LABORATORY MANUAL LAB TOPICS: Amino Acid Analysis

Course: AQU3202/SP4424 Pemakanan dan Tek Makanan Ikan

Level: Bachelor Programme, UMT

NAME OF THE EXPERIMENT: Amino Acid Analysis

METHOD: High Performance Liquid Chromatography (HPLC, Shimadzu
Corp. Kyoto, Japan; Teshima et al., 1986)

SCOPE:
1. This method is used to determine the total amino acid contents in the ingredients and
diets by using high performance liquid chromatography unit with an ion exchange
resin column.

PRINCIPLES:
 After amino acids are separated by cation exchange chromatography, aqueous sodium
hypochlorite solution (to convert prolines, and so on, to primary amines) and o-
phthalaldehyde (OPA) /N-acetylcysteine reagent are added consecutively to the
column eluant, then the resulting fluorescent derivatives are detected.

HOW HPLC WORKS?

 The HPLC system uses detection by post-column fluorescence derivatization, with
o-phthalaldehyde (OPA) / N-acetylcysteine as the reaction reagent, to selectively and
with high sensitivity quantitate the amino acids contained in samples.

 The post-column derivatization method involves separating the amino acids in the
column, then delivering and mixing the derivatizing reagent to let it react with the
amino acids, before finally sending the products to the detector. A flow line diagram
of a typical post-column derivatization process is shown to the right.

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LABORATORY MANUAL LAB TOPICS: Amino Acid Analysis
Course: AQU3202/SP4424 Pemakanan dan Tek Makanan Ikan
Level: Bachelor Programme, UMT

MATERIALS:
1. HPLC (Shimadzu Corp. Kyoto, Japan)
2. Hydrolysis tube.
3. Balance (sensitive to 0.10 mg)
4. Funnel
5. Drummond pipette
6. Beaker
7. Bottle
8. Erlenmeyer flask
9. pH meter
10. Chemicals and reagents (as described below)

Fig. High Performance Liquid Chromatography (Shimadzu Corp, Kyoto, Japan)

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LABORATORY MANUAL LAB TOPICS: Amino Acid Analysis
Course: AQU3202/SP4424 Pemakanan dan Tek Makanan Ikan
Level: Bachelor Programme, UMT

Balance (sensitive to 0.10 mg) Hydrolysis tube

Funnel Drummond Pipette

Light sensitive reagents keeping bottle Erlenmeyer Flask

pH Meter Chemicals

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LABORATORY MANUAL LAB TOPICS: Amino Acid Analysis
Course: AQU3202/SP4424 Pemakanan dan Tek Makanan Ikan
Level: Bachelor Programme, UMT

PROCEDURE

1. PREPARATION OF REAGENTS:

# Sodium citrate buffer solution (pH 2.2) (0.2N trisodium citrate)

1. Weigh 9.8g trisodium citrate dehydrate (C6H5Na3O72H2O).
2. Dissolve in 250mL pure water in 500 mL capacity Erlenmeyer flask.
3. Add 8mL 60% perchloric acid (HClO4).
4. Dilute to 500mL volume and adjust pH to 2.2 using 4N NaOH or 30% perchloric
acid.

# Preparation of Internal Standard (IS) solution for total amino acid analysis:
1. Using electric balance, weigh 6.0 mg DL norleucine (white powdery material). Use
the aluminium dish in the electric balance kit in weighing the IS.
2. With the use of small funnel, transfer IS to 10mL volumetric flask. Wash down with
4N methanesulfonic acid (MSA). Add MSA to 10mL volume. When nearing to 10mL
volume, wash tip to funnel with MSA to collect all IS. Resulting IS concentration is
0.06 mg IS/100 µL MSA.
3. Mix.

# Washing of hydrolysis tube

1. Put 3:1 sulfuric acid (95% H2SO4): nitric acid (60% HNO3) into the hydrolysis tubes.
2. Let stand overnight.
3. Throw the acid to the proper container, wash tubes with water.
4. Fill with pure water and sonicate for 15 minutes. Repeat.
5. Oven dry.

# Preparation of Mobile Phase Solution

a. Mobile Phase Solution A (pH 3.2)

Prepare for 3L using 3L Erlenmeyer Flask.
1. Weigh 58.8 g trisodium citrate dehydrate. Place inside 3L Erlenmeyer falsk.
2. Add ca 2000 mL pure water.
- 210 mL ethanol (99.5% HPLC grade)
- 50mL perchloric acid (60%)
3. Add pure water to 3L volume capacity.
4. Adjust pH to 3.2 using 30% perchloric acid or 4N NaOH.
5. Filter using membrane filter (cellulose nitrate 0.45 µm; 47 mm diameter).

b. Mobile Phase Solution B (pH 10.0)

Prepare for 1L in 1L Erlenmeyer Flask
1. Weigh 58.8 g trisodium citrate dihydrate. Place inside 1 L Erlenmeyer flask.
2. Add ca 700 mL pure water.
- 12.4 g boric acid
- 30 mL 4 N NaOH

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LABORATORY MANUAL LAB TOPICS: Amino Acid Analysis
Course: AQU3202/SP4424 Pemakanan dan Tek Makanan Ikan
Level: Bachelor Programme, UMT

3. Add pure water to 1L volume capacity.

4. Adjust pH to 10.0 using 30% perchloric acid or 4 N NaOH.
5. Filter using membrane filter (cellulose nitrate 0.45 µm; 47 mm diameter).

c. Mobile Phase Solution C

1. Weigh 4g NaOH, place inside 0.5 L Erlenmeyer flask.
2. Dissolve in pure water up to 0.5 L volume capacity.
3. Filter using membrane filter (cellulose nitrate 0.45 µm, 47mm diameter).

