How does an amino acid analyzer work?
Amino acid analysis is a technique based on ion exchange liquid chromatography, with a wide range of applications to provide qualitative and quantitative component analysis. The basic principle of the operation is the continuous flow chromatography procedure developed by Spackman, Moore, and Stein in 1958. This basic principle has now been improved to allow fully automated, high-speed, and sensitive analysis. Sometimes, this is referred to as classical amino acid analysis. A sample containing a mixture of amino acids is loaded onto a cation exchange resin column. Then, buffers with different pH values and ionic strengths are pumped into the chromatography column to separate the various amino acids. The temperature of the chromatography column is precisely controlled and can be changed as needed to achieve the desired separation. The column effluent is mixed with ninhydrin reagent, and the mixture passes through a high-temperature reaction coil. In the reaction coil, ninhydrin reacts with the amino acids in the effluent to form colored compounds. The amount of colored compound produced is proportional to the amount of amino acid present in the effluent. From the reaction coil, the effluent/ninhydrin mixture is sent to a photometer unit, where the amount of each colored compound is determined by measuring the amount of light absorbed. Light absorption is measured at two wavelengths (570 nm and 440 nm), as the colored compounds produced by amino acids absorb light at wavelengths of 440 nm or 570 nm. The photometer output is connected to a dual-channel chart recorder, which plots the amino acid concentration as a series of peaks, or to an appropriate integration system. The retention time of the peaks on the chart determines the amino acid, and the area under the peaks indicates the amount of amino acid present. Since the amino acid analyzer is a comparative instrument, a calibration analysis must be performed before starting a series of analyses to generate a standard trace for comparison.
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