Enantioselective separation of racemates using CHIRALPAK IG amylose-based chiral stationary phase under normal standard, non-standard and reversed phase high performance liquid chromatography

We have previously reported on the solvent versatility of immobilized amylose and cellulose-based chiral stationary phases in enantioselective liquid chromatographic separation of racemates. The studies were mainly focusing on the tris substituted 3,5-dimethylphenylcarbamate polysaccharide-based chiral stationary phases namely CHIRALPAK IA [Amylose tris (3,5-dimethylphenylcarbamate)] or ADMPC and CHIRALPAK IB [Cellulose tris (3,5-dimethylphenylcarbamate)] or CDMPC. Here we focus on the application of the recently introduced amylose tris (3-chloro-5-methylphenylcarbamate) or ACMPC and brand name CHIRALPAK IG with a chlorine substituent replacing the methyl group in CHIRALPAK IA . This was investigated for the enantioslective separation of different classes of pharmaceuticals namely and -blockers, anti-inflammatory and antifungal drugs, norepinephrine-dopamine reuptake inhibitor, catecholamines, sedative hypnotics, anti-histaminics, anticancer drugs, antiarrhythmic drugs, flavonoids, amino acids, alpha-2 adrenergic agonist, adrenaline and miscellaneous.A brief comparison between CHIRALPAK IG and CHIRALPAK IA under normal standard, non-standard and reversed mobile phase is demonstrated. The results revealed the versatility of the CHIRALPAK IG column, its compatibility with a wide ranges of solvent and operation modes and its ability to separate chiral compounds not separated chir with other amylose based


Introduction
Many pharmaceutics and herbicides are chiral. They exist as two incongruent stereoisomers called enantiomers. As optical isomers, they rotate linearly polarized light in opposite directions although they are generally known to have similar physical properties (eg, melting point, hydrophobicity, etc) and they can behave quite differently to one another in a chiral (asymmetric) environment. Since biological processes tend to involve chiral chemicals (eg. enzymes), chirality constitutes an important topic in drug development [1]. The United States Food and Drug Administration (FDA) requires toxicology testing for racemates only, regardless of industry plans to market a single isomer. In case of unexpected or significant toxicity is found in the racemate, FDA suggests querying the agency on whether similar studies are required for individual enantiomers. In such case, the FDA requires that only the active drug enantiomer (the eutomer) is produced by an enantioselective access (e.g., via asymmetric synthesis, resolution via diastereomers, kinetic resolution, enzyme catalysis or chirality pool approach). The inactive enantiomer (the distomer) constitutes 'isomeric ballast' or it may be highly toxic. In the case of thalidomide, one enantiomer possessed the required therapeutic effect, while the other was eventually shown to be teratogenic causing birth defects in the unborn babies. While the use of enantiomerically pure drugs may appear to be a viable solution to such a problem, configurationally unstable stereoisomers like thalidomide may interconvert (known variously as enantiomerization, enantiomeric inversion or racemisation) [2]. The thalidomide tragedy was entirely avoidable, had the physiological properties of the individual thalidomide forms been identified, separated and tested prior to commercialization.
Enantioselective chromatography has been well documented as a powerful, contemporary and practical technique for the chiral separation of racemic drugs, food additives, agrochemicals, fragrances and chiral pollutants [1,2]. This technique is several steps ahead of other previously reported methods to access pure enantiomers; including synthesis from a chirality pool, asymmetric synthesis from pro-chiral substrates and the resolution of racemic mixtures [3]. The separation of racemic mixtures has been considered as the most feasible method for industrial applications compared to the time consuming and expensive synthetic approaches [4]. Remarkable developments have occurred in enantioselective chromatography since the first chiral separation of enantiomers using optically active stationary phase in the midsixties [5]. Following this development, several subclasses have emerged as well established chromatographic techniques with outstanding applications in chiral separation like electrochromatography (EC), supercritical fluid chromatography (SFC), counter current chromatography (CCC), gas chromatography (GC), and high performance liquid chromatography (HPLC) [6]. The chiral selectors used as stationary phases in liquid chromatography play a crucial role in the separation efficiency and the column backpressure governing the entire separation [1]. Most enantioselective separations are performed by direct resolution using a chiral stationary phase (CSP) where the chiral selector is adsorbed, attached, bound, encapsulated or immobilized to an appropriate support to make a CSP. The enantiomers are resolved by the formation of temporary diastereomeric complexes between the CSP and the analyte. Yet, thousands of CSPs have been reported, with more than one hundred commercialized [7]. Among the existing CSPs, those prepared from polysaccharides such as cellulose and amylose, attract more attention due to their powerful separation capability [8][9][10][11][12][13][14][15][16][17][18]. In general, the developments of chemically post-modified polysaccharides are the mainstream trend in the commercial and non-commercial chiral stationary phases. Out of the commercially available polysaccharide-based chiral stationary phases, cellulose and amylose were adsorbed, bonded, encapsulated or immobilized [19][20][21][22][23][24][25][26]. Of the amylose derivatives, the coated tris (3,5-dimethylphenylcarbamate) known as CHIRALPAK AD ® has been widely and effectively used in chiral separation. However, it is not compatible to all eluents solvents, in particular, non-standard organic solvents such as ethyl acetate (EtOAc), tetrahydrofuran (THF), methyl tert-butyl ether (MtBE), dichloromethane (DCM) and chloroform, in which the polysaccharide derivatives can be dissolved or swollen. To widen the selection of solvents, the polysaccharide derivatives have been immobilized/bonded onto a silica matrix and have been extensively used as chiral stationary phases in non-standard organic solvents. Such immobilization of the polymeric chiral selector is considered as an efficient approach to confer a uni-versal solvent versatility [27][28][29][30][31][32]. Several immobilized phases have been commercialized (Fig. 1 showing the effect of chlorine substituent in CHIRALPAK IG ® on the enantiomeric separation of racemates is also demonstrated.

