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METHOD)
1. Principle
SCH3CH2OH
CHO
HSCH3CH2OH
(2-mercaptoethanol)
+ H2NR NR
Room Temperature
CHO
OPA 1 amino acid Fluorescent Isoindole
derivatives
2. Apparatus
2.1 HPLC system with Waters 472, Scanning Fluorescence detector. Wavelength at
338nm.
2.2 Analytical column: Zorbax ODS (Eclipse-AAA) 3.5 m, 4.6 mm id, 15 cm.
2.3 Sonicator
2.4 Vortex mixer
2.5 Magnetic stirrer
2.6 Plastic centrifuge tubes, 150 ml
2.7 Finn pipette, 100 l, 200 l
2.8 Waters injection vial, 2 ml
2.9 Sep Pak C18
2.10 Test tubes, 15 ml
2.11 Beaker, 250 ml
2.12 Volumetric flask, 10 ml, 200 ml
2.13 Pasteur pipettes
2.14 Komagome pipettes , 5 ml
3. Reagents
4. Reagent solutions
Mix o-phtalaldehyde and 2- Mercapto-ethanol (1:1 v/v). Prepare fresh every 2-3
days. Store in amber bottle.
5. Standard solution
The concentration of Wako mixed amino acid standards are given as 2.5
mol/ml.
Pipette 500 l of the mixed amino acid solution from the ampoule to a 10 ml
volumetric flask. Make up to the mark with 0.1 M HCI. (20 x dilution).
Pipette 1 ml of standard working solution 1 into an injection vial and followed with
1 ml of 0.1 M HCI. (2 x dilution).
6. Sample preparation
6.1.1 Weigh 10 g of minced fish meat into a 250 ml beaker and add 100 ml of
cold MilliQ water.
6.1.3 Remove from sonicator and cool in ice water bath for 5 min. Transfer
contents to a plastic centrifuge tube.
6.1.4 Centrifuge for 10 min at 8,000 rpm and filter with Whatman Paper No.1
into a 200 ml volumetric flask.
6.1.5 Wash the filter paper with 10 ml of cold water 3 times and collect the
remaining solution into the same volumetric flask. Then make up to the
mark with cold MilliQ water. (This is the sample stock solution)
6.1.9 Pipette 200 l of the eluted solution into an injection vial. Add 100 l of
0.5 NaOH, mix for a few seconds.
NB. If sample solution is turbid, filter again with 0.45 m filter before HPLC
injection.
Weigh 1 g of fermented product into a 250 ml beaker and add 100 ml of cold
MilliQ water. Follow steps (b) to (g) of the determination for minced fish meat.
Proceed with the same procedure for OPA-derivatization method.
6.2.1 Pipette 0.1 ml of fish sauce into 100 ml volumetric flask and make up with
MilliQ water. (1000x dilution)
6.2.2 Use 1 ml of MilliQ water to condition Sep Pak C18. Next, add in 1 ml of
diluted fish sauce and elute through the Sep Pak slowly.
6.2.3 The eluant collected will be derivatized using the same OPA-
derivatization method.
7. Determination
7.1 HPLC
Gradient Conditions:
(10 ml / 5 ml) = 2
Fish sauce
J.W. Henderson, R.D. Richker, B.A. Bidlingmeyer (2000). Rapid, Accurate, Sensitive
and Reproducible HPLC analysis of amino acids. Agilent Technologies Co. (USA) part
no. 598 1193E.