Page 58 - Application Handbook - Liquid Chromatography

LAAN-A-LC-E210

Application High Performance Liquid Chromatography

News High Speed, High Resolution Analysis (Part 44)

Analysis of Pre-Column Derivatized Amino Acids by
No.L432 SIL-30AC Autosampler

Amino acid analysis is required in a wide range of fields, Application News articles No. L292 and L299A.
including foods and pharmaceuticals, and various Here, using the RF-20Axs fluorescence detector and the
methods of derivatization have been devised to improve SIL-30AC autosampler with its automated pretreatment
sensitivity and selectivity when conducting amino acid functions, we introduce the analysis of amino acids
analysis by HPLC. Previously, amino acid analysis by using pre-column derivatization with OPA and FMOC.
post-column derivatization using OPA was introduced in

n Simultaneous Determination of 22 Amino Acids
Using the automated pretreatment functions of the wavelengths, simultaneous analysis was conducted
Nexera SIL-30AC autosampler, primary and secondary utilizing the automatic wavelength switching feature.
amino acids were automatically derivatized into Table 1 shows the derivatization reagents used in this
fluorescent substances within the autosampler using method, and Fig. 1 shows the reagent addition and
o-phthalaldehyde (hereafter, OPA) and 9-fluorenyl mixing steps used to conduct the automated
methyl chloro formate (hereafter, FMOC), respectively. derivatization using the SIL-30AC autosampler. Since a
After separation of the derivatized amino acids using constant reaction time can be maintained with the
the ultra-high speed YMC-Triart C18 column (1.9 μm, automated derivatization using the autosampler,
YMC Co., Ltd.), high-sensitivity detection was excellent repeatability can be obtained compared with
conducted using the RF-20Axs fluorescence detector. pre-column derivatization by manual operation.
Since the OPA-derivatized amino acids and FMOC- Table 2 shows the analytical conditions, and Fig. 2
derivatized amino acids are detected at different shows the chromatogram.

Table 1 Derivatization Reagents
Vial ● Mercaptopropionic Acid
MercaptoPropionicAcid 45 µL 3-Mercaptopropionic Acid 10 μL in 0.1 mol/L Borate Buffer (pH 9.2) 10 mL
● o-Phthalaldehyde Solution
OPA 22 µL
o-Phthalaldehyde 10 mg in 0.1 mol/L Borate Buffer (pH 9.2) 5 mL
Sample 7.5 µL
● Fluorenyl Methyl Chloro Formate - Acetonitrile Solution
Mix 9-Fluorenyl Methyl Chloro Formate 4 mg in Acetonitrile 20 mL
Wait 1.0 min Table 2 Analytical Conditions
FMOC 10 µL Column : YMC-Triart C18, 1.9 μm
(75 mmL. × 3.0 mmI.D., 1.9 μm, YMC Co., Ltd.)
Mix Mobile Phase : A : 20 mmol/L Phosphate Potussium Buffer (pH 6.9)
B : 45/40/15 Acetonitrile/Methanol/Water
Time Program : B Conc.11 % → 13 % (0.00-3.00 min)
Wait 2.0 min → 31 % (5.00 min) → 37 % (7.5 min)
→ 70 % (10.00 min) → 100 % (10.50-13.50 min)
→ 11 % (14.00 min)
Injection to HPLC 1 µL Flow Rate : 0.8 mL/min
Column Temp. : 35 °C
Injection Volume : 1 μL
Fig. 1 Flowchart of Automated Pre-Column Derivatization with SIL-30AC Detection : RF-20Axs Ex. at 350 nm, Em. at 450 nm
→ Ex. at 266 nm, Em. at 305 nm (9.0 min)
Cell Temp. : 20 °C
Flow Cell : Conventional cell