Electrochromatographic enantioseparation using chiral ligand exchange monolithic sol–gel column

Analytica Chimica Acta 501 (2004) 17–23

Electrochromatographic enantioseparation using chiral ligand exchange monolithic sol–gel column Z. Chen∗ , T. Nishiyama, K. Uchiyama, T. Hobo Department of Applied Chemistry, Graduate School of Engineering, Tokyo Metropolitan University, 1-1 Minamiohsawa, Hachioji, Tokyo 192-0397, Japan Received 28 April 2003; received in revised form 26 August 2003; accepted 15 September 2003

Abstract This paper describes the development of a monolithic sol–gel column modified with l-hydroxyproline as a ligand exchange chiral stationary phase. It has been demonstrated that the monolithic chiral stationary phase can be used for the enantioseparation of dansyl amino acids, free amino acids, hydroxy acids, and dipeptides by capillary electrochromatography and micro-liquid chromatography. The recommended mobile phase was acetonitrile/0.50 mM Cu(Ac)2 –50 mM NH4 Ac (7:3) adjusted to pH 6.5. The characteristics of the monolithic column using hydroxyproline as chiral selector in CEC have been discussed. © 2003 Elsevier B.V. All rights reserved. Keywords: Chiral separation; Monolithic column; Capillary electrochromatography; Ligand exchange; Chiral stationary phase; Micro-liquid chromatography

1. Introduction Chromatographic science plays more and more important roles in the study of life, environmental and pharmaceutical sciences. In recent decades some chromatographic scientists have paid great attention to the miniaturization of columns. Nano- or micro-liquid chromatography (␮-LC) and capillary electrochromatography (CEC) have become attractive fields. However, conventional capillary columns packed with stationary phases in LC have inherent limitations such as slow mass transfer, large void volume between the packed particles, and difficulty in compatibility with CEC due to bubble formation and frit-making difficulty. In recent years, monolithic columns have attracted increasing attention since they possess many advantages over conventional LC columns. A number of papers concerned with monolithic columns have been published, especially in last decade. Recent progress in monolithic columns has been described in several review papers [1–3]. In general, monolithic columns can be classified into two categories, silica-based and polymer-based mono∗ Corresponding author. Present address: Biosensing Research Group, Ubiquitous Interface Laboratory, NTT Microsystem Integration Laboratories, 3-1 Wakamiya, Morinosato, Atsugi, Kanagawa 243-0198, Japan. Tel.: +46-240-2106; fax: +46-240-4728. E-mail address: [email protected] (Z. Chen).

0003-2670/$ – see front matter © 2003 Elsevier B.V. All rights reserved. doi:10.1016/j.aca.2003.09.012

lithic columns. Schmid et al. reported a polymeric ligand exchange-monolithic column for resolving amino acid and hydroxy acid enantiomers [4,5]. Several groups have demonstrated that monolithic sol–gel columns are very promising separation media for ␮-LC and CEC [6–14]. Recently, our efforts have been directed toward the development of chiral monolithic columns, prepared by a sol–gel processing and further chemical modifications for chirally resolving amino acids [10–14], hydroxy acids [11,13], and positional isomers [12] by CEC and ␮-LC. Ligand exchange-liquid chromatography (LE-LC) has extensively been used for resolving various ␣-amino acids since the pioneering work of Davankov and Rogozhin [15]. In general, Cu(II) complexes of ␣-amino acids and their derivatives have been employed in enantioseparations as chiral additives in mobile phase [16–18] or chiral stationary phases (CSPs) [19–21]. In recent decades, chiral ligand exchange has also been used for enantioseparation by the modes of capillary electrophoresis (CE), micellar electrokinetic chromatography (MEKC) and CEC. Davankov published a review paper titled with ‘30 years of chiral ligand exchange’ [22]. In his paper, he briefly described the history of discovery and some benchmark achievements of chiral ligand exchange chromatography. Recently, review papers [23–25] described on the advances in LE-CE, -MEKC and -CEC.

