Combined effect of temperature and organic modifier concentration on the retention under single mode gradient conditions in reversed‐phase HPLC

Abstract

The combined effect of temperature, T, and organic modifier concentration, phi, on the retention under gradient conditions in RPLC is studied by considering, both theoretically and experimentally gradients, of phi at constant T and gradients of T at constant phi. Two approaches are examined: in the first approach the prediction of the elution time of a sample solute is based on the isocratic/isothermal properties of this solute. The second approach is based on a direct fitting procedure of a proper retention model to 2-D isocratic/T-gradient or isothermal/phi-gradient retention data. These approaches were tested using alkylbenzes in eluting systems modified by ACN. We found that both approaches can give excellent predictions under certain prerequisites. However, the first approach exhibits the notable advantage that it can be used effectively to predict retention times under any kind of phi-gradients at constant T or T-gradients at constant phi. The second approach has the advantage that it is relatively simple but its applicability is very restricted since its predictions are satisfactory only if the gradients are of the same kind with those used in the fitting procedure and the conditions lie within those used for fitting.

Multimode Gradient Elution in Reversed-Phase Liquid Chromatography: Application to Retention Prediction and Separation Optimization of a Set of Amino Acids in Gradient Runs Involving Simultaneous Variations of Mobile-Phase Composition, Flow Rate, and Temperature

“…However, due to hysteresis phenomena that appeared when we used conventional chromatographic columns, the oven temperature is not the effective temperature experienced by the analyte. This temperature is estimated by means of Newton’s law, which yields , T e f = false( a 0 + a 1 t + a 2 t 2 + ··· + a n t n false) − 1 h ( a 1 + 2 a 2 t + ··· + n a n t n − 1 ) + 1 h 2 false( 2 a 2 + ··· + n ( n − 1 ) a n t n − 2 false) ··· ( − 1 h ) n false( n ! a n false) + c e − h t …”

“…In multimode gradient elution, a composite gradient ( x 1 , x 2 ,..., x m ) versus t is formed in the mixer of the chromatographic system, where t is the time variable and the coordinates x 1 , x 2 ,..., may be volume fractions of the mobile-phase constituents, φ 1 , φ 2 ,..., the pH of this phase, the flow rate F , or the column temperature T . Since the temperature that the analyte feels inside the column may be not the column temperature T but an effective one, T ef , we should consider T ef in place of T . , The ( x 1 , x 2 ,..., x m ) versus t profile is transformed to a new one, ( x 1 ′ , x 2 ′ ,..., x m ′ ) versus t , at the inlet of the chromatographic column, because any variation in F or T in the mixer reaches the column almost instantaneously, whereas any change in φ i or pH needs some time to cover the same distance. Moreover, if F varies, there is a distortion of the φ i or pH versus t profiles when they reach the inlet of the chromatographic column since any change in φ i or pH that takes place at the time t p * in the mixer is transferred to the beginning of the column at t p , where t p * and t p are interrelated through the following equation , t normal D ( F = 1 ) = ∫ t p * t p F ( t ) d t …”

Isolation and Purification of a Hydrophobic Non-Ribosomal Peptide from an Escherichia coli Fermentation Broth

“…We found that all three factors had a significant effect on the resolution (Supplementary Materials). The optimization of HPLC methods often involves the evaluation of pH [23][24][25][26][27] and temperature [23,[28][29][30][31]. The temperature affects viscosity [28] as well as hydrophobic interactions by influencing enthalpy and entropy [32,33].…”

Competitional hydrophobicity driven separations under RP‐LC mechanism: Application to sulfonylurea congeners

“…. ., n; usually n is not greater than 2) in the mobile phase and their corresponding concentrations (C i ) [23,24]. This influence on retention (represented as the ten-base logarithm of the capacity factor, k) is described via a second order (j = 2) polynomial equation, according to relationship:…”