Instrumentation
Conventional HPLC analysis was carried out using a Prominence Shimadzu System that consists of an LC-20 AD VP pump (Kyoto, Japan), SIL-20AHT auto sampler, a GL Science UV-vis detector model MU 701 UVVIS (Tokyo, Japan), and a Shimadzu CDM-20A communications bus module (Kyoto, Japan). All analyses were performed at room temperature. CHIRALPAK IG ® (4.6 mm ID × 250 mm, 5 m silica gel) was supplied by Daicel (Tokyo, Japan).

Chemicals and reagents
All solvents were HPLC grade purchased from Sigma-Aldrich (St. Louis, MO, USA). Most of the tested compounds ( Classification of the investigated racemates and their purities are as listed below:

Sample preparations
Stock solutions of the racemic analytes at concentrations of 1 mg/mL in filtered HPLC-grade 2-propanol were prepared, filtered through Sartorius Minisart RC 15 0.2-m pore size filters (Goettingen, Germany) and further used for analysis without dilution; the injection volume was 1 L.

HPLC conditions
The enantioselective analyses were conducted using standard normal mobile phase comprised of n-hexane in combination with 2-propanol (2-PrOH) or ethanol (EtOH) and non-standard normal phase namely tetrahydrofuran (THF), dichloromethane (DCM) and methyl tert-butyl ether (MtBE). Reversed mobile phase consisted of acetonitrile (ACN) and water (H 2 O) mixture. The additives TEA and TFA were added in both normal and reversed mobile phases. UV analyses were performed at fixed wavelength (254 nm) for all compounds.

Results and discussion
The well-known coated amylose tris (3,5dimethylphenylcarbamate) ADMPC (CHIRALPAK AD ® ) in which the amylose derivative is physically coated on 5 or 10 m silica particles has been widely and effectively used in chiral separation of racemates in high performance liquid chromatography. Its immobilized version namely CHIRALPAK IA ® introduced ten years ago showed excellent solvent versatility and enantioselectivity in normal standard and non-standard organic mobile phases [27][28][29][30][31][32]. More recently, this phase showed promising enantioselectivity under HILIC and reversed phase modes as well [33]. In CHIRALPAK IA ® , the chiral selector is immobilized/bonded onto

Chiral separation under normal standard and non-standard organic mobile phase
The initial mobile phase selected for the enantioselective separation of racemates 1-28 (Fig. 1) (Table 1). Comparing 2-PrOH with ethanol in mobile phase composition and in terms of enantioselective separation, resolution Rs and separation factor ␣, ethanol in mobile phase composition was superior than 2-PrOH. Thus, 1, 3, 5, 14, 26 and 28 were all separated under n-hexane/ethanol which wasn't the case in n-hexane/2-PrOH implying that ethanol works better with the 3-chloro substituted amylose in amylose tris (3-chloro-5-methylphenylcarbamate) or CHIRALPAK IG ® . It is noteworthy that the retention is generally shorter with ethanol than 2-PrOH or when using higher alcohol contents in relation to n-hexane in mobile phase composition ( Table 2 and Fig. 3). To widen the choice of solvents in an attempt to enhance the separation or resolve the unresolved compounds under standard solvents above; dichloromethane (DCM), tetrahydrofuran (THF) or methyl tert-butyl ether (MtBE) were used before combination with standard organic solvent. The addition of nonstandard solvents in mobile phase composition enhanced the resolution R s and separation factor ␣ of several tested racemates (     Fig. 3). One can conclude that polarity plays a role in the chiral recognition of CHIRALPAK IG ® . For example, ethanol with polarity index 5.2 works well in combination with nhexane or MtBE while 2-PrOH with polarity index 3.9 is less sensible in terms of enantioseparation under standard and non-standard organic solvents. Another factor might be the amendment of the stereo environment of the chiral cavities in amylose derivatives is favourable in presence of ethanol for the enantioseparation of the investigated racemates.

Chiral separation under reversed phase
Although the use of reversed phase in amylose and cellulosebased as CSPs in enantioselective liquid chromatography is limited, there are few recently reported studies [22,[34][35][36][37][38][39]. The choice of reversed phase was based on its economic and environmental benefits. Thus, the enantioselective separation was investigated using reversed phases including acetonitrile (ACN) and water (H 2 O) mixture ranging from 10-90% (v/v) (  Table 2).

Conclusions
The solvents versatility of CHIRALPAK IG ® has been demonstrated. The results revealed that solvents known as prohibited non-standard LC solvents such as MtBE, DCM and THF in which the amylose derivatives CSP can be dissolved/swollen can be used as eluents in mobile phase compositions. The addition of these solvents will be also beneficial when used as diluents to directly monitor organic reactions online. Several tested racemates that were not separable under normal standard organic solvents were separated under non-standard organic solvents in mobile phase composition. The use of reversed phase consisting of ACN/H 2 O broaden the application of CHIRALPAK IG ® with enhanced resolution R s and separation factor ␣ comparing to similar separation under standard and non-standard organic solvents. Compared with CHIRALPAK IA ® and in terms of resolution Rs and separation factor ␣, CHIRALPAK IG ® appears to be superior under standard and nonstandard solvents for the tested compounds. Overall, for the tested compounds, CHIRALPAK IG ® appears to be superior to CHIRALPAK IA ® and it may offer an alternative to CHIRALPAK IA ® .