Separation of liquid crystalline racemic mixtures obtained on the basis of (R,S)-2-hexanol on amylose tris(3-chloro-5-methylphenylcarbamate) covalently immobilised on silica in high-performance liquid chromatography

ABSTRACT The separation on the enantiomers of thirteen newly synthesised liquid crystalline racemic mixtures obtained on the basis of (R,S)-2-hexanol was studied in HPLC on a chiral column prepared by covalent immobilisation of the chiral selector, namely amylose tris(3-chloro-5-methylphenylcarbamate) on silica. The mobile phases (MPs) consisting of n-heptane/isopropanol (IPA) or acetonitrile (ACN)/water (H2O) at different volume ratios were used. All measurements were made at room temperature. The order of the elution was also determined. The chromatographic parameters such as: resolution - (R), selectivity - (α) and number of theoretical plates - (N) were determined. On the basis of the obtained results, optimal conditions were indicated for the best separation of the tested racemic mixtures.


Introduction
The racemic mixture (also called the racemate) has equal amounts of left-and right-handed enantiomers of the chiral molecule, as shown in Figure 1.This kind of mixture is difficult to separate due to the similar properties of the two chiral enantiomers, however chemical and chromatographic methods have been developed for this purpose.One method for separating the racemic mixture is the reaction of that racemate with an enantiomerically pure chiral reagent produces the mixture of the diastereomers that have different physical properties meaning they can be separated easily.
Chromatographic techniques [1] can also be used to separate the racemic mixture.The enantiomers can be separated on conventional achiral stationary phases by adding an appropriate chiral selector to the mobile phase.These additives can be cyclodextrins, chiral crown ethers, chiral counter-ions or chiral ligands which are capable of forming ternary complexes with the solute enantiomers in the presence of a transition metal ion.We can also use the chiral stationary phases (CSPs), which are capable to resolve the racemic analyte via interactions of different strength with individual enantiomers [2][3][4][5][6][7][8].CSPs can be grouped in several ways.Depending on their separation principles, the main classes are as follows: Pirkle CSPs, modified polysaccharides, mainly cellulose and amylose, cavity phases, e.g.crown ethers (the chiral separation based on inclusion are achieved through a mechanism by which the guest molecule is accepted into the cavity in a host molecule), macrocyclic antibiotic phases, protein-CONTACT Magdalena Urbańska magdalena.urbanska@wat.edu.plThis article has been corrected with minor changes.These changes do not impact the academic content of the article.
Supplemental data for this article can be accessed online at https://doi.org/10.1080/02678292.2023.2221669.
Polysaccharides are naturally-occurring polymers and their derivatives were found to exhibit the ability of chiral recognition as CSPs.Chiral high-performance liquid chromatography (HPLC) utilising chiral stationary phases based on modified polysaccharides coated or immobilised on silica particles is a very effective tool for the characterisation of a wide variety of newly synthesised materials [10][11][12][13][14].The polysaccharide-based chiral stationary phases are also suitable for the separation of the enantiomers in ultra-performance liquid chromatography (UPLC) and supercritical fluid chromatography (SFC) [15][16][17][18][19][20][21][22].The separation with these CSPs is carrying out in normal-(NP-HPLC) and reverse-phase conditions (RP-HPLC) [23][24][25][26][27]. RP-HPLC is economical method because it allows water to be used in the composition of the mobile phase with other solvents.The solvents are also necessary for dissolving the sample as it is injected in the HPLC system, as well as for other processes involved in sample preparation prior to HPLC analysis.In HPLC, the solvents may be used with a constant composition (isocratic elution) or with variable composition during the chromatographic run (gradients).
The separation of the enantiomers by chromatographic techniques is one of the most important tasks in modern analytical chemistry, especially in the analysis of compounds of biological and pharmaceutical interest.
The aim of the work was to demonstrate the high potential of chiral HPLC in the characterisation of newly synthesised structurally similar racemic mixtures and to compare the results obtained using the different mobile phases and the same chiral column.The analysis of obtained results is presented.

