A general method for optimizing gradient-elution separations is described and applied to the specific separation of the 20 PTH-amino acid derivatives. The optimum separation of these compounds is shown to be performed in 25 min using a PE column with a ternary gradient containing phosphate buffer, methanol, and tetrahydrofuran as the mobile phase. The specific optimized gradient-elution separation is compared to a previously reported optimized isocratic separation, and advantages of each system are compared. Additional influences on separation selectivity for the PTH-amino acids, such as ionic strength and the use of serially connected columns, are also investigated.
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Glajch et al. (1987) studied this question.