AMINO ACID- DETERMINATION

Materials and Methodology

(can choose freeze dry or spray drying)

Materials
Sample(fermented chilli)

Freeze Drying Method

Fermented chili was freezed at -20 ⁰C prior to the freeze drying process. The sample
was dried using Vacuum Freeze Dryer (Labconco, USA) and the dried fermented
chili was kept in desiccator until further analysis.

Spray Drying Method

Spray drying process was carried out in the spray dryer machine). Ultrasonic
frequency was applied which was 40 kHz. The liquid sample was run at 70-80 ⁰C
with a flow rate of 100 ml/hour.

Hydrolysis of sample

1. Two hundred milligram of the sample were weight accurately and filled into a
hydrolysis0tube followed by an addition of012 ml HCl 6M.
2. The tube then aerated for01 minute with nitrogen gas and0immediately sealed
with a Teflon-lined0cap.
3. The tube was placed in an0electric oven for 24 hours at0110℃ for sample
hydrolysis and was cooled0to room temperature.
4. The content of0the tube was quantitatively transferred to050 ml clean
volumetric flask and0diluted to 50 ml with distilled0water.
5. After mixing, 1 ml of0diluted sample was filtered, and 100 µl0filtrate placed in
a limited0volume insert (LVI) of an auto-sampler0vial.

Running HPLC
1. The analysis of amino acids0will is carried out in an0HPLC system equipped
with an auto0sampler.
2. The injection volume will be five μL, and a 436 nm Uv detector will be used.
The column will be a SUPELCOSIL LC-DABS, 15cm x 4.6 mm ID, 3 µm
particles.
3. The mobile phase used was (A) 25mM potassium dihydrogen phosphate with
pH 6.8 and (B) acetonitrile:2-propanol, 75:25 with a flow rate of 2 mL min -1.
Figure 1: Hydrolysis of the sample