Measurements
In the present study, the separation of the enantiomers on the chiral stationary phase (CSP) made by covalent immobilisation of amylose tris(3-chloro-5-methylphenylcarbamate) on silica was investigated.The ReproSil Chiral MIG column with a particle size of 5 μm, dimensions of 250 mm × 4.6 mm i.d and pore size of 1000 Å (Dr.Maisch, Germany), was used for the chiral separation.The Shimadzu LC-20AP HPLC system (Kyoto, Japan) consisting of a binary solvent delivery pump, an autosampler (SIL-10AP), a communications bus module (CBM-20A), a diode array detector (SPD-M20A) and a fraction collector (FRC-10A) were used for the separation and detection of analytes.Data acquisition was performed by Shimadzu software.The injection volume of samples was 15 or 20 μL.The measurements were carried out at room temperature.The mobile phase flow rate was 0.3-1.0mL•min −1 and the detection wavelength was set at 254 nm.
The mobile phases consisted of n-heptane/isopropanol or acetonitrile/water at different volume ratios.Elution was performed in an isocratic mode and a gradient mode.The sample concentrations were about 0.5-0.6 mg•mL −1 .The samples were dissolved in ACN or IPA.All solvents were used as purchased (IPA -J.T. Baker TM ; ACN -POCH S.A., n-heptane -Sigma-Aldrich).In addition, ultrapure water was used.

Chiral HPLC experiments
The separation conditions (MP composition and flow rate) were optimised on an ongoing basis for the tested racemic mixtures.Figure 3(a-c) show the chromatograms obtained in the isocratic elution for one of the tested racemic mixtures and ACN/H 2 O as the MP.The chromatographic parameters were also determined (see Table 1 and Figure 3(d)).All chromatographic parameters were calculated on the basis of the equations presented in Ref [42].
Figure 4(a-c) show the chromatograms obtained in the isocratic elution for one of the tested racemic mixtures and n-heptane/IPA as the MP.The chromatographic parameters are presented in Table 2 and in Figure 5(a).Reducing the flow rate from 1.0 mL•min −1 to 0.5 mL•min −1 more than doubles the retention times, as shown in Figure 5(b).
Analysing the chromatograms, it can be stated that the best resolution in ACN/H 2 O as the MP was obtained where the flow rate is 0.3 mL•min −1 and the concentration of water is the highest.In the case of n-heptane/IPA as the MP, the reduction of the flow rate does not significantly affect the resolution, therefore the following were adopted as the optimal conditions for separating the racemic mixtures: • ACN/H 2 O 90/10 (ν/ν), 0.3 mL•min −1 , • n-heptane/IPA 85/15 (ν/ν), 1.0 mL•min −1 .
Table 3 summarises the resolution (R) obtained on the ReproSil Chiral MIG column under the indicated separation conditions.The comparison of the retention times for used mobile phases is shown in Figures 6 and 7.All chromatograms are included in Appendicle A. Table 1.The chromatographic parameters determined on the basis of the chromatograms in Figure 3(a-c).Table 2.The chromatographic parameters determined on the basis of the chromatograms in Figure 4(a-c).The results obtained for ACN/H 2 O were much better than for n-heptane/IPA, therefore two more types of gradient were tested: • a decreasing gradient, ACN concentration changed linearly from 99 (v) to 90 (v), the flow rate: 0.3 mL•min −1 , • an increasing-decreasing gradient, ACN concentration at the beginning increased from 95 (v) to 99 (v) and later decreased to 95 (v), the flow rate: 0.25 mL•min −1 .
Table 3.The resolution data obtained for the tested racemic mixtures under the optimal separation conditions.The resolution (R) obtained for the applied gradients is presented in Table 4.
The racemic mixture with the acronym 3.(FF) (R,S) was not separated in any gradient elution.In this type of elution, shorter retention times are observed (the mixture 2.(HF) (R,S) has the best separation in the increasing-decreasing gradient and in this case the retention times are longer than in the isocratic elution).Longer times in this gradient are also observed for the mixture 5.(HH) (R,S), see Figures 8 and 9.
A worse separation is observed for almost all racemic mixtures in the decreasing gradient than in the isocratic elution, except for three unsubstituted mixtures with the oligomethylene spacer equal to 4, 5 and 7 for which complete separation is observed.
In the increasing-decreasing gradient complete separation is observed for most of the racemic mixtures, except for the racemic mixtures:   obtained for the increasing-decreasing gradient, but for this elution mode the flow rate was slower (0.25 mL•min −1 ).
To determine the order of the elution, non-equimolar mixtures were prepared by mixing the racemic mixture and the (S) enantiomer in equal amounts.The results for two such mixtures are shown in Figure 10(a,b).
It was found that the (S)-enantiomer elutes first, and the (R)-enantiomer is retained more strongly.For other mixtures, the situation is analogous.

