This application note describes amino acid analysis methods using Agilent ZORBAX Eclipse Plus C18 columns for a variety of Agilent LC instrumentation and separation goals. The methods utilize a proven ortho-phthalaldehyde/9-fluorenyl- methyl chloroformate (OPA/FMOC) derivatization and take advantage of the Agilent 1200 Series SL and Agilent ZORBAX Eclipse Plus C18 stationary phase columns.
This document presents improved High-Performance Liquid Chromatography (HPLC) methods for amino acid analysis, utilizing the Agilent ZORBAX Eclipse Plus C18 stationary phase. The protocols are designed to offer better retention of aspartic and glutamic acids and higher resolution for closely eluting pairs compared to previous iterations.
The note details ten specific column configurations ranging from traditional high-resolution methods to rapid high-throughput analyses. These methods are optimized for various Agilent LC models, including the 1100 and 1200 Series, and cover particle sizes from 1.8 to 5 micrometers. The document provides specific instructions for mobile phase preparation, derivatization reagents, and automated liquid sampler programs.
| Column dimensions (Traditional) | 4.6 x 250 mm, 5 um |
|---|---|
| Column dimensions (Rapid Resolution) | 4.6 x 150 mm, 3.5 um |
| Column dimensions (High Throughput) | 4.6 x 100 mm, 1.8 um |
| Mobile phase A composition | 10 mM Na2HPO4, 10 mM NaB4O7, pH 8.2 |
| Mobile phase B composition | Acetonitrile: Methanol: Water (45:45:10) |
The methods are developed for Agilent 1100 with quaternary or binary pumps, and Agilent 1200 with binary pumps (400 bar) or 1200 SL with binary pumps (600 bar).
The 2.1 x 150 mm, 3.5 um Rapid Resolution method is the preferred replacement, offering comparable analysis time and similar column dimensions.
Once opened, the OPA reagent is potent for about 7-10 days.