To detect amount of 20 types of amino acids in the sample

Sample Preparation:

1. Hydrolyze the protein sample by heating it with 6N HCl at 110°C for 24 hours in an evacuated sealed tube to break down proteins into individual amino acids.

2. Remove HCl by evaporation under vacuum.

3. Dissolve the dried hydrolysate in a suitable solvent like 0.1N HCl.

4. Filter the sample through a 0.45 μm membrane filter.

HPLC Analysis:

5. Choose a reverse-phase C18 analytical column and compatible mobile phases like:

Mobile phase A: 0.1M sodium acetate buffer, pH 6.5
Mobile phase B: Acetonitrile/methanol/water (45:45:10 v/v)

6. Set up a gradient elution program to separate amino acids, e.g. 0-5 min 100% A, 5-30 min 0-100% B, 30-35 min 100% B.

7. Detect amino acids with a UV detector at 254 nm or a fluorescence detector after derivatization with reagents like o-phthalaldehyde (OPA).

8. Identify amino acids by matching retention times with those of standard amino acid mixture run under same conditions.

9. Quantify each amino acid by integrating peak areas and using calibration curves prepared from standard amino acid solutions of known concentrations.