|Budget Amount *help
¥2,200,000 (Direct Cost : ¥2,200,000)
Fiscal Year 1995 : ¥300,000 (Direct Cost : ¥300,000)
Fiscal Year 1994 : ¥300,000 (Direct Cost : ¥300,000)
Fiscal Year 1993 : ¥1,600,000 (Direct Cost : ¥1,600,000)
Amino acid racemization dating is known as an unique dating method that can cover the generation field less than 1,000 years and from fifty thousands to hundreds of thousands years which are determination domain outside of radiocarbon and K-Ar dating methods. It is one of advantage that a quantity of fossil sample necessary for 1 determination answers the purpose by approximately 1 g. Howere, weak point of this method is pre-treatment of the fossil sample takes very long time for 1 determination. In this study, the experiment method has been improved for faster dating of the fossils as describing follows. First, fossil has been dissolved in dilute hydrochloric acid of necessary minimum amount. Next, about 5 times amount of ethanol has been added to the hydrochloric acid solution and cooled down to -20ﾟC for 1 hour to precipitate proteins. Collecting the precipitate by means of centrifugation, inorganic salts, free amino acids, low molecular weight peptides, and most of dissolved organi
c impurities have been removed simultaneously. White precipitate of proteins has been turned into free amino acids by hydrochloric acid hydrolysis. After evaporated off the excess hydrochloric acid, the free amino acids have been derivatized into their N-isopropyloxycarbonyl methyl esters within 1 min. The derivatives have been separated into their enantiomeric pairs by gas chromatography using capillary column coated with modified cyclodextrin as chiral stationary phase. The outcome of this study is as follows : 1) Ion-exchange purification of the hydrolyzed sample could be eliminated, and the time required for derivatization became rapid. Therefore, total analysis time has been shortened by less than 1/2 compared with conventional method. 2) Recoveries of proteins in fossils have increased considerably by employing ethanol precipitation method of proteins in fractionation. According to this method, amount of fossil sample necessary for 1 determination could be reduced to 100 mg result in contribution for preservation of valuable fossil samples. 3) Analytical precision for determination of the D/L ratio of aspartic acid in fossil sample could be hold down to less than 3 % of RSD( relative standard deviation).