Amino Acids

Amino acid derivative: n-trifluoroacetyl-n-butyl ester (TAB)

 

Reagents:

1.      0.4N HCl in butanol (Regis, discontinued.  BF3-butanol-Supelco )

2.      Trifluoroacetic anhydride (TFA Supelco 33165-U)

3.     Methylene chloride (Dichloromethane, Fisher D151-4)

 

Standards: unlabeled amino acids.

 

Procedure:

1.     To a dried amino acid sample, add 200 mL butanolic HCl.  Leave on heating block at 100oC for at least one hour.  Dry with nitrogen.

2.      Add 100 mL methylene chloride and 25 mL TFA.  Leave at room temp for 20 min. Dry under nitrogen.  Transfer to ALS vials with methylene chloride.

 

GC/MS:

n-trifluoroacetyl-n-butyl esters of amino acids used to be separated on a packed TAB-sorb column, made by Regis.  It is now discontinued, and the HP-5, or equivalent capillary columns can be used for the same application.  A detailed method with masses and retention times is described for glutamate.

 

Comments:

Ref. 1 describes the general method for TAB derivatives of all amino acids.  Ref. 2 is an example of the use of isotopomer analysis to study glutamine oxidation.

 

References:

1.      Leimer K.H., R. H. Rice and C.W. Gehrke.  Complete mass spectra of n-trifluoroacetyl-n-butyl esters of amino acids.  Journal of chromatography, 141 121-144 (1977)

2.     Lee W.N.P., J. Edmond, S. Bassilian and J.W. Morrow, Mass isotopomer study of glutamime oxidation and sythesis in primary culture of astrocytes.  Dev Neurosci 18: 469-477 (1996)

 

 

 

Glutamate

Glutamate derivative: n-trifluoroacetyl-n-butyl ester

 

Reagents:

1.     0.4N HCl in butanol (Regis, discontinued.  BF3-butanol-Supelco )

2.      Trifluoroacetic anhydride (TFA Supelco 33165-U)

3.      Methylene chloride (Dichloromethane, Fisher D151-4)

 

Standards: unlabeled glutamate.

 

Procedure:

1.     To a dried glutamate sample, add 200 mL butanolic HCl.  Leave on heating block at 100oC for at least one hour.  Dry with nitrogen.

2.      Add 100 mL methylene chloride and 25 mL TFA.  Leave at room temp for 20 min. Dry under nitrogen.  Transfer to ALS vials with methylene chloride..

 

GC/MS: 

GC column: HP5, length 30m, 250mm.

Inlet temp. 250oC, split ratio 1:100 or adjusted depending on sample concentration.

Oven temperature programmed as follows: 205oC for 3min, then a 3oC/min ramp to 215oC and hold for 1min. Retention time: 3.20 min

Relevant ion clusters for SIM analysis: 

CI mode: m/z 356 cluster for C1-C5. (monitor 355 to 362)

EI mode: m/z 198 cluster for C2-C5 fragment, and m/z 152 cluster for C2-C4 fragment.

 

 
 

Web Guider

Ch 1.General Lab Techniques

Ch 2.Molecular Separation

Ch 3.DNA and RNA

Ch 4.Genetics

Ch 5.PCR Serials

Ch 6.Protein

Ch 7.DNA Protein Interactions

Ch 8.Immunohistoch / immunology

Ch 9.Cellular Biology

Ch 10.GC/MS, NMR and Proteomics

Ch 11.Animal Experiments

Ch 12.Worm: C. Elegans

Ch 13.HPLC and TLC

Ch 14.Buffers formats in Lab.

Ch 15.Other Resources

Free eBooks at Library Online

Cinema Online,Free Movies-(1)

Progresses in Life Science

Free eBooks in biomedicine

Pathway databases

Biological Educational Resources

Textbooks and Lab Manuals

Lab-Manual.Com
Home || Biosocial || Videos || Recipes || Resource