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A novel chiral column for the HPLC separation of a series of dansyl amino and arylalkanoic acids
Talanta 76 (2008) 1261–1264
Contents lists available at ScienceDirect
Talanta journal homepage: www.elsevier.com/locate/talanta
Short communication
A novel chiral column for the HPLC separation of a series of dansyl amino and arylalkanoic acids Yves-Claude Guillaume, Claire Andre´ ∗ Equipe des Sciences S´eparatives et Biopharmaceutiques (2SB)-EA 3924, Laboratoire de Chimie Analytique, Facult´e de M´edecine Pharmacie, Place Saint Jacques, 25030 Besanc¸on Cedex, France
a r t i c l e
i n f o
Article history: Received 29 October 2007 Received in revised form 12 May 2008 Accepted 14 May 2008 Available online 21 May 2008 Keywords: Separation CLHP Cyclic hexapeptide
a b s t r a c t In a previous paper [C. Andre, M. Thomassin, A. Umrayami, L. Ismaili, B. Refouvelet, Y.C. Guillaume, Talanta 71 (2007) 1817] a novel cyclic hexapeptide molecule dissolved in the mobile phase was evaluated as a chiral selector (CS) for the enantiomer separation of a series of dansyl amino and arylalkanoic acids using high performance liquid chromatography (HPLC). In this paper, this CS was immobilized to the surface of a monolithic support and the enantioselectivity and the performance of this novel column are discussed. © 2008 Elsevier B.V. All rights reserved.
1. Introduction The separation and analysis of chiral compounds is an area of increasing interest and high performance liquid chromatography columns containing immobilized chiral selector (CS) [1] have a great potential for optical resolution of such compounds. The chiral selector commonly used include amino acids [2,3], proteins [4–7], crown ethers [8–9], oligo and polysacharides [10–12] or macrocyclic antibiotics [13–15]. In a previous paper, a novel macrocyclic chiral selector synthesized in our laboratory was dissolved in the mobile phase [1]. This CS demonstrated to be effective in the separation of a great number of enantiomer pairs [1]. In this paper, this novel CS was immobilised on a monolithic support and the enantioselective properties of the resulting material was investigated using d,l-dansyl amino and arylalkanoic acids as model compounds. 2. Materials and methods 2.1. Apparatus The HPLC system for these measurements consisted of a Merck Hitachi pump L7100 (Nogent sur Marne, France), an Interchim Rheodyne injection valve model 7125 (Montluc¸on, France) fitted
∗ Corresponding author. Tel.: +33 3 81 66 55 44; fax: +33 3 81 66 56 55. E-mail address: [email protected] (Y.-C. Guillaume). 0039-9140/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.talanta.2008.05.014
with a 20 L sample loop injection, and a Merck L4500 diode array detector. The column used was an Interchim C1 monolithic (100 mm × 4.6 mm i.d.) under controlled temperature conditions (25 ◦ C) in an interchim TM 701 oven (Montluc¸on, France). The detection wavelength was fixed at 254 nm. 2.2. Solvents and samples Water was obtained from an Elgastat option I water purification system (Odil, Talant, France) fitted with a reverse osmosis cartridge. Methanol, analytical grade, was provided by Prolabo (Paris, France). Sodium hydrogen phosphate and potassium dihydrogen phosphate were obtained from prolabo (Paris, France) and the mobile phase consisted of a phosphate buffer (at pH 5.60). This phosphate buffer was prepared by mixing 10 mL of sodium hydrogen phosphate (0.06 M) and 190 mL of potassium dihydrogen phosphate. The chiral selector was synthesized as described in a previous paper [1]. Sodium nitrate was used as a dead time marker (Merck, Saint Quentin Fallavier, France). Dansyl amino acids (i.e., dansyl-alanine (dan-ala), dansyl-valine (dan-val), dansyl-norvaline (dan-nor), dansyl-leucine (dan-leu), dansyl-phenylalanine (danphe), dansyl-tryptophane (dan-try)) and arylalkanoic acids, i.e., etodolac (eto), flobuphen (flo), ibuprofen (ibu), flurbiprophen (flu), naproxen (nap), sulindac (sul), racemic standard compounds and Denantiomers were purchased from Sigma–Aldrich (Saint Louis, MO, USA). The molecular structure of the arylalkanoic acids is given in Fig. 1. Sample solutions were separately prepared at concentrations of 5 mM in the phosphate buffer. All samples and mobile phases
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Y.-C. Guillaume, C. Andr´e / Talanta 76 (2008) 1261–1264 Table 1 Retention factor, k, of the D-enantiomer, and enantioselectivity ˛ for the compound analysed in this study Compound
k
dan-ala dan-val dan-nor dan-leu dan-phe dan-try Eto Flo Flu Ibu Nap Sul
4.71 5.04 8.24 7.00 7.95 6.10 4.01 13.10 14.89 12.50 10.20 6.58
˛ 1.32 1.31 1.34 1.33 1.20 1.18 ≈0 1.44 1.41 1.43 1.54 ≈0
Mobile phase: phosphate buffer (pH 5.60, M/15), T = 25 ◦ C, mobile phase flow-rate 1 mL/min.
room temperature, the interchim C1 monolithic stationary phase (100 mm × 4.6 mm i.d.) was connected to the HPLC system. The chiral selector solution (2 g/L) in (M/15) phosphate buffer (pH 5.60) containing 0.5 M of sodium chloride was recycled through the column at a flow rate of 0.5 mL/min until saturation. Thereafter, the column was washed with (M/15) phosphate buffer (pH 5.60). The amount of CS bound to the column was determined from the HPLC analysis of the fractions collected at the column outlet during the percolation of the CS and the washing steps. 3. Results and discussion
Fig. 1. Arylalkanoic acid molecular structure.
