Purpose

Samples containing peptides and proteins can be hydrolyzed to release the bound amino acids. In order to preserve Tryptophan, samples are put through an alkaline hydrolysis. This method is for accurate determination and quantification of amino acids in proteins.

Summary

Samples are prepared using an alkaline hydrolysis method to release and preserve bound Tryptophan in proteins. Samples are then diluted to an appropriate concentration based on the protein content of the sample. A solution of 0.2M phosphate buffer is added to the sample to achieve the target dilution. The mixture is then filtered into an HPLC vial

Alkaline hydrolyzed tryptophan amino acid analysis is performed using a reversed phase C-18 column with an aqueous buffer containing organic solvents using an Agilent 1100 HPLC analyzer.  Peak detection uses a combination of in-line, programmable, and scanning fluorescence detectors. Appropriate standards are run to ensure each sequence is running properly and used in the quantification of samples using peak areas and applying an external standard technique utilizing a calibration curve.

Amino acids quantified

Tryptophan