Design and Assembly of an Inexpensive, Automated Microbore Amino Acid Analyzer: Separation and Quantitation of Amino Acids in Physiological Fluids

Abstract

An amino acid analyzer capable of separating and quantitating 0.5 to 20 n moles of each ninhydrin-positive compound in physiological fluids has been designed and assembled from commercially available components. The buffer sequence, column temperature change and sample application are controlled by an automatic programmer constructed from a series of timers. The liquid delivery portion of the instrument consists of a series of polystyrene and stainless steel chambers pressurized with argon and connected through valves and manifolds to conventional positive displacement pumps. The column is highly polished stainless steel tubing (0.21-cm ID) packed with 9-µ cation exchange resin. Micro colorimeters, equipped with appropriate interference filters and small-volume (2–8 µ1) flow cells, are used as detectors. The sample loader is a dual 20-port automatic valve containing 25-µl sample loops. Small-bore teflon tubing (32 AUG), interconnected with tubing adapters and connectors, is used for buffer lines and reaction coil (100° C); ninhydrin lines are 1/16-inch stainless steel tubing. Separation of 42 ninhydrin positive compounds, including column equilibration, is accomplished in 5 hours. Procedures for the extraction of amino acids from physiological fluids and tissues as well as the preliminary clean-up of these extracts are also described.