Protein Hydrolysis with Formic Acid and Analysis of Amino Acid Using Butylthiocarbamyl - trimethylsilyl (BTC - TMS) Derivatives by Gas Chromatography

  • Published : 2003.04.01


The protein hydrolysis with 6 M formic acid containing 0.3% tryptamine was a superior method for amino acid analysis of standard amino acid and protein than 6 M HCI containing 0.3% tryptamine. The recoveries of standard amino acid after acid hydrolysis were more accurate in the 6 M formic acid hydrolysis than 6 M HCI hydrolysis, especially recovery of tryptophan showed higher values of 1.5 times than that of 6 M HCI hydrolysis. The results of analysis on the standard protein, bovine serum albumin, showed very similar values compared to the sequence analysis reported in the literature for the 6 M formic acid hydrolysis than 6 M HCI hydrolysis, especially in the tryptophan recovery as standard amino acid recovery. Butylthiocarbamyl - trimethylsilyl (BTC - TMS) derivatives of 22 standard amino acids were successfully resolved DB-17 capillary column. Excellent reproducibility of standard amino acid recovery and composition of bovine serum albumin were obtained with BTC-TMS derivatives.


  1. Hirs CHW, Stein H, Moore S. 1954. The amino acid composition of ribonuclease. J Biol Chem 211: 941-949.
  2. Moore S, Stein WH. 1963. Methods of Enzymology 6. Colowick SP, Kaplan NO, eds. Academic Press, New York. p 819.
  3. Hare PE. 1977. Subnanomole-range amino acid analysis. In Method of Enzymology 47. Hirs CHW, Timasheff SN, eds. Academic Press, New York. p 3-18.
  4. Zumwalt RW, Absheer JS, Kaiser FE, Gehrke CW. 1987. Symposium on chromatography of amino acids. 99th annual international meeting of AOAC. Acid hydrolysis of protein for chromatographic analysis of amino acids. J Assoc Off Anal Chem 70: 147-151.
  5. Woo KL, Lee DS. 1995. Capillary gas chromatographic determination of proteins and biological amino acids as N(O)-tert.-butyldimethylsilyl derivatives. J Chromgatogr B 665: 15-25.
  6. Favian V, Szakacs MP, Perl IM. 1990. Gas chromatography of tryptophan together with other amino acids in hydrochloric acid hydrolysates. J Chromatogr 520: 193-199.
  7. Perl IM, Sazakacs MP, Khalifa M. 1993. High-performance liquid chromatography of tryptophan and other amino acids in hydrochloric acid hydrolysates. J Chromatogr 632: 57-61.
  8. Woo KL, Jang DK, Kim JI, Woo DK, Lee YW. 2002. Effective hydrolysis and clean-up methods for the analysis of amino acid composition in food proteins using N(O)-tert.-butyldimethylsilyl derivative by GC. Food Sci Biotechnol 11: 205-214.
  9. Gajewski E, Dizdaroglu M, Simic MG. 1982. Kovats indices of trimethylsilylated amino acids on fused-silica capillary columns. J Chromatogr 249: 41-55.
  10. Mawhinney TP, Robinett RSR, Atalay A, Madson MA. 1986. Analysis of amino acids as their tert-butyldimethylsilyl derivatives by gas chromatography and mass spectrometry. J Chromatogr 358: 231-242.
  11. Portier G, Tenaschuk D, Meckenzie SL. 1986. Capillary gas chromatography micro-assay for pyroglutamic, glutamic and aspartic acids, and glutamine and asparagine. J Chromatogr 361: 253-261.
  12. Woo KL, Chang DK. 1993. Determination of 22 protein amino acids as N(O)-tert.-butyldimethylsilyl derivatives by gas chromatography. J Chromatogr 638: 97-107.
  13. Woo KL, Lee SH. 1994, Determination of protein amino acids as butylthiocarbamyl derivatives by reversed-phase highperformance liquid chromatography with precolumn derivatization and UV detector. J Chromatogr A 667: 105-111.
  14. Hirayama K, Akashi S, Furuya M, Fukuhara KI. 1990. Rapid confirmation and revision of the primary structure of bovine serum albumin by ESIMS and Frit-FAB LC/MS. Biochem Biophys Res 173: 639-646.