Amino Acid Analyzer

YL Amino Acid Analyzer is to analyze the first or second amino acids with a variety of solutions in an easy way.
With a superior sensitivity, this enables to detect derivatized natural amino acids in animal tissues, broths, fruits and
Food

beverage juices and hydrolyzed amino acids in protein, collagen, peptides and processed foods.

• The need of derivatization

a. Except for tryptophane, tyrosine and phenylalanine that have an absorbance at UV/Vis wavelength range, amino
acids formed in single bond have no absorbance at UV/Vis wavelength range so they are needed to be derivatized
to have fluorescence by derivatization reagents.
b. Derivatization Reagents for Amino Acids
- Ninhydrin
- OPA (ortho-phthalaldehyde)
- PITC (phenylisothocyanate)
- FMOC (fluorenylmethoxycarbonyl chloride)

Post-column derivatization
This procedure is a method to detect the derivatized amino acids that are separated in a cation exchange column by
adding the derivatization reagent at a reactor online in front of a detector .

a. Ninhydrin Method (Reaction condition under temperature control)

: This procedure uses Ninhydrin to indirectly detect
1° Amino Acid
amino acids with UV/VIS spectrophotometry after the CO 130, 50 sec
2

amino acids have been separated by the cation exchange H NH 2

column. The Ninhydrin reagent, which constructs Ninhydrin

R

derivatives of both the first and second amino acid Ruhemann's Purple
570, 405 nm
groups. The amino acids of the first group would have Hydrindanth
reacted with the Ninhydrin reagent at 130 °C as it passed
130°C
through Hydrindatin in the column. The derivatives of
the first group produce purple products, with a UV/VIS
Ninhydrin 2° Amino Acid λmax 440, 405 nm
absorbance values at 570 nm and 405 nm.
b. OPA Method
: This method utilizes the OPA reagent to react with a free
amino acid that has been separated by cation exchange OPA 1° Amino Acid NMe 2
-
in the column and construct a derivative of an amino CHO CO 2
pH ≥ 9, RT S
H NH H
acid that is easily detected and measured by either UV/ CHO R
2

H CO 2
-

Vis detector or Fluorescence detector. The amino acid R

derivative is constructed at room temperature which HS
NMe 2 N-Substiruted 2-Alkylthio Isoindole
is different from Ninhydrin method. Isoindole, which Thiofluor
fluoresces under UV light, is produced as the first amino
acid reacts with the OPA reagent and Thioflour, allowing for the detection of amino acids by UV/VIS fluorescent
methods. (OPA method can be used for both post and pre-column derivatization.)

Sample preparation procedure

a. Decide the sample amount (Ex: Place 200 mg of soy sauce in a vial)
b. Add 30 ml of 6 N HCl to the vial. Incubate the sample at 130 °C for 24 hours to hydrolyze the amino acids.
c. Dilute the hydrolyzed sample by adding 50 ml of purified water.
d. Take 1 ml in the vial and vaporize it. (Pre-column Derivatization)
e. Put 1 ml of pH 2.2 Borate buffer (or 0.02N HCl) into the vial and dissolve it.
d. Filter the sample with a 0.45 µm aqueous syringe filter
e. Mix 20 ml of HCl and 20 µl of the hydrolyzed sample

• Application
• Life Sciences: Protein structure, Synthesized peptide qualification
• Pharmaceutical: Medicine for Injection, Medicine for Nutrients
• Food and beverages: Measurement of the essential amino acids found in food
• Clinical: Diagnosis of amino acid levels and deficiencies

8 Dedicated Analyzer

전용분석_영문2012.indd 8 2012-10-05 오후 3:45:30

 ■ Post-Column Derivatization

•Column : High efficiency Sodium 8

1. Aspartic acid
Cation-exchange (4.0 mm, 150 mm) Standard

Food
2. Threonine
•Temperature : 42 °C 3. Serine
4. Glutamic acid
•Flow rate : 0.4 ml/min 10
5. Proline
9 6. Glycine
•Mobile phase : 1700-0112 : 23 6
12
15 17 7. Alanine
1 11 8. Cystine
Na740 : RG011 (Gradient) 7 13 14
4 9. Valine
•Detector : UVD 570 nm 19 10. Methionine
5 16 18 11. Isoleucine
•Injection volume : 20 µL 12. Leucine
13. Tyrosine
14. Phenylalanine
Post-column Conditions 0 10 20 30 40 50 60
15. Lysine
Time (min) 16. Ammonia
•System : Pinnacle PCX with 0.5 ml reactor 17. Histidine
•Reagent : TRIONE (Derivatizing reagent) 16 18. Tryptophan
19. Arginine
•Reactor condition : 130 °C, 0.3 ml/min Sample
12

15

79
1 4 6
11
14 17

23 5 10 13
18 19

0 10 20 30 40 50 60
Time (min)

■ Pre-Column Derivatization
•Column : C18 (4.6 mm, 150 mm, 5 µm)
•Temperature : 40 °C
•Flow rate : 2.0 ml/min 7 8 9 11

•Mobile phase : A : 40 mM Na2HPO4 pH7.8 3 6

10 16 17
12 14
B: ACN:MeOH:Water (45:45:10), 15
2
Gradient 1 5
13
4
•Detector : UVD 338 nm
•Injection volume : 20 µL

Action Type From To Amount (µl)

1 Add Sample Destination 20
2 Add Reagent A Destination 30
0 5 10 15 20 25
3 Mix 3 35 Time (min)
4 Add Reagent B Destination 20
5 Mix 5 0 50
1. Aspartic acid 2. Glutamic acid 3. Serine, 4. Histidine
5. Glycine 6. Threonine 7. Arginine 8. Alanine
9. Tyrosine 10. Cystine 11. Valine 12. Methionine
13. Tryptophane 14. Phenylalanine 15. Isoleucine 16. Leucine
17. Lysine

YL INSTRUMENT CO., LTD. 9

전용분석_영문2012.indd 9 2012-10-05 오후 3:45:30