FACTORS AFFECTING RESOLUTION AND REPRODUCIBILITY OF GRADIENT ELUTION AUTOMATIC AMINO ACID CHROMATOGRAMS.
ARMY MEDICAL RESEARCH LAB FORT KNOX KY
Pagination or Media Count:
Reduction of the total volume of the gradient did not reduce proportionately the elution time of each amino acid. Titration of the resin was an important factor. The elution position of cystine relative to valine was shifted by changes in the column length. Trials with many different gradients showed that the elution positions of all the amino acids could be accelerated or retarded, and to a lesser extent, contracted or expanded, but the relative positions remained fairly constant, owing in part to limitations of the nine-chambered gradient device. The early part of the chromatogram was sensitive to salt and pH of the sample and to variations in equilibration, especially when the column was equilibrated at one pH and the gradient was started at another. Increasing the flow rate from 0.5 to 1.2 mlmin resulted in a demonstrable increase in peak width and loss in resolution. Increasing the flow rate to 0.7 mlmin decreased analysis time to less than 16 hours without loss in resolution. Runs could be made overnight conveniently and with good reproducibility. A 0.636 x 11 cm column eluted with pH 6.00 buffer gave a convenient method of separating four basic amino acids in less than 2 hours at low flow rates and low pressures. Principal causes for disturbances in the baseline, other than the ammonia plateau, were precipitation of hydrindantin and formation of contaminants in the buffers at room temperature. Author