The degree of substitution affects the enantioselectivity of sulfobutylether-β-cyclodextrin chiral stationary phases

Denisa Folprechtová1, Květa Kalíková1, Petr Kozlík2, Eva Tesařová1

1 Department of Physical and Macromolecular Chemistry, Faculty of Science, Charles University, Prague, Czech Republic
2 Department of Analytical Chemistry, Faculty of Science, Charles University, Prague, Czech Republic
Correspondence: Květa Kalíková, Department of Physical and Macromolecular Chemistry, Faculty of Science, Charles University, Prague, Czech Republic, Albertov 2030, 12843

Abbreviations : AA, amino acid; AMAC, ammonium acetate; CS, chiral selector; CSP, chiral stationary phase; FAC, DS, degree of substitution; formic acid; MeOH, methanol; MP, mobile phase; SBE-β-CD, SP, stationary phase; sulfobutylether-β-cyclodextrin; t-Boc, tert-butyloxycarbonyl

degree of substitution, dynamic coating, enantioseparation, chromatography, sulfobutylether-β- cyclodextrin


Three chiral stationary phases were prepared by dynamic coating of sulfobutylether-β-cyclodextrin (SBE-β-CD) with different degrees of substitution, onto strong anion-exchange stationary phases. The enantioselective potential and stability of newly prepared chiral stationary phases were examined using a set of structurally different chiral analytes. Measurements were performed in reversed-phase high-performance liquid chromatography. Mobile phases consisted of methanol/formic acid, pH 2.10, and methanol/10 mM ammonium acetate buffer, pH 4.00, in various volume ratios. SBE-β-CDs with degrees of substitution (DS) 4, 6.3 and 10 proved suitable for the enantioseparation of 14, 11 and 8 analytes, respectively. The SBE-β-CD DS 4 based chiral stationary phase enabled the enantioseparation of the nearly all basic and neutral compounds. Chiral stationary phases with higher sulfobutylether-β-cyclodextrin substitution (especially DS 10) yielded higher enantioresolution values for acidic compounds. Additional supporting information may be found in the online version of this article at the publisher’s web-site.

1 Introduction

Selecting a suitable chiral stationary phase (CSP) is crucial for chiral separations. Although many different types of CSPs [1-7] are currently available for the separation of enantiomers of various chiral compounds, research and development continuously aims to increase the efficiency, the stereoselectivity and, particularly, the versatility of new CSPs [8]. Several different approaches can be used to prepare CSPs. For example, click chemistry can be used to prepare cyclodextrin-based CSPs [9-11], in addition to the chemical incorporation of a chiral selector into the monolithic stationary phase (SP) [5, 12]. Dynamic coating is an alternative approach for SP preparation, which is characterized by efficiency, simplicity and speed of preparation [13-17]. Pittler et al. showed that a dynamically coated CSP can be stable for several months, depending on the type of chiral selector [15]. The main advantage of a dynamically coated CSP over chemically bonded SPs is the possibility to exchange a chiral selector (CS) for another because CS can be removed with a suitable washing process and then recoated [16].

Cyclodextrins (CDs) are cyclic oligosaccharides consisting of six or more D-glucopyranose units linked in α(1–4). Due to their availability, inexpensiveness and ability to form inclusion complexes [18], CDs are among the most commonly used chiral selectors for enantioselective separation in various separation techniques, such as high-performance liquid chromatography (HPLC) [10, 17-21], capillary electrophoresis (CE) [22, 23], gas chromatography [24, 25] or supercritical fluid chromatography (SFC) [26, 27]. Especially derivatized CDs often show excellent separation performance [28-30].Accordingly, HPLC using CSPs based on derivatized CDs are an excellent tool for enantioselective separations [18]. CD derivatives can be neutral or positively or negatively charged. Electrostatic interactions with the oppositely charged analyte molecule help to stabilize the formed inclusion complex [31]. In this work, we focused on negatively charged sulfobutylether-β-CD (SBE-β- CD)because of its polyanionic structure, which can be used to prepare dynamically coated CSPs. The presence of negatively charged sulfate groups allows interactions with the positively charged surface of a SP [13, 32].Based on the previous study [13], the main objective of this work was to prepare three new CSPs by dynamic coating of SBE-β-CD with varying degrees of substitution (DS 4, DS 6.3 and DS 10) on a strong anion exchange SP. Furthermore, structurally different chiral analytes were separated to characterize the enantioselective potential of newly prepared CSPs, assessing the effect of the degree of substitution on retention and enantioselectivity.

