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B-17 AMINO ACID ANALYSIS HPLC (PRE COLUMN DERIVATIZATION OPA

METHOD)

1. Principle

o-phtalaldehyde (OPA) pre-column derivatization method is utilised for analysing primary


amino acids. These amino acids are Aspartic acid (Asp), Glutamic acid (Glu), Serine
(Ser), Histidine (His), Glycine (Gly), Threonine (Thr), Arginine (Arg), Alanine (Ala),
Tyrosine (Tyr), Cysteine (Cys), Valine (Val), Methionine (Met), Phenylalanine (Phe),
Isoleucine (Ile), Leucine (Leu) and Lysine (Lys). The reaction of OPA reagent and
primary amino acid group in the presence of 2-mercaptoethanol will yield fluorescent
isoindole derivatives. These derivatives have an excitation wavelength, Ex = 340 nm and
emission wavelength, Em = 455 nm. The absorbance wavelength, max = 338 nm. These
derivatives are only stable for 6 to 7 hours.

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

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Revised on: - Issued on: 27 April 2006
Revised & issued by: Tan Lu Hsia
2.15 Measuring cylinder, 250 ml, 500 ml
2.16 Whatman filter paper No. 1
2.17 Glass rod

3. Reagents

3.1 Amino acid standards:


Wako Amino acids Standard Solution Type H, 1ml x 5 ampoule, Lot HCJ9933
NIST Standard Reference Material 2389, Amino acids in 0.1 mol/L HCI, US Dept
of Commerce National Institute of Stds & Technology Gaithersburg, MD 20899.
3.2 0.1M Hydrochloric acid
3.3 0.5M Sodium hydroxide
3.4 2M Sodium hydroxide
3.5 40mM Sodium Acetate
3.6 5% Trichloroacetic acid
3.7 Borate buffer (0.4M in Water)
3.8 Methanol, HPLC grade
3.9 Acetonitrile, HPLC grade
3.10 o-phtalaldehyde (OPA)
3.11 2-Mercapto-ethanol
3.12 Diluted acetic acid (For pH adjustment)
3.13 Cold Ultrapure (MilliQ) water

4. Reagent solutions

4.1 0.1 M Hydrochloric acid, HCl

Add 1 ml of concentrated HCl to 120ml measuring cylinder and make up to


volume with distilled water.

4.2 0.5 M Sodium hydroxide, NaOH

Weigh 2 g of sodium hydroxide in 100 ml MilliQ water.

4.3 2 M Sodium hydroxide, NaOH

Weigh 8 g in 100 ml MilliQ water. Use for pH adjustment. Recommended further


dilution when necessary.

4.4 40 mM Sodium Acetate, CH3COONa (pH 7.8)

Weigh 3.2812 g in 1000 ml of MilliQ water.


Or
Dilute from stock solution, 400 mM CH3COONa.

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Revised on: - Issued on: 27 April 2006
Revised & issued by: Tan Lu Hsia
4.5 5% Trichloroacetic acid

Dissolve 5 g in 100 ml MilliQ water.

4.6 Borate buffer (0.4 M in water)

i. 0.8M Boric acid (H3BO3) Mol. wt: 61.8

Weigh 2.472 g in 50 ml of MilliQ water.

ii. 0.8M Sodium Hydroxide (NaOH) Mol. wt: 40.05

Weigh 1.602g in 50 ml of MilliQ water.

Add approximately 46 ml of solution (ii) into 50 ml of solution (i) in a 100 ml


beaker. Adjust the pH to pH 10.2. Transfer the solution to a 100 ml volumetric
flask. Make up to the mark of the flask with MilliQ water.

4.7 Reagent solution

Mix o-phtalaldehyde and 2- Mercapto-ethanol (1:1 v/v). Prepare fresh every 2-3
days. Store in amber bottle.

5. Standard solution

Preparation of standard solution

5.1 Standard stock solution

The concentration of Wako mixed amino acid standards are given as 2.5
mol/ml.

E.g. Aspartic acid (Asp)

m.w. = 133.1 g/mol.


In the ampoule, concentration of Asp = 2.5 mol/ml.
2.5 mol/ml x 133.1 g/mol = 332.75 g/ml = 332.75 ppm.

5.2 Standard working solution 1 (0.125 mol/ml) / (16.6 ppm)

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).

5.3 Standard working solution 2 (0.0625 mol/ml) / (8.3 ppm)

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).

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Revised on: - Issued on: 27 April 2006
Revised & issued by: Tan Lu Hsia
5.4 Standard working solution 3 (0.0125 mol/ml) /(1.66 ppm)

Pipette 1 ml of standard working solution 1 into a 10 ml volumetric flask and


make up to the mark with 0.1 M HCI. (10 x dilution).