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Z. Chen et al. / Analytica Chimica Acta 501 (2004) 17–23

l-Hydroxyproline and l-proline are the most well known ligands used for chiral separations by conventional LC [19,20], CE [26], and MEKC [27–33]. l-Hydroxyproline derivatives have also been applied for enantioseparation by CE [34,35]. However, in conventional LE-LC the stationary phase must be synthesized on the surface of silica support and packed into the column. In addition, a large amount of mobile phase must be consumed. In LE-CE, a high concentration of Cu(II) complex must be added in the electrolyte, resulting in consuming large amount of chemical regents and having difficulty for highly sensitive detection. Now CEC has been known as a promising technique having advantages like high chromatographic efficiency and low regent consumption. Therefore, the development of chiral monolithic column using l-hydroxyproline as chiral selector will add a new dimension of column technique for chiral separations and make CEC very promising in the future application. Gübitz’s group once tried the CEC using 3 ␮m l-proline-bonded silica packed inside a capillary, but the results have not been published due to ‘extremely high retention times’ [4]. Their work indicated that a packed column using l-proline as stationary phase would not suit for the CEC. Our previous works have demonstrated that monolithic column could make a promising approach to chiral CEC. In order to extend the application of hydroxyproline selector for chiral separation by CEC, we have developed l-hydroxyproline-immobilized monolithic columns for enantioseparation of dansyl amino acids, hydroxy acids, and dipeptides by both CEC and micro-LC. The properties of monolithic column, for examples, the electroosmotic flow (EOF) and enantioselectivity, have been discussed.

from Wako Pure Chemical Industries Ltd. (Tokyo, Japan). Poly(ethylene glycol) (PEG) (Mw : 10,000), dansyl amino acids (Dns-AA), and hydroxy acids were obtained from Sigma (Saint Louis, USA). Dehydrated toluene, dehydrated N,N-dimethylformamide (DMF), acetonitrile for HPLC, copper acetate monohydrate, and ammonium acetate were purchased from Kanto Chemical (Tokyo, Japan). Fused silica capillary (0.375 mm o.d., 0.10 mm i.d.) was purchased from GL Sciences (Tokyo, Japan). 2.3. Preparation of monolithic sol–gel columns and chemical modifications The pre-treatment of capillary, the preparation of monolithic sol–gel columns and the modification of spacer 3-GPTM were carried out as the same procedures described in our previous works [10–14]. The modification of l-hydroxyproline selector was carried out as follows. A 300 mg/ml l-hydroxyproline solution was adjusted with Na2 CO3 to pH 10–12, and used for the further modification of the monolithic column having modified with 3-GPTM. The l-hydroxyproline solution was introduced through the column with a pump. After the liquid drops were observed at the outlet end of column, the pumping was kept for 30 min. The column was kept at room temperature for one week. Then, it was washed with methanol and water. After loading Cu(II) ion on the surface of CSP with 16 mM CuSO4 for 30 min, we conditioned the column by the mobile phase. A detection widow was made right after the monolithic CSPs by burning out the polyimide coating on the surface of capillary with a lighter. 2.4. Separation conditions of CEC and µ-LC

2. Experimental 2.1. Instrumentation CEC was carried out on a home-built instrumental setup, involving an HCZE-30PNO25-LD high voltage power supply (Matsusada Precision Devices, Tokyo, Japan), a CE-1570 intelligent UV-VIS detector (JASCO, Tokyo, Japan) and a C-R7A plus Chromatopac (Shimadzu, Tokyo, Japan). A ␮-LC instrumental system was set-up by a LC-10ADvp pump (Shimadzu, Tokyo, Japan), a CTO-10ACvp column oven (Shimadzu, Tokyo, Japan), an integrator (Chromatopac C-R7A plus, Shimadzu, Tokyo, Japan), a Rheodyne 7520 injector with a 0.2 ␮l sample rotor (Supelco, Bellefonta, USA), and a CE-1570 intelligent UV/VIS detector (JASCO, Tokyo, Japan). Programmed temperature heating was performed within a GC-17A oven (Shimadzu, Tokyo, Japan). 2.2. Chemicals

The monolith columns were prepared inside capillaries with 37 cm effective length (EL) of LE-CSP and 45 cm total length (TL), unless otherwise stated. Sample solutions were injected by an electrokinetic method for 3–5 s. UV detection wavelengths were kept at 254, 214 or 208 nm. Mobile phases were prepared by mixing acetonitrile and the electrolyte solution containing ammonium acetate and copper acetate, then adjusting the pH with ammonia water or acetic acid. Before use, all solutions were filtered through a 0.45 ␮m membrane (Nihon Milipore Ltd. Japan) and degassed by vacuum and ultrasonication. Water was purified by distillation apparatus (Advantec Tokyo, Japan). Sample solutions were dissolved in the mobile phase at the range of 5.0 × 10−4 to 1.0×10−4 M. The calculations of resolution (Rs ), separation factor (α) and EOF are the same as previous works [10–14].

