Amino Acid Analysis by Automated Pre-column OPA Derivatization | JASCO

Download PDF August 15, 2017


amino acid analysis
LC-2000 automated HPLC system with fluorescence detector

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.



Quaternary low pressure gradient mixerLG-2080-04
Autosampler (with sample cooling)AS-2057
Column OvenCO-2060
Fluorescence DetectorFP-2020


ColumnCrestPak C18S
4.6mm x 150mm 5um
Solvent ASodium acetate pH 6.0, Methanol, THF (89:10:1)
Solvent BMethanol, 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 rate1mL/min
Injection volume10uL
Column temperature20 deg C
WavelengthEx 345 Em 455
Standards18 amino acids - each 1nmol/mL in 0.01N HCl



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


Figure 1. Chromatogram of a standard mixture of amino acids. 1: Asparatic acid, 2: Glutamic acid, 3: Asparagine, 4: Histidine, 5: Serine, 6: Glutamine, 7: Arginine, 8: Glysine, 9: Threonine, 10: Taurine, 11: Alanine, 12: Tyrosine, 13: Methionine, 14: Valine, 15: Phenylalanine, 16: Isoleucine, 17: Leucine, 18: Lysine

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.

Figure 2. Pre-column derivatization using an AS-2057 Autosampler.

Conditions for Pre-column Derivatization

About the Author

DJ Tognarelli received his B.S. from the University of Richmond where he studied Analytical Chemistry. He is a Chromatography Applications Scientist at JASCO.