Analytical Enantioseparation of β-Substituted-2-Phenylpropionic Acids by High-Performance Liquid Chromatography with Hydroxypropyl-β-Cyclodextrin as Chiral Mobile Phase Additive

Abstract

Analytical enantioseparation of five β-substituted-2-phenylpropionic acids by high-performance liquid chromatography with hydroxypropyl-β-cyclodextrin (HP-β-CD) as chiral mobile phase additive was established in this paper, and chromatographic retention mechanism was studied. The effects of various factors such as the organic modifier, different ODS C₁₈ columns and concentration of HP-β-CD were investigated. The chiral mobile phase was composed of methanol or acetonitrile and 0.5% triethylamine acetate buffer at pH 3.0 added with 25 mmol L⁻¹ of HP-β-CD, and baseline separations could be reached for all racemates. As for chromatographic retention mechanism, it was found that there was a negative correlation between the concentration of HP-β-CD in mobile phase and the retention factor under constant pH value and column temperature.

Introduction

Chiral separations have attracted considerable attention since stereochemical isomers usually exhibit different bioactivities and biotoxicities. β-Substituted-2-phenylpropionic acids are important pharmaceutical precursors that can be used as starting materials for the synthesis of chiral drugs. Taking tropic acid and 2-phenylbutyric acid as examples, they carry anti-fungal properties or can be used to treat breast cancer by producing tamoxifen derivatives (1, 2).

There are several reported methods for the enantioseparation of β-substituted-2-phenylpropionic acids, such as enantioseparation by high-performance liquid chromatography (HPLC) (3–7), capillary electrophoresis (8–11) and subcritical fluid chromatography or supercritical fluid chromatography (12, 13), in which most of the enantioseparations are conducted with an expensive chiral stationary phase. In contrast, the method of chiral mobile phase additive shows a wider applicability with less cost. It can be performed on a conventional achiral column with a different chiral mobile phase additive to apply for diverse samples (14–16).

Hydroxypropyl-β-cyclodextrin (HP-β-CD), being one of the synthetically modified derivatives of β-cyclodextrin, has been widely exploited in enantioseparations by HPLC (17–19). In this study, HP-β-CD was selected as the chiral mobile phase additive for enantioseparation of five racemic β-substituted-2-phenylpropionic acids on a conventional achiral ODS C₁₈ column, including 2-phenylbutyric acid; 2,3-diphenylpropionic acid; phenylsuccinic acid; tropic acid and 2-phenyl-3-methylbutyric acid (Figure 1). Meanwhile, chromatographic retention mechanism was investigated in order to explore the relationship between chiral selector and enantiomers.

Figure 1.

Chemical structures of racemic β-substituted-2-phenylpropionic acids.

Experimental

Chemicals and reagents

Racemic 2-phenylbutyric acid was purchased from Tokyo Chemical Industry Co., Ltd, Tokyo, Japan. Phenylsuccinic acid was purchased from Xiangfan Nuoer Chemical Co., Ltd, Hubei, China. 2,3-Diphenylpropionic acid was purchased from J&K chemical Scientific Co., Ltd, Shanghai, China. Tropic acid and 2-phenyl-3-methylbutyric acid were purchased from Alfa Aesar, A Johnson Matthey Company. HP-β-CD (degree of substitution 7.5) was purchased from Qianhui Fine Chemical & Co. Inc., Shandong, China. Acetic acid and triethylamine with analytical grade were purchased from local commercial chemical store. Methanol and acetonitrile used for HPLC analysis were of chromatographic grade. The water used for HPLC study was redistilled.

Apparatus

The HPLC studies were performed on a LabSolutions Ver.5.54 system (Shimadzu, Japan) comprising of a Shimadzu CBM-20Alite controller, a Shimadzu SPD-20A UV/Vis detector, two Shimadzu LC-20AT pumps, a Shimadze CTO-10ASvp column oven and a LabSolutions Ver.5.54 workstation.

The pH value for buffer solutions was determined with a portable Delta 320-s pH meter (Mettler-Toledo, Greifensee, Switzerland).