2 Materials and Methods

2.1 Chemicals and reagents

Methanol (MeOH, Chromasolv®, ≥ 99.9%), formic acid (FAC, reagent grade ≥ 95%), acetic acid (AAC, ReagentPlus® ≥ 99%), ammonium acetate (AMAC, ≥ 99.00%) were supplied by Sigma-Aldrich (Steinheim, Germany). Deionized water was purified with a Rowapur and Ultrapur system from Watrex (Prague, Czech Republic). The testing set of chiral analytes, namely oxazepam (analytical standard), lorazepam (analytical standard), promethazine (analytical standard), thioridazine (≥ 99%), carprofen (analytical standard), fenoprofen (analytical standard), flurbiprofen (analytical standard), indoprofen (analytical standard),6-hydroxyflavanone (≥ 99%), 7-hydroxyflavanone (≥ 98%), tert-butyloxycarbonyl-D-tryptophan (t- Boc-D-Trp, analytical standard), tert-butyloxycarbonyl-L-tryptophan (t-Boc-L-Trp, analytical standard), Tröger’s base (98%), propranolol (≥ 99%) and 5-fluor-DL-tryptophan (5-F-DL-Trp, analytical standard), was purchased from Sigma-Aldrich (Steinheim, Germany). DL-tryptophan butylester (DL-Trp butylester, analytical standard) was purchased from Pfaltz & Bauer, Inc. (Waterbury, CT, USA). Sulfobutylether-β-cyclodextrins (SBE-β-CD, degree of substitution (DS) 4, DS
6.3 and DS 10) were purchased from CycloLab LTD. (Budapest, Hungary). The chemical structures of the analytes are shown in Fig S1, in Electronic Supplementary material.

2.2 Instrumentation

All chromatographic measurements were performed on an Agilent Technologies HPLC system a with 1200 series quaternary pump, a 1290 Infinity column and autosampler thermostats and a 1260 Infinity diode array detector, controlled by the OpenLab® software, Agilent Technologies (Waldbronn, Germany).

The Spherisorb® column, a strong anion exchange stationary phase used for dynamic coating with SBE-β-CD, was purchased from Waters (Milford, MA, USA). The dimensions of the column were 100
× 4.6 mm i.d.; 5 µm silica particle size.

2.3 Procedure

2.3.1 General conditions and procedures

The column and autosampler temperatures were maintained at 25°C and 20°C, respectively. The flow rate was 1 mL/min. The sample injection volume was 5 µl. UV detection was performed at 254 nm. The column dead time was determined using the solvent peak. All measurements were performed in triplicate.The stock solutions of samples were prepared at the concentration of 1 mg/mL using MeOH as a solvent. An aqueous solution of FAC, pH 2.10, (365 mM) was prepared by adding an appropriate amount of concentrated FAC to deionized water. 10 mM AMAC buffer was prepared by dissolving an appropriate amount of AMAC in deionized water and by adding the calculated amount of AAC until pH 4.00. Program PeakMaster was used to calculate the required concentration of FAC and AMAC ( The final pH values of the aqueous parts of mobile phases were verified by measurement. Methanol was used as an organic modifier. Minisart syringe filters, 0.2 µm and 0.45 µm, Sartorius Stedim Biotech (Göttingen, Germany), were used to filtrate all the prepared samples and aqueous parts of the mobile phases (MPs), respectively.

2.3.2 Coating procedure

Based on preliminary results, the coating procedure was optimized and performed as follows. SBE-β-

CD dissolved in 1 mg/mL 40/60 (v/v) MeOH/deionized water

was coated on a commercial strong anion exchange Spherisorb® column containing a silica-based quaternary ammonium bonded sorbent. The flow rate was 0.6 mL/min, and the coating time was 2
h. The same dynamic coating procedure was applied for SBE-β-CD DS 4, DS 6.3 and DS 10. Fig S2 in Electronic Supplementary material shows a schematic representation of the coating procedure.

Gravimetric analysis was used to determine the amount of SBE-β-CD deposited on the SP surface during the procedure. The average amounts of 0.09 g, 0.06 g and 0.07 g of SBE-β-CD DS 4, DS 6.3 and DS 10 were deposited on the SP surfaces during the coating procedures. Other parameters describing individual selectors/stationary phases are summarized in Table S1, in Electronic Supplementary material. No significant difference in the average coated amount of SBE-β-CD DS 6.3 and DS 10 was observed within the margin of error. The prepared SPs were stable after more than 800 injections, and the measurements were repeatable. The relative standard deviations (RSDs) of the retention factor and resolution were lower than 1% for three consecutive injections. The RSDs of the retention factors and resolutions of selected chiral compounds increase up to 4% only, during one month of measurements, which indicates good column stability. To test the repeatability of the column preparation, the same coating procedure of SBE-β-CD DS 6.3 was used for another

3 Results and discussion

The set of fifteen structurally different chiral compounds was used to evaluate the enantioselective potential of three newly prepared SBE-β-CD based CSPs. Differences in the chromatographic behavior of individual CSPs were examined and discussed in detailed. All measurements were performed in MP compositions MeOH/aqueous part ranged from 90/10 to 10/90, with a step of 10% (v/v). Two different aqueous parts of MP at different pHs were used for the measurements: FAC, pH
2.10 and 10 mM AMAC buffer, pH 4.00. MeOH was used as an organic modifier in all cases.

