Hold‐up volume and its application in estimating effective phase ratio and thermodynamic parameters on a polysaccharide‐coated chiral stationary phase

Abstract

As an "unretained" marker, 1,3,5-tri-tert-butylbenzene (TTBB) has been commonly used to measure the hold-up volume. Despite many racemates have been resolved on Chiralcel OJ column, the hold-up volume of the column is still not well characterized. The aim of this work was to evaluate the chromatographic behavior of TTBB on the OJ column, and its application in estimating the effective phase ratio and thermodynamic parameters. The hold-up volume was affected not only by the mobile phase composition but also the solvents used for dissolving TTBB. A higher concentration of TTBB (0.500 mg/mL) showed a better reproducibility than when used at a lower concentration. After correction for thermal expansion of the mobile phase, TTBB was found to have slight retention on the OJ phase. The effective phase ratio increased with an increase in the temperature and decrease in the strength of the mobile phase. The enthalpy and entropy of enantiomers of imidazolinone herbicides were independent of the temperature in a linear van't Hoff plot when the effective phase ratio was changed. This study shows that, based on the hold-up volume from TTBB, thermodynamic evaluation with parameters derived from the distribution constant is valuable for understanding chromatographic retention and enantioseparation mechanisms of chiral analytes.

Van't Hoff analysis in chiral chromatography

“…Early researchers recommended TtBB as a void volume marker both for polysaccharide and brush‐type CSPs with normal phase (NP) or polar organic mode eluents. Later, weak retention of TtBB have been observed on polysaccharide Chiralcel OJ and Chiralpak IA columns with hexane‐isopropanol mobile phases and on a brush‐type Chiris Chiral AX:QD1 column with hexane/ethyl acetate solutions as eluents .…”

Temperature effects on a doubly tethered diproline chiral stationary phase: Hold‐up volume, enantioselectivity and robustness

“…Increasing temperature can cause expansion of stainless steel column tube, while this small change of dimension is usually negligible in conventional chromatography [29]. Moreover, increasing temperature can also make thermal expansion of MB so that the hold-up volume measured by the detector is smaller than the actual value [11]. If correction for thermal expansion of the MB was made, the corrected elution times of TTBB at elevated temperatures should be even longer.…”

“…If correction for thermal expansion of the MB was made, the corrected elution times of TTBB at elevated temperatures should be even longer. The upward trend of TTBB elution time as dissolving in 100% Hex implied there was additional factor accounting for temperature dependence of the holdup time [11,28]. In the normal phase mode, the stationary phase is expected to be covered and solvated by the MB.…”

“…pycnometry, is simple to be performed, it is hardly ever used in temperature program [28]. Therefore, dynamic method employing TTBB as the maker was selected [10][11][12]. Increasing temperature can cause expansion of stainless steel column tube, while this small change of dimension is usually negligible in conventional chromatography [29].…”

“…Since then, TTBB was utilized as a primary hold-up volume marker in HPLC. However, its slight retentions on Chiralcel OJ and Chiralpak IA columns have been qualitatively reported [10][11][12]. As quantitative retention time of TTBB is somehow unknown till now, it prompts uncertainty of measured hold-up volume should be concerned.…”