Chromatographic condition

The chiral separations were performed on a Shimpak CLC-ODS (150 × 4.6 mm i.d., 5 μm) or a Boston Green ODS (150 × 4.6 mm i.d., 5 μm) column. The mobile phase was a mixture of organic modifier and 0.5% triethylamine acetate buffer at pH 3.0 containing 25 mmol L⁻¹ of HP-β-CD. The flow rate was 0.5–1.0 mL min⁻¹. The mobile phase was filtered through a 0.45-μm filter and sonicated for 20 min prior to use. The wave length of the UV detector was set at 254 nm for all the analytes. The column was operated at 25°C. The sample injection volume was 20 μL. All the enantioseparations were performed with above-mentioned chromatographic conditions unless otherwise specified.

Preparation of stock solution

Each of racemic β-substituted-2-phenylpropionic acid was accurately weighed, transferred to volumetric flasks and dissolved in the aqueous phase of the mobile phase to make an individual stock solution of 1.0 mg mL⁻¹. All the solutions were stored at 5°C and brought to room temperature before use.

Optimization of chromatographic condition

The effects of organic modifier, different columns and concentration of HP-β-CD on the enantioseparation of the five β-substituted-2-phenylpropionic acids were studied. The performance of chiral separation was evaluated by peak resolution along with retention time.

Results

Chromatographic conditions for the method development

According to our previous studies, it was found that HP-β-CD showed chiral recognition only for an enantiomer of free molecule instead of an ionic one (17, 20). All solutes in the present research belong to the organic acids and each of them tends to dissociate in the mobile phase. Therefore, a mobile phase with low pH is necessary to restrain its ionization. Meanwhile, it was indicated that a low column temperature benefited for enantioseparation, because the interaction process between an enantiomer and the cyclodextrin was most likely to be an exothermic process and low temperature would improve its enantioselectivities. Therefore, a buffer solution with pH 3.0 and the column temperature of 25°C were selected for our present studies without repetitive investigation.

Two different ODS C₁₈ columns, namely Shimpak CLC-ODS column (150 × 4.6 mm i.d., 5 μm) and Boston Green ODS column (150 × 4.6 mm i.d., 5 μm), were investigated under otherwise the same chromatographic conditions. Compared with Shimpak CLC-ODS column, Boston Green ODS column showed a higher peak resolution with a much higher column pressure. Taking the enantioseparation of 2-phenylbutyric acid as an example, both of the two columns gave a higher peak resolution (R_s ≥3.0) with the mobile phase composed of 0.5% triethylamine acetate buffer at pH 3.0 containing 25 mmol L⁻¹ of HP-β-CD and methanol (70 : 30, v/v), but different column pressure was observed between Shimpak CLC-ODS column (12.7–12.9 MPa) and Boston Green ODS column (17.8–18.2 MPa). So, Shimpak CLC-ODS column was selected.

Generally, the type and percentage of organic modifier in the mobile phase would greatly affect both the peak resolution and retention time. The two frequently used organic modifiers, methanol and acetonitrile, did not show much difference with regard to peak resolution and retention time under otherwise the same chromatographic conditions. And, methanol was selected as an organic modifier. The lower percentage of organic modifier generally resulted in a better peak resolution, but the retention times drastically increased. With a flow rate of 1.0 mL min⁻¹, optimum percentage of methanol in the mobile phase was ∼40% for 2-phenylbutyric acid and 2,3-diphenylpropionic acid, and 20% for phenylsuccinic acid in order to achieve an ideal peak resolution along with a suitable retention time. However, no complete enantioseparation could be achieved for tropic acid and 2-phenyl-3-methylbutyric acid even with 10% of methanol in the mobile phase with a flow rate of 1.0 mL min⁻¹. Generally, high enantiorecognition could be obtained by HP-β-CD with low percentage of organic modifier in the mobile phase where a higher peak resolution could be achieved. However, it was found that large fluctuation of the baseline was caused by a very low percentage (≤5%) of organic modifier due to unknown reasons, in which all peaks of enantiomer could not be detected. As a result, the flow rate was changed to 0.5 mL min⁻¹ for enantioseparation of tropic acid with a 10% of methanol. As for 2-phenyl-3-methylbutyric acid, it was difficult to obtain a baseline separation with the Shimpak CLC-ODS column, but the Boston Green ODS column provided an ideal enantioseparation with a flow rate of 1.0 mL min⁻¹ and 10% of acetonitrile in the mobile phase.