Conlusions
Suitable chiral HPLC separation methods of the racemic mixtures obtained on the basis of (R,S)-2-hexanol were developed.The methods were optimised with respect to the composition of the MP, the flow rate and the type of the elution.
All racemic mixtures were separated on the ReproSil Chiral MIG column in the reverse-phase liquid chromatography with the mobile phase composed of acetonitrile/water and in the isocratic elution.In the case of the gradient elution the better separation was obtained for the decreasing-increasing gradient and slower flow rate.The best resolution was obtained for the tested racemic mixtures with the MP composed of acetonitryle/water in the ratio of 90/10 (υ/υ) and the flow rate of 0.3 mL•min −1 .
In the normal phase mode with the mobile phase composed of n-heptane/isopropanol the results of the separation were worse than in the reversed phase mode.
It was found that the separation and the retention times of the (R)-and (S)-enantiomers strongly depend on the structure of the liquid crystalline racemic mixtures.
The possibility of the direct separation of chiral isomers is extremely important in chemistry, both from the analytical and preparative point of view.In further analyses, the separation of these racemic mixtures on other chiral polysaccharide-based columns [43] will be tested.

Figure 2 .
Figure 2. The general formula of the separated racemic mixtures.

Figure 3 .
Figure 3. (Colour Online) (a-c) The chromatograms of enantioseparation of the racemic mixture 4.(HH) (R,S), injection volume 15 µl.(d) The change of the resolution depending on the composition of the mobile phase for the racemic mixture 4.(HH) (R,S).
Acronym of the racemic mixture Resolution Selectivity Number of theoretical plates 4.(HH) (R,S) a) 1.340 b) 1.677 c) 2.210 a) 1.125 b) 1.164 c) 1.170 a) 2800 b) 3700 c) 4400 All racemic mixtures were separated on enantiomers on the used column in ACN/H 2 O as the MP.In all cases, the separation and the retention times depend on the type of the substitution of the benzene ring and the length of the oligomethylene spacer in the achiral part of the molecule.Two mixtures with the same substitution with the acronyms 5.(FH) (R,S) and 6.(FH) (R,S) have the longest retention times.Partial separation is observed for the mixtures with the acronyms: 2.(HF) (R,S) and 3.(FF) (R,S).In the case of the mixture 2.(HF)

Figure 5 .
Figure 5. (Colour Online) (a) The change of the resolution depending on the flow rate for the racemic mixture 5.(FH) (R,S).(b) The comparison of the retention times for the different flow rates (n-heptane/IPA).

Figure 6 .
Figure 6.(Colour Online) The comparison of the retention times for ACN/H 2 O as the MP.

Figure 7 .
Figure 7. (Colour Online) The comparison of the retention times for n-heptane/IPA as the MP.

Figure 9 .
Figure 9. (Colour Online) The comparison of the retention times in the increasing-decreasing gradient.

Figure 8 .
Figure 8. (Colour Online) The comparison of the retention times in the decreasing gradient.

Table 4 .
The resolution data obtained for the tested racemic mixtures in the gradient elution.