Page 12 - Application Handbook - Liquid Chromatography

LAAN-A-LC-E235

Application High Performance Liquid Chromatography

News High Speed, High Resolution Analysis (Part 47)

Analysis of Pre-Column Derivatized Amino Acids by
No.L458 the Nexera SIL-30AC Autosampler (Part 3)

Amino acid composition analysis has traditionally been Table 1 Derivatization Reagents
conducted for protein quantitation and peptide ● Mercaptopropionic Acid
structure prediction. Its use has also extended in recent 3-MercaptopropionicAcid 10 µL in 0.1 mol/L Borate Buffer (pH9.2) 10 mL
years to the quantitation of such functional
components as branched chain amino acids (BCAA). ● o - phthalaldehyde - Ethanol Solution
o - Phthalaldehyde 10 mg in 0.1 mol/L Borate Buffer (pH9.2) 5 mL
Previously, in Application News No. L432 and L437, we
introduced examples of analysis of amino acids ● Fluorenyl Methyl Chloro Formate - Acetonitrile Solution
subjected to fluorescence derivatization using 9-Fluorenyl Methyl Chloro Formate 4 mg in Acetonitrile 20 mL
o-phthalaldehyde (OPA) using the SIL-30AC. Here, we
introduce an example of fast analysis of amino acids in
proteins.

■ Simultaneous Determination of 17 Amino Acids Table 2 Analytical Conditions
This method utilizes the pretreatment functions of the Column : YMC-Triart C18 1.9 µm
Nexera SIL-30AC to automatically derivatize amino acid （50 mm L. × 3.0 mm I.D., 1.9 µm, YMC CO., LTD.）
samples with OPA. The sample rack is set up with the Mobile Phase : A : 20 mmol/L Phosphate Potassium Buffer (pH 6.2)
B : 60/40 Acetonitrile/Methanol

underivatized samples in one section and empty vials in Time Program : Gradient Elution
another section that will contain the sample and Flowrate : 1.2 mL/min
reagent(s) delivered by the SIL-30AC prior to injection. Column Temp. : 40 ˚C
Injection Volume : 1 µL
Table 1 shows the derivatizing reagents used with this Detection : RF-20AXS Ex. at 350 nm, Em. at 450 nm
method. Fig. 1 shows the chromatogram obtained from → Ex. at 266 nm, Em. at 305 nm (8.5 min)
measurement of a standard mixture of seventeen Cell Temp. : 30 ˚C
amino acids in solution using the analytical conditions Flow Cell : Conventional Cell
shown in Table 2. The total analysis time can be
shortened by using the overlapping injection feature
that was described in Application News No. L437. This
feature permits derivatization and injection preparation
of the sample to follow the sample that is currently
being analyzed.

mV
225
■ Peaks 17
200 1. Aspartic Acid, 2. Glutamic Acid, 3. Serine, 4. Histidine, 5. Glycine, 6. Threonine,
7. Argininge, 8. Alanine, 9. Tyrosine, 10. Valine, 11. Methionine, 12. Cystine,
175 13. Phenylalanine, 14. Isoleusine, 15. Leucine, 16. Lysine, 17. Proline
150

125
100 7 8
2
1 3 15
75 9 14
5
50 6 11 13 16
10
4
25
12
0
2.5 5.0 7.5 min
Fig. 1 Chromatogram of 17 Amino Acids in Standard Solution (25 μmol/L each)