Effect of the concentration of chiral mobile phase additive

The effect of the concentration of HP-β-CD on the peak resolution of the five β-substituted-2-phenylpropionic acids is shown in Figure 2. With the increasing concentration of HP-β-CD within a range of 15–55 mmol L⁻¹, the peak resolutions for 2,3-diphenylpropionic acid and 2-phenyl-3-methylbutyric acid decreased, but the peak resolutions for 2-phenylbutyric acid, phenylsuccinic acid and tropic acid reached a maximum value when 25–35 mmol L⁻¹ of HP-β-CD was used. As expected, the retention time decreased and the column pressure increased with the increasing concentration of HP-β-CD. Since a good enantioseparation with appropriate retention time could also be achieved for 2,3-diphenylpropionic acid and 2-phenyl-3-methylbutyric acid with a low concentration of HP-β-CD, a concentration of 25 mmol L⁻¹ of HP-β-CD was finally selected. Table I summarizes the optimized HPLC conditions for enantioseparation of five β-substituted-2-phenylpropionic acids and Figure 3 shows the typical chromatograms for enantioseparations.

Figure 2.

Effects of the concentration of HP-β-CD on peak resolution. Experimental conditions: mobile phase: 0.5% triethylamine acetate buffer at pH 3.0 containing different concentration of HP-β-CD and methanol (60 : 40, v/v); flow rate: 1.0 mL min⁻¹ for 2-phenylbutyric acid and 2,3-diphenylpropionic acid; (80 : 20, v/v), 1.0 mL min⁻¹ for 2-phenylpropionic acid and phenylsuccinic acid and (90 : 10, v/v), 0.5 mL min⁻¹ for tropic acid and 2-phenyl-3-methylbutyric acid; wavelength: 254 nm; column temperature: 25°C.

Table I.

Optimized Chromatographic Conditions for Enantioseparation of Racemic β-Substituted-2-Phenylpropionic Acids

Racemates	Ratio of mobile phase	Flow rate (mL min−1)	Resolution (Rs)	Retention factor k′	Enantioselectivity α
2-Phenylbutyric acid	60 : 40	1.0	2.24	6.18; 7.16	1.16
2,3-Diphenylpropionic acid	60 : 40	1.0	3.06	12.56; 15.41	1.23
Phenylsuccinic acid	80 : 20	1.0	2.18	1.54; 2.18	1.42
Tropic acid	90 : 10	0.5	1.58	1.79; 2.16	1.21
2-Phenyl-3-methylbutyric acid	90 : 10	1.0	1.60	10.37; 11.65	1.12

Mobile phase: 0.5% triethylamine acetate buffer at pH 3.0 containing 25 mmol L⁻¹ of HP-β-CD: methanol or acetonitrile, acetonitrile only for 2-phenyl-3-methylbutyric acid; wavelength: 254 nm; column temperature: 25°C; column: Shimpak CLC-ODS or Boston Green ODS, Boston Green ODS column only for 2-phenyl-3-methylbutyric acid.

Figure 3.

Chromatograms of enantioseparation of the six racemic β-substituted-2-phenylpropionic acids with the optimized HPLC chromatographic conditions. For chromatographic conditions, see Table I.

Discussions

Chromatographic retention mechanism contributes to understanding the retention behavior of enantiomer with HP-β-CD as the mobile phase additive. Each solute tends to ionize in the mobile phase since all of them are organic acids:

$$\text{HA}\stackrel{k_{\mathrm{a}}}{⟷}{\text{H}}^{+}+{\text{A}}^{-}$$

where HA, H⁺ and A⁻ donate the free form of the monobasic acid, hydrogen ion and acid radical ion, and dissociation constant k_a could be defined as follows:

$$k_{\mathrm{a}}=\frac{[{\text{H}}^{+}][{\text{A}}^{-}]}{[\text{HA}]}$$ (1)

