The Moore and Stein method applied to the automatic analyzer

The sample to be analyzed to autmatically loaded in to the analyzer in the form of small capsules (the sample is injected into the capsule prior to loading). The sample is then 'washed' onto the ion exchange column by a buffer (the weakest) after being kept at a specific temperature by the fridge. The system, prior to loading, is flushed through with the weakest buffer to extinguish any impurities, such as ammonia (common in all walks of life) which is extremely detrimental to the amino acid.

Having been 'washed' onto the column, the amino acids 'stick' in varying strengths to the resin. Buffers of increasing strength 'knock' the amino acids off of the column. The eluate from the column is mixed with ninhydrin and passed into a reaction coil. This reaction heats the mixture to a specific temperature so that the ninhydrin mixture reacts forming differing dye compelxes. These complexes are passed into a photometer, the mixture is then placed in a flowecell, where the light absorbed is the detected (at 570nM and 440nM). This quantity is the correlated and can be output as a chromatogram on a chart recorder or a special integrator which prints out a small chromatogram and also the relevant information.

This whole function is controlled via a programmer (or computer). The programmer makes the whole system far more flexible - i.e. the type of buffer can be changed at any point abd for varying lengths of time, different capsules can be loaded at will and many other functions can be performed by reprogramming.

The reason why light is passed through the mixture at two different wavelengths is that most amoni acids are detected at 570nM but not all, the other remaining amino acids are detected at 440nM.