This technical note demonstrates the performance of amino acids analysis on the Agilent 1100 Series modules and systems for LC. Detailed information regarding instrumental and chromatographic conditions and performance are given. The precision for retention times over 10 runs is as low as 0.2 % RSD, and the RSD of areas is between 0.6 and 5 %. Since the introduction of automated amino acid analysis on the HP 1090 Series HPLC system using a two step precolumn derivatization, this technique has become a well accepted and routine method for the analysis of primary and secondary amino acids.
This document details the performance of amino acid analysis on the Agilent 1100 Series modules and systems for Liquid Chromatography (LC). It demonstrates that this technique can achieve better performance than the previous HP 1090 Series system. The note covers instrumental and chromatographic conditions, including the use of binary pumps, online vacuum degassers, autosamplers, thermostatted column compartments, and detectors (DAD and FLD).
The document provides specific experimental procedures for preparing mobile phases, standards, and derivatization reagents using ortho-phthalaldehyde (OPA) and 9-fluorenylmethyl chloroformate (FMOC). It includes detailed instructions for preparing calibration standards for both standard and high sensitivity analyses, as well as extended amino acid kits. Results are presented for both UV and fluorescence detection methods, highlighting the precision of retention times and areas, and the limits of detection.
| Limit of Detection (DAD) | below 5 pmol |
|---|---|
| Limit of Detection (FLD) | below 100 fmol |
| Linearity Correlation Factor | 0.99998 to 0.99999 |
| Retention Time Precision | 0.2 % RSD |
| Area Precision (RSD) | 0.6 to 5 % |
| Flow Rate | 0.45 ml/min |
| Oven Temperature | 40 C |
The fluorescence detector (FLD) is recommended for concentrations below 100 pmol due to better selectivity.
Ortho-phthalaldehyde (OPA) is used for primary amino acids and 9-fluorenylmethyl chloroformate (FMOC) is used for secondary amino acids.
The flow rate is set to 0.45 ml/min.