During the enantioseparation by HPLC, the molecular form and the ionization form of each solute would interact with stationary phase and the chiral additive in the mobile phase. Supposing the molar ratio of the inclusion complex between enantiomer and solute was 1 : 1, these processes could be described as follows:

$$\text{HA}+\text{S}\stackrel{k_1}{⟷}\text{HA}-\text{S}$$

$${\text{A}}^{-}+\text{S}\stackrel{k_2}{⟷}{\text{A}}^{-}-\text{S}$$

$$\text{HA}+\text{CD}\stackrel{k_3}{⟷}\text{HA}-\text{CD}$$

$${\text{A}}^{-}+\text{CD}\stackrel{k_4}{⟷}{\text{A}}^{-}-\text{CD}$$

where S denotes the stationary phase, CD means the chiral selector and HA − S, A⁻− S, HA − CD, A⁻− CD as the binding forms, and the combination constants, k₁, k₂, k₃ and k₄, could be defined as follows:

$$k_1=\frac{[\text{HA}-\text{S}]}{[\text{HA}][\text{S}]}$$ (2)

$$k_2=\frac{[{\text{A}}^{-}-\text{S}]}{[{\text{A}}^{-}][\text{S}]}$$ (3)

$$k_3=\frac{[\text{HA}-\text{CD}]}{[\text{HA}][\text{CD}]}$$ (4)

$$k_4=\frac{[{\text{A}}^{-}-\text{CD}]}{[{\text{A}}^{-}][\text{CD}]}$$ (5)

The definition of the retention factor k′ is as follows:

$$k^{\mathrm{\prime }}=\frac{Q_{\mathrm{s}}}{Q_{\mathrm{m}}}$$ (6)

where Q_s represents the quantity of solute in the stationary phase and Q_m represents the quantity of solute in the mobile phase. So, the retention factor of each solute could be expressed as follows:

$$k^{\mathrm{\prime }}=\frac{[\text{HA}-\text{S}]+[{\text{A}}^{-}-\text{S}]}{[\text{HA}-\text{CD}]+[{\text{A}}^{-}-\text{CD}]+[\text{HA}]+[{\text{A}}^{-}]}$$ (7)

The following equation could be obtained by the combination of Equations (1–7):

$$\frac{1}{k^{\mathrm{\prime }}}=\frac{k_3[{\text{H}}^{+}]+k_{\mathrm{a}}{k}_4}{k_1[{\text{H}}^{+}]+k_{\mathrm{a}}{k}_2}\times \frac{1}{[\text{S}]}[\text{CD}]+\frac{[{\text{H}}^{+}]+k_{\mathrm{a}}}{k_1[{\text{H}}^{+}]+k_{\mathrm{a}}{k}_2}\times \frac{1}{[\text{S}]}$$ (8)

The correlation between the reciprocal retention factor and the concentration of chiral mobile phase additive CD could be obtained as Equation (8). It was indicated that, using the buffer solution with a definite pH value and column temperature, there was a negative correlation between the concentration of HP-β-CD in mobile phase and the retention factor, which indicated that with an increase in the concentration of HP-β-CD, the peak retention time would decrease accordingly. The results of the present studies, as summarized in Table II, showed the relationship between retention factor and the concentration of HP-β-CD in the mobile phase, which supported the model of the chromatographic retention mechanism.

Table II.

Variation in the Retention Factor (k) for Each Enantiomer with Increasing Concentration of HP-β-CD in the Mobile Phase

Racemates	Concentration of HP-β-CD (mol L−1)
	0.015	0.025	0.035	0.045	0.055
2-Phenylbutyric acid	8.08; 9.09	6.18; 7.16	5.09; 6.02	4.14; 4.98	3.71; 4.52
2,3-Diphenylpropionic acid	12.66; 22.40	12.56; 15.41	9.83; 12.16	7.66; 9.53	6.65; 8.33
Phenylsuccinic acid	2.19; 2.94	1.54; 2.18	1.20; 1.76	0.92; 1.42	0.84; 1.29
Tropic acid	2.31; 2.68	1.79; 2.16	1.38; 1.70	1.12; 1.38	0.84; 0.99
2-Phenyl-3-methylbutyric acid	31.26; 35.43	21.70; 24.56	15.13; 16.82	11.06; 12.54	9.58; 10.86

For the chromatographic conditions, see Figure 2.

Conclusions

Analytical enantioseparation of five β-substituted-2-phenylpropionic acids was investigated by reversed-phase HPLC with HP-β-CD as the chiral mobile phase additive. Baseline separations for all racemates could be achieved under the optimized chromatographic conditions. Investigation of chromatographic retention mechanism showed a negative correlation between the concentration of HP-β-CD in the mobile phase and the retention factor under the definite pH value and column temperature, which was consistent with the results of separation studies.

Funding

This work was financially supported by the National Natural Science Foundation of China (21105090) and the Department of Education of Zhejiang Province (pd2013031).