3. Results and discussion 3.1. General of monolithic LE-CSPs

Tetramethoxysilane (TMOS) and 3-glycidoxypropyltrimethoxysilane (3-GPTM) were obtained from Shin-Etsu Chemicals (Tokyo, Japan). l-Hydroxyproline was obtained

The preparation of monolithic LE-CSP includes following steps: (1) the preparation of monolithic silica columns

Z. Chen et al. / Analytica Chimica Acta 501 (2004) 17–23

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Fig. 1. Chemical structures of monolithic LE-CSPs.

3.2. EOF characteristic of the LE-CSP modified with l-hydroxyproline EOF is one of the most important characteristics in CEC. If LE-CSP takes the structure in Fig. 1A, it possesses a net positive charge and generates anodic EOF. If LE-CSP takes a neutral structure in Fig. 1B, it may not generate EOF. We measured the velocity of the EOF by using acetone as a neutral marker. However, the EOF was too weak to be determined, since the acetone peak was not observed within 2 h. When the pH of the mobile phases was changed, EOF still could not be determined. This fact suggests that the silanol groups on the surface of monolithic matrix had almost been modified with the spacer of 3-GPTM and chiral selector. On the other hand, the fact of weak EOF indicates the complexes of LE-CSPs in Fig. 1 do not possess much charge (almost present in the form of Fig. 1B) or much less positive charges (in the form of Fig. 1A). Although the EOF of LE-CSPs modified with l-hydroxyproline was too weak to be determined, the enantioseparation can be achieved.

This result suggests that the migration of analytes in CEC was mainly driven by the electrophoretic mobility of analytes themselves in this system. To enhance the velocity of EOF, we investigated the dynamic effect of cationic surfactants added in the mobile phases. Since a negative voltage was applied at the inlet end of column for the separation, the addition of cationic surfactants would enhance the anodic EOF, if cationic surfactants could dynamically adsorb on the CSPs and increase the amounts of positive charges. We investigated the effect of cetyltrimethylammonium bromide (CTAB) on the separation behaviors of Dns-dl-Phe; the results are shown in Fig. 2. As migration times had been shortened by adding CTAB, EOF had to some extent been enhanced. Unfortunately, enhanced EOF was not strong enough to be determined by using acetone as a marker.

25 t1

Migration time (min)

by sol–gel process, (2) the modifications of 3-GPTM and l-hydroxyproline, and (3) the loading of Cu(II) ion on the surface of LE-CSP. The chemical structures of LE-CSP are shown in Fig. 1. The morphology, permeability, and the mechanical strength have been examined by scanning electron microscope (SEM) and chromatographic test, and shown that there was insignificant difference compared to previous works [10–13]. In LE-CEC, analytes are driven through the monolithic stationary phase by the electroosmotic flow (EOF) and/or the electrophoretic mobility. The chiral resolution results from ligand exchange interaction between the CSP and analyte.

t2

20

15

0

0.2

0.4

0.6

0.8

Concentration of CTAB (mM) Fig. 2. Effect of CTAB on the migration times of Dns-dl-Phe. Mobile phases: acetonitrile/0.50 mM Cu(Ac)2 –50 mM NH4 Ac (7:3), pH 6.5. Effective and total lengths of monolithic CSP were 33 and 41 cm. Applied voltage, −13.6 kV; current, 32 ␮A. Detection wavelength, 214 nm.

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Z. Chen et al. / Analytica Chimica Acta 501 (2004) 17–23 Table 1 Enantioseparations by CEC using l-hydroxyproline modified monolithic columns

Separaton factor (α)

2

1.5

1 Dns-DL-Phe 0.5 Dns-DL-Trp 0

4

5

6 pH

7

8

Fig. 3. Effect of pH on the separation factor of Dns-dl-Phe and Dns-dl-Trp. Mobile phases: acetonitrile/0.50 mM Cu(Ac)2 –50 mM NH4 Ac (7:3), pH 6.5. Effective and total lengths of monolithic CSP were 47 and 57 cm. Applied voltage, −17.8 kV. Detection wavelength, 214 nm.

