from the solution by the application of chiral silica gel. Therefore we separated the amino acids with the LKB-4101 analyser and the LKB fractioner. This was followed by the drying of the test tubes containing the amino acids by lyophilization. The remainders containing the D- and L-amino acids were diluted in a solution of methanol-water whose ratio was 1:1, which resulted in the obtaining of an amino acid of 5-10 ul for an approximately a 1% solution. The silica gel thin layer of the Macheiey-Nagel company (Düren, GFR) with catalogue number 811055 was treated with a chiral reagent and with Cu 2+ ions and was then activated for 15 minutes at 100 °C. This was followed by dropping a solution of 1% in a quantity of 2 ul, and the separation was conducted with a chemical containing methanol-water—acetonitrile in a 50:50:200 ratio. This lasted approximately for 14—15 minutes. Following the drying of the thin layer it was sprayed with a reagent solution containing 0.1% ofninhydrin,and it was then dried in a cabinet at 105 °C for 10 minutes. The area of the spot and the intensity of the colour were measured by a Vitatron densitometer at 570 nm, and the result was evaluated by a recorder connected to the densitometer and by an integrator. With this technique the amino acids the D-and L-enantiomers of all amino acids of protein can be separated. The exceptions are Gly, which is asymmetric, the Trp which totally decomposes under acidic conditions, and the Thr and Ser. On the silica gel thin layer the serine and threonine spots of D and L overlap which does not allow quantitative evaluation. All other amino acids can, however, easily be evaluated at a precision equivalent to the densitometric method.
