Introduction
Amino acid analysis by pre-column derivatization has been widely used in reversed-phase HPLC, offering high selectivity and sensitivity for multiple components.
Several derivatization reagents are commercially available and one can choose the most suitable reagent for the application. Among them, orthophtalaldehyde (OPA) is one of the most commonly used reagents because it reacts with amino acids very quickly (seconds) at room temperature and derivatized amino acids can be detected by a uv-visible or fluorescence detector offering increased sensitivity.
In this application, the OPA pre-column derivatization is demonstrated by using the automated pre-column derivatization function of JASCO’s autosampler for greater reproducibility than manual sampling.
Experimental
Instrument
| Pump | PU-2080 |
| Degasser | DG-2080-54 |
| Quaternary low pressure gradient mixer | LG-2080-04 |
| Autosampler (with sample cooling) | AS-2057 |
| Column Oven | CO-2060 |
| Fluorescence Detector | FP-2020 |
Conditions
| Column | CrestPak C18S 4.6mm x 150mm 5um |
| Solvent A | Sodium acetate pH 6.0, Methanol, THF (89:10:1) |
| Solvent B | Methanol, THF (90:10) |
| Gradient | (A/B%) 0min - 85/15, 7min - 80/20, 19min - 56/44, 23min - 48/52, 29min - 48/52, 30min - 0/100, 35min - 0/100, 35.1min - 85/15, 60 minutes 1 cycle |
| Flow rate | 1mL/min |
| Injection volume | 10uL |
| Column temperature | 20 deg C |
| Wavelength | Ex 345 Em 455 |
| Standards | 18 amino acids - each 1nmol/mL in 0.01N HCl |
Keywords
430012H, amino acid analysis, reversed phase HPLC, precolumn derivitization, OPA, orthophtalaldehyde, Asparatic acid, Glutamic acid, Asparagine, Histidine, Serine, Glutamine, Arginine, Glysine, Threonine, Taurine, Alanine, Tyrosine, Methionine, Valine, Phenylalanine, Isoleucine, Leucine, Lysine
Results
Pre-column Derivatization using the AS-2057 Autosampler
The operating principle for pre-column derivatization using the AS-2057 autosampler is shown in figure 2. This function can be used to perform pre-column derivatization automatically.
Conditions for Pre-column Derivatization