3.3. Examination on separation conditions pH and the content of organic solvent are two most important separation conditions in the CEC. We investigated their effects on the separation factors by using Dns-dl-Phe and Dns-dl-Trp as test samples. As showed in Fig. 3, separation factor increased with the increase of pH in the range of 4.5–6.5, and then slightly decreased with the further increase of pH to 7.5. When pHs were higher than the isoelectric point (pI) values of analytes and hydroxyproline, both analytes and the selector of l-hydroxyproline were negatively charged. The pI values of Phe, Trp and hydroxyproline are 5.5, 5.9, and 5.7, respectively. Negatively charged analytes preferentially interact with positively charged CSPs in Fig. 1A to form ternary Cu(II) complexes, resulting in the increase of separation factor in the pH range from 4.5 to 6.5. Effects of acetonitrile content on the separation fac-

Samples

tD

tL

α

Rs

Dns-dl-Leu Dns-dl-Glu Dns-dl-NorVal Dns-dl-ABA Dns-dl-Met Dns-dl-Val Dns-dl-Asp Dns-dl-Ser Dns-dl-Thr Dns-dl-Phe Dns-dl-Trp Dns-dl-NorLeu dl-p-Hydroxyphenyl-lactic acid dl-Indole-3-lactic acid dl-␤-Phenyl-lactic acid dl-Leu-dl-Phe dl-Leu-dl-Trp dl-Phe dl-Trp

17.91 N 16.51 17.53 19.70 15.89 N 30.33 24.92 19.68 25.82 16.93 34.14 17.40 21.26 37.69 40.30 N N

17.91

1.0

0

17.34 17.53 20.90 16.44

1.05 1.0 1.06 1.03

0.53 0 0.43 0.44

33.00 25.59 31.71 45.40 18.74 52.05 18.60 24.90 39.16 42.87

1.09 1.03 1.61 1.76 1.11 1.52 1.07 1.17 1.04 1.06

0.31 0.31 8.13 8.40 1.34 5.07 0.99 1.70 0.45 0.30

Notes: (1) α = tl /td ; (2) separation conditions are the same as in Fig. 5; (3) N means that no sample perks were observed for running more than 60 min. (4) ABA: ␣-amino-n-butyric acid.

tor and the migration time of d-enantiomers are shown in Fig. 4 by using Dns-dl-Phe and Dns-dl-Trp as test samples. With the increase of acetonitrile content in the mobile phase from 60 to 80%, the migration times were shortened, but the separation factors increased. 3.4. Enantioseparation by CEC Based on above examinations, the optimum separation conditions of mobile phase were recommended as 40

1.6

35 30 1.5

25

1.45

20 15

1.4

α (Dns-DLPhe)

1.35

1.3

60

α (Dns-DL-Trp)

10

t (Dns-D-Phe) t (Dns-D-Trp

5

70 Acetonitrile content (%)

80

Migration time (t: min)

Separation factor (α)

1.55

0

Fig. 4. Effect of acetonitrile content on separation factors and migration times of Dns-dl-Phe and Dns-dl-Trp. Conditions are the same as in Fig. 3.

Z. Chen et al. / Analytica Chimica Acta 501 (2004) 17–23

acetronitrile/0.50 mM Cu(Ac)2 –50 mM NH4 Ac (7:3) adjusted to pH 6.5. The results of enantioseparation by CEC are summarized in Table 1. Representative electrochromatograms are shown in Fig. 5. It has been demonstrated that LE-CSP modified with l-hydroxyproline can be applied for the enantioseparations of dansyl amino acids, hydroxyl acids and dipeptides by the CEC mode. d-Enantiomers migrated faster than l-enantiomers. For the resolution of dipeptide such as dl-Leu-dl-Phe in Fig. 5, only two peaks were observed, because only the C-terminals of dipeptides can provide the sites of ligand exchange interaction. The

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migration orders of dipeptide enantiomers were not identified due to the lack of standard samples. We tried the enantioseparations of free amino acids such as dl-Phe, dl-Trp and dl-Glu by CEC; but the peaks of analytes were not observed for running about 2 h. However, these free amino acids can be resolved well and shown excellent enantioselectivities by ␮-LC mode, as shown in Fig. 6 and Table 2. These results indicate that free amino acids cannot be driven to the detection end by EOF or their electrophoretic mobility in the mode of CEC, probably because they show extremely strong retention on CSPs.

