Elsevier

Journal of Chromatography A

Volume 1317, 22 November 2013, Pages 167-174
Journal of Chromatography A

On the effect of basic and acidic additives on the separation of the enantiomers of some basic drugs with polysaccharide-based chiral selectors and polar organic mobile phases

https://doi.org/10.1016/j.chroma.2013.08.029Get rights and content

Highlights

  • Unusual effects of acidic additives on separation of basic chiral β-blocker drugs.

  • Increase of separation factor of enantiomers by addition of acidic additive.

  • Adjustment of enantiomer elution order with minor additives to the mobile phase.

Abstract

This article reports the systematic study of the effect of basic and acidic additives on HPLC separation of enantiomers of some basic chiral drugs on polysaccharide-based chiral columns under polar organic mobile-phase conditions. In contrary to generally accepted opinion that the basic additives improve the separation of enantiomers of basic compounds, the multiple scenarios were observed including the increase, decrease, disappearance and appearance of separation, as well as the reversal of the enantiomer elution order of studied basic compounds induced by the acidic additives. These effects were observed on most of the studied 6 chiral columns in 2-propanol and acetonitrile as mobile phases and diethylamine as a basic additive. As acidic additives formic acid was used systematically and acetic acid and trifluoroacetic acid were applied for comparative purposes. This study illustrates that the minor acidic additives to the mobile phase can be used as for the adjustment of separation selectivity and the enantiomer elution order of basic compounds, as well as for study of chiral recognition mechanisms with polysaccharide-based chiral stationary phases.

Introduction

The separation of enantiomers of chiral compounds represents one of the hot topics of the current research in the field of high-performance liquid chromatography [1], [2], [3]. This interest is based on the need for enantioseparations for the analysis and production of chiral compounds for various applications, and also on the lack of proper understanding the underlying molecular mechanisms of enantiomer separation in chromatography. Although many research papers describe the use of high-end (and expensive) instrumentation or state of the art theoretical calculations for this purpose [1], [2], [3], [4], [5], [6], [7], [8], [9], [10], [11], [12], [13], [14], [15], [16], [17] there is still no plausible theory that allows to either explain most experimental observations or to predict a separation result based on analyte structure, nature of chiral selector and separation conditions. This is the most important issue not only in separation science but also for the better understanding of the driving forces of non-covalent interactions in chemical and biological systems, in general. The primary question in this field is to understand the nature of forces causing the preferential binding of one enantiomer to the chiral selector over the other. Therefore, revealing the critical structural features of chiral analytes, selectors and separation conditions leading to this preferential binding is of especial interest. The various tunable factors, such as temperature and the composition of the medium (mobile phase) can cause in few cases a complete reversal in the chiral recognition pattern. This makes these variables of great interest from a practical point of view (in some cases allowing for the adjustment of the enantiomer elution order, EEO), as well as for a better understanding of enantiomer recognition mechanisms. The reversal of EEO with polysaccharide-based chiral stationary phases (CSP) has been reported based on separation temperature [1], [18], [19], [20], [21], [22], [23], [24], the nature of the major component of the mobile phase [1], [25], [26], [27], [28], [29], [30], [31], [32], its percentage [1], [19], [30], [32], [33], [34], and the minor additives in the mobile phase [1], [20], [34], [35], [36], [37], [38], [39], [40], [41], [42], [43]. The influence of minor additives to the mobile phase on the chiral recognition ability of polysaccharide-based CSPs has been mostly studied under normal-phase conditions [20], [34], [35], [36], [37], [39], [41], [42], [43], supercritical fluid chromatography [38], [40] and to some extent under reversed-phase conditions [34]. The effect of mobile-phase additives on the separation of enantiomers in polar organic mobile phase conditions is less studied [44]. In our recent work we reported some interesting cases of reversal of EEO with polysaccharide-based CSPs under normal-phase [20], [34] and polar organic mobile phase [34], [35] conditions induced by minor additives (formic acid) for both acidic [20], as well as for basic compounds [34], [35]. The effect of acidic additives on the EEO of acidic compounds can be explained conveniently in line with the current understanding of chiral recognition. However, the reversal of EEO of basic compounds caused by minor acidic additives is rather unexpected. Therefore, the goal of the present study was a systematic investigation of the effect of acidic additives (primarily formic acid) on the separation of selected chiral basic drugs under polar organic mobile phase conditions with six different polysaccharide-based CSP.

Section snippets

Materials

The commercially available chiral basic drugs carazolol, carvedilol, pindolol, propranolol and cis-tramadol (Fig. 1), as well as the enantiomers of propranolol were purchased from Sigma Aldrich Fluka (Taufkirchen, Germany). S-carvedilol was a kind gift of Roche Pharmaceuticals (Frankfurt, Germany). The enantiomers of other basic drugs in this study were collected in-house by micropreparative chromatography on appropriate analytical columns. Purified enantiomers were used for spiking racemates

Decrease of enantioselectivity by addition of an acidic component to the mobile phase

The decrease of separation selectivity of basic analytes due to the presence of an acidic additive may be expected. Basic additives in the mobile phase are believed to mask non-enantioselective strong interactions with acidic sites on the CSP surface (suspected to be residual silanol groups) and bring about decreased nonselective retention and improved enantioresolution [37]. Additionally, basic additives may also suppress the ionization (if possible at all) of basic chiral analytes and thus

Conclusions

As shown in this study, the presence of minor additives in the mobile phase can bring about major changes in the resolution of enantiomers in HPLC with polysaccharide-based chiral stationary phases. Their effect is much more complex than it is currently considered since disappearance, decrease, increase or appearance of chiral resolution, as well as a reversal of enantiomer elution order can be observed, depending on the case under observation. Furthermore, numerous cases of reversal of chiral

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