This paper describes a new methodology for amino acid analysis using recently developed columns with an engineered particle size of 1.8 µm. These patented particles are specifically designed to create less backpressure than other sub-two-micron particles on the market; therefore, they can be used on 400 bar HPLC systems as well as higher pressure limit instruments.
The document presents a new methodology for Amino Acid Analysis (AAA) designed to improve precision, run time, column longevity, and ruggedness compared to previous methods. It utilizes recently developed 1.8 micron engineered particle columns that allow operation on standard 400 bar HPLC systems while maintaining high sensitivity.
The application details the use of sequential OPA/FMOC chemistry first introduced by Hewlett-Packard/Agilent in 1988. The text provides specific instrument configurations, including pump parameters, flow rates for various column sizes (2.1 mm, 3.0 mm, and 4.6 mm), gradient timetables, and detector settings for both Diode Array Detectors (DAD) and Fluorescence Detectors (FLD). It also includes instructions for preparing low-sensitivity amino acid standard solutions.
| Column Particle Size | 1.8 pm |
|---|---|
| Maximum System Pressure | 400 bar |
| Flow Rate (2.1 mm id) | 0.42 mL/min |
| Flow Rate (3.0 mm id) | 0.85 mL/min |
| Flow Rate (4.6 mm id) | 2.00 mL/min |
| Column Temperature (TCC) | 30 C to 40 C |
The columns can be used on 400 bar HPLC systems as well as higher pressure limit instruments.
The method uses OPA/FMOC chemistry to derivatize primary and secondary amino acids.
The analysis is performed on a Rapid Resolution HT Eclipse Plus C18, 4.6 x 50 mm column.