Analytical Method for 18 Amino Acids

Table of contents

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I. Instruments and Reagents

Instruments:

Analytical balance (precision: 0.1 mg)
Thermostatic water bath
Volumetric flasks
Test tubes (1.5 × 15 cm or 1.5 × 10 cm)
Micro-syringe (5 μL or 10 μL)
Adjustable micropipette (1000 μL and 200 μL) with multiple tips
Vortex mixer
HPLC system with a dedicated amino acid analysis column (4.6 × 250 mm, 5 μm)

Reagents:

Ultrapure water (≥18 MΩ·cm)
Acetonitrile (HPLC grade)
Sodium acetate trihydrate (analytical grade)
Glacial acetic acid (analytical grade)
Derivatization reagents A and B (store in the refrigerator; use proper protection as these reagents are hazardous)
Hexane (HPLC grade)
0.1 mol/L hydrochloric acid solution: Add 9.0 mL concentrated HCl to deionized water and dilute to 1000 mL.
Norleucine internal standard solution: Weigh approximately 10 mg of norleucine, dissolve in 10 mL of 0.1 mol/L HCl solution, and mix thoroughly.

II. Preparation of Mobile Phases

Mobile Phase A:

Composition: 0.1 mol/L sodium acetate solution (pH 6.5): acetonitrile = 93.0:7.0
Preparation: Dissolve 13.6 g of sodium acetate trihydrate in 1000 mL of water. Adjust the pH to 6.5 using glacial acetic acid or sodium hydroxide solution. Measure 930 mL of this solution and mix it with 70 mL of acetonitrile. Filter through a 0.22 μm membrane.

Mobile Phase B:

Composition: Water: acetonitrile = 20.0:80.0
Preparation: Mix 200 mL of water with 800 mL of acetonitrile. Filter through a 0.22 μm membrane.

III. Derivatization Reaction

1. Standard Solution Concentrations

Name	M.W.	C (μmol/mL)	Name	M.W.	C (μmol/mL)
Aspartic Acid	133.10	2.50	Cystine	240.30	1.25
Glutamic Acid	147.13	2.50	NH₄Cl	53.49	2.50
Serine	105.09	2.50	Tyrosine	181.19	2.50
Glycine	75.067	2.50	Valine	117.15	2.50
Histidine	155.15	2.50	Methionine	149.21	2.50
Arginine	174.20	2.50	Isoleucine	131.17	2.50
Threonine	119.12	2.50	Leucine	131.17	2.50
Alanine	89.093	2.50	Phenylalanine	165.19	2.50
Proline	115.13	2.50	Lysine	146.19	2.50

Name	M.W.	C (μmol/mL)
Aspartic Acid	133.10	2.50
Glutamic Acid	147.13	2.50
Serine	105.09	2.50
Glycine	75.067	2.50
Histidine	155.15	2.50
Arginine	174.20	2.50
Threonine	119.12	2.50
Alanine	89.093	2.50
Proline	115.13	2.50
Cystine	240.30	1.25
NH₄Cl	53.49	2.50
Tyrosine	181.19	2.50
Valine	117.15	2.50
Methionine	149.21	2.50
Isoleucine	131.17	2.50
Leucine	131.17	2.50
Phenylalanine	165.19	2.50
Lysine	146.19	2.50

2. Preparation of Sample Solutions

Accurately measure or weigh an appropriate amount of the sample and prepare it into a solution of the desired concentration for use.

3. Derivatization Steps

Dilute derivatization reagents A and B with diluent to 1/5 of their original concentrations.
Pipette 160 μL of the standard solution into a test tube. Add 100 μL each of diluted reagents A and B. Mix well and react at room temperature for 60 minutes. Then, add 400 μL of hexane, seal the tube, and shake for 5–10 seconds. Let it stand at room temperature to separate layers.
Take 200 μL of the lower layer and mix with 800 μL of water. Take 200 μL of the mixture and mix with another 800 μL of water. Filter through a 0.22 μm organic membrane for analysis.
For the sample, follow the same procedure as the standard. Adjust the dilution volume based on the amino acid content.

Note: If acid hydrolysis is used for sample pretreatment, ensure the acid is evaporated completely before derivatization. Derivatization requires an alkaline environment for complete reaction, as excess acidity can neutralize the reaction.

IV. Chromatographic Conditions

Column	Ultisil Amino Acid, 4.6×250 mm, 5 μm
Mobile Phase	Phase A: 0.1 mol/L sodium acetate solution (pH 6.5): acetonitrile = 93.0:7.0 Phase B: Water: acetonitrile = 20.0:80.0
Gradient Program	Time (min)	Phase A	Phase B
	0.01	100.0%	0.0%
	11	93.0%	7.0%
	13.9	88.0%	12.0%
	14	85.0%	15.0%
	29	66.0%	34.0%
	32	30.0%	70.0%
	35	0.0%	100.0%
	42	0.0%	100.0%
	45	100.0%	0.0%
	60	100.0%	0.0%
Flow Rate	1.0 mL/min
Temperature	40 °C
Wavelength	254 nm
Injection Volume	10 μL

Chromatogram of 18 amino acids

1	Aspartic Acid	8	Alanine	15	Isoleucine
2	Glutamic Acid	9	Proline	16	Leucine
3	Serine	10	NH₄Cl	17	Norleucine
4	Glycine	11	Tyrosine	18	Phenylalanine
5	Histidine	12	Valine	19	Lysine
6	Arginine	13	Methionine
7	Threonine	14	Cystine

1	Aspartic Acid	11	Tyrosine
2	Glutamic Acid	12	Valine
3	Serine	13	Methionine
4	Glycine	14	Cystine
5	Histidine	15	Isoleucine
6	Arginine	16	Leucine
7	Threonine	17	Norleucine
8	Alanine	18	Phenylalanine
9	Proline	19	Lysine
10	NH₄Cl

V. Procedures and Precautions

1. Procedures:

Set the column temperature to 40 °C and the flow rate to 1.0 mL/min.
Degas the channels A and B using a solution of acetonitrile: water = 20:80 (if channel B does not contain a buffer, degassing with this solution is not required). Then degas channel B with mobile phase B.
Wash the system with acetonitrile: water = 20:80 (channel A) for 20 minutes to prevent buffer salt precipitation.
Use mobile phase A to degas channel A, then run the baseline for 30 minutes with mobile phase A to equilibrate the column.
Perform a blank gradient run.
Inject samples for analysis.
After analysis:
- Replace mobile phase A with acetonitrile: water = 20:80 and inject water samples to clean the autosampler. Perform gradient elution.
- Rinse the column with 90% acetonitrile for at least 40 minutes.

2. Precautions:

Inject the standard solution before the sample solution.
Prepare buffer solutions fresh daily.
Beware of buffer salt precipitation.

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