Fig. 5. Electrochromatograms of dansyl amino acids, hydroxy acids, and dipeptides. Mobile phases: pH 6.5; acetronitrile/0.50 mM NH4 Ac–50 mM Cu(Ac)2 (7:3). Total length of column: 45 cm, effective length of CSP: 37 cm. Applied voltage: −13.5 kV. Injection: electrophoretic method for 3-5 s. Detection wavelength: 214 nm. d-enantiomers migrate faster than l- ones.

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Z. Chen et al. / Analytica Chimica Acta 501 (2004) 17–23

3.5. Enantioseparation by µ-LC Samples employed in the CEC have also been resolved by ␮-LC mode, the results are shown in Fig. 6 and Table 2. It has been known that the LE-CSP modified with l-hydroxyproline showed good enantioselectivity for dansyl amino acids, free amino acids, hydroxyl acids and dipeptides. l-Enantiomers were eluted as second peaks. Free amino acids like Phe and Trp were not resolved by CEC, but they were well resolved by ␮-LC. As shown in Table 2, the retention and the separation factors of free amino acids such

as dl-Trp and dl-Phe are higher than their dansylated derivatives, which suggest that dansyl groups probably offer steric hindrance in the ligand exchange interaction, especially for the molecules containing big substituents like phenyl and indole groups of Phe and Trp. Gubitz et al. reported that the glycyl-dipeptides were resolved well, but dipeptides containing two stereogenic centres fairly, by conventional LC on l-proline or l-hydroxyproline CSP [36]. Interestingly, dl-Leu-dl-Phe and dl-Leu-dl-Trp were not resolved by ␮-LC, but they were poorly resolved in CEC as described in Section 3.4. Gly-dl-Phe was resolved well by ␮-LC.

Fig. 6. Representative chromatograms resolved by ␮-LC. Separation conditions are the same as in Table 2.

Z. Chen et al. / Analytica Chimica Acta 501 (2004) 17–23 Table 2 Resolutions by ␮-LC using l-hydroxyproline modified monolithic CSP Samples

kD 

kL 

α

Rs

Dns-dl-Phe∗

0.39 0.44 4.21 1.96 3.50 2.74 4.05 3.77 1.60 1.94 2.53

0.99 1.27 7.14 8.40 15.64 5.55 17.16 13.82 3.22 3.86 5.67

2.54 2.90 1.70 4.28 4.47 2.02 4.24 3.66 2.01 1.99 2.24

0.99 0.81 0.74 1.12 1.03 0.60 1.05 0.93 0.83 0.75 0.90

Dns-dl-Trp∗ Dns-dl-Glu∗ dl-Phe o-dl-Tyr o-F-dl-Phe dl-Trp 6-CH3 -dl-Trp dl-p-Hydroxyphenyl-lactic acid∗ dl-Indole-3-lactic acid Gly-dl-Phe∗

 ; k  = (t −t )/t ; (2) mobile phase: pH 6.5, acetoniNotes: (1) α = kl /kd r 0 0 r trile/0.5 mM Cu(Ac)2–50 mM NH4Ac (7:3); flow rates: 5 and 2 ␮l/min marked with symbol ∗ ; injection volume: 0.2 ␮l; detection: 254 nm for dansyl amino acids and 208 nm for others.

4. Conclusions We have successfully demonstrated that l-hydroxyproline can be immobilized on the surface of monolithic column prepared by sol–gel process and chemical modifications. This monolithic LE-CSP can be used for resolving amino acids, hydroxy acids and dipeptides by both CEC and ␮-LC modes. Enantiomer migration orders were identified as d-enantiomers were faster than l-ones. Ligand exchange monolithic column integrates both advantages of high enantioselectivity of l-hydroxyproline selector and high chromatographic performance of monolithic bed. This work further makes monolithic column technology and LE-CEC very promising for the future applications.

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