3.1 Enantioselectivity
Table 1 summarizes the results from the chromatographic measurements (i.e., retention factor (k), resolution of enantiomers (R) and enantioselectivity (α)) of the fifteen compounds tested under mobile phase compositions optimized for each SBE-β-CD based CSP. As shown in Table 1, SBE-β-CD DS 4 CSP exhibits higher enantiorecognition than the other CSPs tested. Fourteen of fifteen analytes tested in this study were at least partly enantioseparated on SBE-β-CD DS 4 based CSP, whereas the SBE-β-CD DS 10 based CSP was able to separate only eight enantiomeric pairs. Conversely, the SBE- β-CD DS 10 based CSP provided the highest values of enantioresolution for five analytes, i.e., oxazepam, lorazepam, thioridazine, DL-Trp butylester, and t-Boc-DL-Trp.Chromatographic data of all compounds tested on all three new CSPs obtained in MeOH/aqueous part ratios ranging from 40/60 to 10/90 (v/v) are listed in Electronic Supplementary material, Tables S2-S7. The use of high methanol content in the mobile phase led to very low or even no retention on the stationary phases. Therefore, we observed very low or no resolution due to weak interactions between analytes and stationary phases and presumably not due to poor stereoselectivity.

3.2 Retention

Retention is affected by the interactions between the SBE-β-CD and the analyte and between the uncoated SP surface and the analyte. The analytes mainly showed an RP mode behavior. 5-F-DL-Trp and t-Boc-DL-Trp revealed a mixed-mode interaction mechanism: hydrophilic interaction liquid chromatography (HILIC) and RP behavior (see Fig. S3 in Electronic Supplementary material). This interaction complexity is demonstrated with two Trp derivatives, in Fig. S3, in Electronic Supplementary material, showing the variation in retention as a function of the MeOH content in the MP. Trp butylester (carrying positive charge) exhibits a typical RP behavior, whereas the negatively charged t-Boc-DL-Trp shows mixed-retention behavior (HILIC vs RP). The longer retention times of t- Boc-DL-Trp (at buffer pH 4.00 compared with pH 2.1) can be attributed to ionic interactions between the uncoated positively charged surface of SP and the negatively charged AA.
The retention factors of both benzodiazepines were similar among all CSPs tested – see Tables S2-S4 in Electronic Supplementary material. The retention of hydroxyflavanones was correlated with the degree of substitution of SBE-β-CD, that is, the retention increased with the degree of substitution of SBE-β-CD (see Tables S2-S7 in Electronic Supplementary material and also Table 1). The use of MP containing 10 mM AMAC buffer, pH 4.00, increased the retention of most analytes tested except for basic analytes. As shown, a low retention of Tröger’s base (only about 3 min) was observed in the MP with FAC, pH 2.10, whereas the retention time increased significantly (to approximately 36 min) when using AMAC buffer, pH 4.00 (Tables S2-S3 in Electronic Supplementary material). In addition, the low retention of propranolol can be explained by repulsive interactions between the uncoated positively charged surface of the anion exchanger and propranolol, which was protonated at both pH values.

4 Concluding remarks

Three new CSPs were successfully prepared by dynamic coating of SBE-β-CD with varying degrees of substitution onto strong anion exchange SPs. The retention and enantioselective potential of the newly prepared chiral stationary phases were tested using
a set of chiral analytes in two types of MPs: MeOH/FAC, pH 2.10, and MeOH/10 mM AMAC, pH 4.00. The results showed that all CSPs prepared have a high enantioseparation potential and that the composition of the mobile phase affects the enantioseparation. From our set of 15 structurally diverse analytes, the SBE-β-CD DS 4 based CSP was suitable for the enantioseparation of 14 analytes, the SBE-β-CD DS 6.3 based CSP was suitable for the enantioseparation of 11 analytes, and the SBE- β-CD DS 10 based CSP was suitable for the enantioseparation of 8 analytes. Our results showed the suitability of SBE-β-CD DS 4 based CSP for the enantioseparation of especially basic and neutral
compounds. CSPs with higher SBE substitution, mainly the SBE-β-CD DS 10 based CSP, showed higher enantioresolution values for acidic compounds.


The authors gratefully acknowledge the financial support of the Czech Science Foundation, Grant No 19-18005Y. Authors thank Carlos V. Melo for proofreading the manuscript.

The authors have declared no conflict of interest.