# Preparation of reaction reagents

a. Carbonic acid – boric acid buffer solution. Prepare 3L

1. Weigh 122.10g sodium carbonate (Na2CO3)
2. Dissolve in ca 2000mL pure water.
3. Add 40.65g boric acid (H3BO3) and dissolve. (Note: can be dissolve with the use
of magnetic stirrer)
4. Add 56.40 g potassium sulfate (K2SO4) and dissolve.
5. Dilute to 3L volume using pure water.

b. O-phthalaldehyde (OPA) solution. (prepare 1L)

1. Prepare 1L Erlenmeyer flask with aluminium foil.
2. Weigh 0.8g OPA, place OPA in 1L Erlenmeyer flask and dissolve in 15.0mL
ethanol (99.5%). Note: OPA is light sensitive.
3. Add 4mL 10% BRIJ and 1.0 g N-acetyl-L (+) cysteine.
4. Dissolve in carbonic acid-boric acid buffer solution (reaction reagent A) and
dilute to 1L carbonic acid –boric acid buffer solution.
5. Filter using cellulose nitrate membrane filter (cellulose nitrate 0.45µm; 47mm
diameter. Note: filter reaction reagent C first then reaction reagent B to avoid
possible contamination of reagent C.
6. Store in brown bottle.

c. Sodium hypochlorite (NaClO) solution. Prepare 1L

1. In 1L Erlenmeyer flask, add 500mL of carbonic acid-boric acid buffer solution.
2. Add 0.70 mL NaClO. Add carbonic acid-boric acid buffer solution to 1L volume.
Mix.
3. Filter using cellulose nitrate membrane filter (cellulose nitrate 0.45 µm; 47 mm
diameter).

# Pump A = Mobile phase A and C

# Pump B = Mobile phase B
# Rotating pump unit number 1= mobile phase A; number 6=mobile phase C

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LABORATORY MANUAL LAB TOPICS: Amino Acid Analysis
Course: AQU3202/SP4424 Pemakanan dan Tek Makanan Ikan
Level: Bachelor Programme, UMT

2. PREPARATION OF SAMPLE:
1. Weight about 2 ± 0.5 mg (1 ± 0.5 mg is better) sample in electric balance by using
aluminium foil dish.

2. Transfer sample to hydrolysis tube as follows:

- Connect small funnel to tube and put the sample foil dish within the funnel then
wash all sample down with diethyl ether. Diethyl ether should be not more than ½
of the tube volume.

3. Put all tubes into a water contained plastic beaker to evaporate diethyl ether using N
gas or in hot water bath (55oC). This step takes around 45 minutes.

4. Add 100µL 0.6353 mol NOR (norleucine), solution (internal standard) using 100 µL
Drummond pipette.
(Note: wipe Drummond pipette with tissue paper after intake of IS. Be sure to drop
the IS into the hydrolysis tube on the side opposite the tube arm. Drummond pipette
with pure water).

5. Add 400 µL 4N methane sulfonic acid (MSA) using 200 µL micro pipette.

6. Degas completely by freezing with acetone bath under reduced pressure. Acetone bath
is prepared by putting about 150 ml acetone (acetone for iced bath) and several pieces
of dried ices into 500ml of stainless steel beaker.

7. Seal tightly (O-ring is revealed) and hydrolyze for 22-h at 105 – 110 oC. After 30 min,
check the temperature whether it is constant or not. When hydrolysis finish, solution
became black or dark brown due to the hydrolyzed carbon.

8. After hydrolysis, transfer to 5 ml test tube (with scale) and rinse using sodium citrate
butter (pH 2.2). Mix using touch mixer. Note: The sample in this stage can not be
stored because acid digestion will proceed.

9. Add 400 µL 4N NaOH. (Note: can be stored in the freezer at this stage).

10. Calibrate the pH meter and adjust pH sample to 2.2±0.05 by using 4N NaOH solution
or 30% perchloric acid (HClO4).

11. Add sodium citrate buffer solution (pH 2.2) to 5mL total volume of sample. (can be
diluted to 5mL or more depending on the on the concentration of amino acids within
the samples and it would not be overload the HPLC).

12. Filter using syringe filter (0.45 µm nitrate membrane filter paper)

13. Collect sample filtrate. The sample is now ready for injection at the HPLC or keep in
freezer until analysis.

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LABORATORY MANUAL LAB TOPICS: Amino Acid Analysis
Course: AQU3202/SP4424 Pemakanan dan Tek Makanan Ikan
Level: Bachelor Programme, UMT

3. ANALYSIS OF AMINO ACIDS USING HPLC

1. Switch on all the units of HPLC including computer, column oven, pumps, auto
injector, detector etc.

2. Transfer the nozzles from pure water to respective reaction reagents and mobile
phages.

3. Run the software for HPLC operation.

4. At first, inject amino acid standard to stabilize the peaks of the chromatograph by
manipulating pH of the mobile phages.

5. Put all the samples in the auto-injector and run the analysis.

6. One sample takes 58 – 70 min depending on the version of HPLC.

7. Calculate the amount of specific amino acids based on the sample weight, retention
time and peak height.

References
Teshima, S., Kanazawa, A., Yamashita, M., 1986a. Dietary value of several proteins and
supplemental amino acids for larvae of the prawn, Penaeus japonicus. Aquaculture
51, 225–235.
Shimadzu Corporation, Kyoto, Japan http://www.shimadzu.com/an/hplc/aplsys/amino.html

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LABORATORY MANUAL LAB TOPICS: Amino Acid Analysis
Course: AQU3202/SP4424 Pemakanan dan Tek Makanan Ikan
Level: Bachelor Programme, UMT

Fig. Sample of Chromatograph for standard (norleucine)