5.5 Standard working solution 4 (0.00125 mol/ml) /(0.166 ppm)

Pipette 1 ml of standard working solution 3 into a 10 ml volumetric flask and


make up to the mark with 0.1 M HCI (10x dilution).

6. Sample preparation

6.1 Determination of Free amino acid in minced fish meat

6.1.1 Weigh 10 g of minced fish meat into a 250 ml beaker and add 100 ml of
cold MilliQ water.

6.1.2 Homogenise for 10 min in an ultrasonic bath(sonicator) using a glass rod.

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)

6.1.6 Pipette 5 ml of the sample stock solution and 5 ml of 5% Trichloroacetic


acid into a 15 ml glass centrifuge tube. (NB. Coagulation of the
contaminants will occur)

6.1.7 Centrifuge for 5 min at 3,000 rpm to remove the coagulated


contaminants.

Derivatization procedure with OPA

6.1.8 Condition a Sep Pak C18 with 1 ml of MilliQ water. Follow by 1 ml of


sample extract from step (g). Slowly put pressure on the syringe so that
the MilliQ water and fish sample are eluted drop by drop. (NB. Sep Pak
C18 is utilised to remove other contaminants that are not of the carboxylic
group and amide group. Normal filters only remove minute solid matter.)

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.

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Revised on: - Issued on: 27 April 2006
Revised & issued by: Tan Lu Hsia
6.1.10 Add 100 l of borate buffer and mix for a few seconds. Add 200 l of
reagent solution (OPA and 3 mercaptoethanol mixture 1:1 v/v) and mix for
a few seconds. (NB. If foam appears, add 1-2 drops of ethanol/methanol)

6.1.11 Proceed for HPLC injection.

NB. If sample solution is turbid, filter again with 0.45 m filter before HPLC
injection.

6.2. Determination of Free amino acid in fermented fish products

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.

Determination of free amino acids for fermented products fish sauce

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

Mobile phase: A 40mM CH3COONa (pH 7.8)


B CH3CN: CH3OH: H2O (45:45:10 v/v/v)
Flow rate: 1 ml/min
Column: Zorbax ODS (Eclipse-AAA) 3.5 m, 4.6 mm id, 15 cm.
Temperature: 24C
Detector: Waters 470 Scanning Fluorescence Detector
Run time: 60 minutes
Wavelength: Ex: 340 nm Em: 450 nm
Injection volume: 10 l

Gradient Conditions:

Time Flow rate A% B% Curve


0.00 1.0 80.0 20.0
10.00 1.0 80.0 20.0 8
20.00 1.0 50.0 50.0 8
30.00 1.0 40.0 60.0 8
35.00 1.0 20.0 80.0 8
40.00 1.0 0.0 100.0 8
45.00 1.0 80.0 20.0 8
60.00 1.0 80.0 20.0 8

Original issue date: 27 April 2006 Page B-17: 5/7


Revised on: - Issued on: 27 April 2006
Revised & issued by: Tan Lu Hsia
8. Calculation:

Minced fish meat and fermented fish product

Amino acid, (g/g)(ppm) = C x m.w. x V x N x 10-6 x 106


W

C: (Sample peak area / standard peak area) x std conc. (mol/ml)


m.w: Molecular weight (g/mol) of amino acid
W: Weight of sample (g)
V: Volume (ml)
N: Dilution factor
(Sample volume taken plus 5% TCA /sample volume taken) / sample solution
for OPA derivatization) i.e.

(10 ml / 5 ml) = 2

Fish sauce

Amino acid, (g/g)(ppm) = C x m.w. x V x N x 10-6 x 106


W

C: (Sample peak area / standard peak area) x std conc. (mol/ml)


m.w: Molecular weight (g/mol) of amino acid
W: Weight of sample (g) where 0.1 ml = 0.1 g
V: Volume (ml)
N: Dilution factor (Sample volume taken) = 1

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Revised on: - Issued on: 27 April 2006
Revised & issued by: Tan Lu Hsia
9. REFERENCE

B.N. Jones, J.P. Gilligan (1983). Journal of Chromatography 266, pp 471.

P. Lindroth, K. Mopper (1979). Analytical Chemistry 51, pp 1667.

B.N. Jones, P. Paabo, S. Stein (1981) Journal of Liquid Chromatography 4, pp 565.

LC Application News, No. 49 (1997.5), No. 61 (1998.6), No. 12 (1992.4) Yokokawa


Analytical Systems.

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.

Original issue date: 27 April 2006 Page B-17: 7/7


Revised on: - Issued on: 27 April 2006
Revised & issued by: Tan Lu Hsia