[1] Fanali, C., Fanali, S., Chankvetadze, B., Chromatographia 2016, 79, 119-124.
*2+ Vozka, J., Kalíková, K., Janečková, L., Armstrong, D. W., Tesařová, E., Anal. Lett. 2012, 45, 2344- 2358.
[3] Shedania, Z., Kakava, R., Volonterio, A., Farkas, T., Chankvetadze, B., J. Chromatogr. A 2018,
1557, 62-74.
[4] Hyun, M. H., J. Chromatogr. A 2016, 1467, 19-32.
[5] Guo, J., Lin, Y., Xiao, Y., Crommen, J., Jiang, Z., J. Pharm. Biomed. Anal. 2016, 130, 110-125.
[6] Hyun, M. H., J. Chromatogr. A 2018, 1557, 28-42.
[7] Péter, A., Lázár, L., Fülöp, F., Armstrong, D. W., J. Chromatogr. A 2001, 926, 229-238.
[8] Chankvetadze, B., Liquid Chromatography, 2nd Edition, Elsevier 2017, pp. 69-86.
[9] Zhou, J., Yang, B., Tang, J., Tang, W., J. Chromatogr. A 2016, 1467, 169-177.
[10] Tang, J., Lin, Y., Yang, B., Zhou, J., Tang, W., Chirality 2017, 29, 566-573.
[11] Huang, G., Ou, J., Zhang, X., Ji, Y., Peng, X., Zou, H., Electrophoresis 2014, 35, 2752-2758.
[12] Pumera, M., Jelínek, I., Jindřich, J., Benada, O., J. Liq. Chromatogr. Relat. Technol. 2002, 25, 2473-2484.
*13+ Kučerová, G., Procházková, H., Kalíková, K., Tesařová, E., J. Chromatogr. A 2016, 1467, 356-362.
*14+ Kučerová, G., Kalíková, K., Procházková, H., Popr, M., Jindřich, J., Coufal, P., Tesařová, E.,
Chromatographia 2016, 79, 529-536.
[15] Pittler, E., Schmid, M. G., Biomed. Chromatogr. 2010, 24, 1213-1219.
[16] Schmid, M. G., Schreiner, K., Reisinger, D., Gübitz, G., J. Sep. Sci.2006, 29, 1470-1475.
[17] Li, L., Cheng, B., Zhou, R., Cao, Z., Zeng, C., Li, L., Talanta 2017, 174, 179-191.
[18] Crini, G., Fourmentin, S., Fenyvesi, É., Torri, G., Fourmentin, M., Morin-Crini, N., Environ. Chem.
Lett. 2018. DOI: 10.1007/s10311-018-00818-0
[19] Gao, J., Qu, H., Zhang, C., Li, W., Wang, P., Zhou, Z., Chirality 2017, 29, 19-25.
*20+ Szabó, Z. I., Szőcs, L., Horváth, P., Komjáti, B., Nagy, J., Jánoska, Á., Muntean, D. L., Noszál, B., Tóth, G., J. Sep. Sci. 2016, 39, 2941-2949.
[21] Armstrong, D., Chen, S., Chang, C., Chang, S., J. Liq. Chromatogr. 1992, 15, 545-556.
*22+ Stavrou, I. J., Agathokleous, E. A., Kapnissi‐Christodoulou, C. P., Electrophoresis 2017, 38, 786- 819.
*23+ Kalíková, K., Riesová, M., Tesařová, E., Open Chem. 2012, 10, 450-471.
*24+ Gus’kov, V. Y., Maistrenko, V. N., J. Anal. Chem. 2018, 73, 937-945.
[25] Menestrina, F., Ronco, N. R., Romero, L. M., Castells, C. B., Microchem. J. 2018, 140, 52-59.
[26] Kalíková, K., Šlechtová, T., Vozka, J., Tesařová, E., Anal. Chim. Acta 2014, 821, 1-33.
[27] Speybrouck, D., Lipka, E., J. Chromatogr. A 2016, 1467, 33-55.
[28] Kalíková, K., Šlechtová, T., Tesařová, E., Curr. Med. Chem. 2017, 24, 829-848.
[29] Menges, R. A., Armstrong, D. W., Acs Sym. Ser. 1991, 471, 67-100.
[30] Han, S. M., Biomed. Chromatogr. 1997, 11, 259-271.
[31] Hui, B. Y., Raoov, M., Zain, N. N. M., Mohamad, S., Osman, H., Crit. Rev. Anal. Chem. 2017, 47, 454-467.
[32] Zhou, J., Tang, J., Tang, W., Trac-Trends Anal. Chem. 2015, 65, 22-29.
*33+ Jaroš, M., Hruška, V., Štědrý, M., Zusková, I., Gaš, B., SBE-β-CD Electrophoresis 2004, 25, 3080-3085.