Separation Of PTH-amino Acids Research Articles

Capillary electrochromatography with gradient elution.

A capillary electrochromatograph incorporating a gradient-forming system generally employed in HPLC is described, and the use of gradient elution in reversed phase electrochromatography is demonstrated by the separation of PTH-amino acids and steroid hormones. The gradient former employs two reciprocating displacement pumps to control the composition of the eluent in the reservoir at the column inlet with time in a controlled manner. Thus, the composition of the mobile phase flowing through the column and driven by electrosmotic forces can be changed with time in a controlled fashion as customary in HPLC with gradient elution. The design of the system allows also for isocratic elution by pumping the eluent of constant composition through the cavity at the column inlet and thus continuously supplying fresh buffer. The eluent gradient is generated by the two pumps and a 10 microL mixer. From there the liquid passes at a flow rate of 0.1-0.2 mL/min through the 17 microL cavity housing the column inlet and an electrode. The flow of the mobile phase was electrosmotic at an effective overall electric field strength of 500-1500 V/cm through a 50 microns x 20/12 cm capillary column packed with 3.5 microns octadecylated silica particles. Gradient profiles generated in this manner were highly reproducible. The same-day and day-today reproducibilities of the electrosmotic flow were found to be better than 3%. The use of the capillary electrochromatographic system was demonstrated with isocratic and gradient elution for the separation of complex mixtures of biologically interesting substances. The influence of the column temperature on the electrosmotic flow velocity and retention of PTH-amino acids was also investigated.

Retention prediction and computer-assisted optimization for the separation of PTH-amino acids in isocratic reversed-phase liquid chromatography

Retention prediction of phenythiohydantoin amino acid derivatives in isocratic reversed-phase liquid chromatography was investigated. The predicted retention data of all derivatives were evaluated by comparing them with actually measured retention data. Excellent agreements between these data were found. The optimized conditions to separate overlapping components can also be predicted using the developed computer-assisted optimization system with the concept of retention prediction.

Separation of PTH-amino acids by Isocratic high-performance liquid chromatography

Separation of PTH-amino acids by Isocratic high-performance liquid chromatography