were filtered with 0.45 M syringe filter discs (Whatman, Clifton, NJ, USA) and degassed by sonification. Twenty microliters of each sample were injected in triplicate and the retention times were measured. 2.3. Chiral selector immobilisation via physical absorption The in situ process, which consists of the attachment of the chiral selector (CS) directly in pre-packed columns, was used to immobilize the CS to create the chiral column. The physical absorption was carried out on the C1 stationary phase by hydrophobic interaction. The immobilisation was carried out at a pH 5.60. At
From the retention time obtained, the retention factor (k) of all the enantiomers were determined for a phosphate buffer pH 5.6 (0.06 M). All the experiments were repeated three times. The precisions of retention times of each enantiomer was characterised by relative standard deviation (R.S.D.) values comprised between 0.10 and 0.30%. The variation coefficients of the k values were less than 1% in most cases, indicating a high reproducibility and good stability for the chromatographic system. The stability was tested by comparing the d-naproxen retention factor during the study and then after more than 4 months under the same conditions. The maximum relative difference between retention time of this compound was never more than 0.6% proving the stability of the column during an extended period of time. The chromatographic parameters – retention factor (k) selectivity (˛) – of the racemic mixtures tested are presented in Table 1. The chromatograms of dan-nor, dantryp, flu and nap are depicted in Fig. 2. For each solute molecule the L-enantiomer was more retained than the D-enantiomer. For the dansyl amino acid series, and for each enantiomer (D or L) the retention factor increased in the order: dan-ala < dan-val < dantry < dan-leu < dan-phe < dan-nor. For the arylalkanoic acids, and for each enantiomer (D or L), the retention order increased in the order: eto < sul < nap < ibu < flo < flu.
Table 2 Chromatographic parameters of naproxen (d-naproxen retention factor (k), enantioselectivity, (˛) asymmetry factor (As )) on the chiral column at different flow-rate Flow-rate (mL/min)
k
˛
As
0.5 0.8 1.0 1.2 1.5 2.0
10.19 10.21 10.22 10.20 10.21 10.23
1.54 1.53 1.55 1.56 1.53 1.53
1.20 1.19 1.22 1.21 1.20 1.19
Mobile phase: phosphate buffer (pH 5.60, M/15), T = 25 ◦ C.
Y.-C. Guillaume, C. Andr´e / Talanta 76 (2008) 1261–1264
1263
Table 3 Retention factor, k, of d-naproxen and enantioselectivity factor, ˛, for the naproxen molecule on the chiral column at different methanol fraction (v/v) in the mobile phase (phosphate buffer (pH 5.60, M/15)) and with different injection number, T = 25 ◦ C Injection number
100 200 300 400 500 600 700 800 900 1000
0.10 (v/v)
0.15 (v/v)
0.20 (v/v)
0.25 (v/v)
0.30 (v/v)
0.40 (v/v)
k
˛
k
˛
k
˛
k
˛
k
˛
k
˛
8.52 8.52 8.53 8.53 8.52 8.53 8.54 8.52 8.54 8.52
1.43 1.43 1.41 1.42 1.43 1.42 1.41 1.42 1.41 1.40
7.21 7.22 7.21 7.23 7.21 7.22 7.21 7.22 7.21 7.22
1.34 1.35 1.33 1.35 1.34 1.33 1.35 1.34 1.35 1.34
6.12 6.11 6.12 6.10 6.12 6.11 6.12 6.12 6.11 6.12
1.20 1.21 1.22 1.20 1.21 1.20 1.22 1.21 1.22 1.21
5.09 5.09 5.08 5.07 5.06 5.06 5.05 5.04 5.05 5.03
1.15 1.14 1.15 1.14 1.13 1.12 1.11 1.13 1.14 1.11
4.10 4.09 4.07 4.05 4.04 3.98 3.97 3.89 3.87 3.86
1.10 1.09 1.08 1.09 1.08 1.09 1.07 1.07 1.07 1.06
3.22 3.22 3.21 3.18 3.15 3.00 2.95 2.90 2.92 2.93
1.03 1.02 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00
Mobile phase flow-rate 1 mL/min.
These results were in accordance with those obtained in a previous paper [1]. Indeed, the overall association of the enantiomer-CS depended, at least in part, on the overall hydrophobic interaction between the enantiomer and the methyl group of the chiral selector. In addition, the shape of the side chain R for the dansyl amino acid series for example participated in the overall association mechanism [1]. From a chiral discrimination point of view, for
the aliphatic amino acids dan-ala, dan-val, dan-nor, dan-leu) the enantioselectivity remained relatively constant around 1.31. As it was observed previously when the CS was dissolved in the mobile phase [1], for the aromatic dansyl amino acids (dan-phe, dan-try) a decrease of the enantioselectivity was observed (around 1.20). For the arylalkanoic acid derivatives, the nap molecule presented the highest separation (1.54). flo, flu and ibu presented an intermediate separation around 1.40. No separation of sul and eto enantiomers was observed in accordance with previous results obtained when the CS was dissolved in the bulk solvent [1]. An important feature of monolithic supports is their ability to operate at high flow-rate regardless of column back pressure, this is intrinsically not possible with particulate columns because by operating at flow-rate higher than 1.0 mL/min a high column back pressure would result. Focus was therefore given to the evaluation of this column with respect to speed of eluent flow. d-naproxen was used as a probe to demonstrate that this novel chiral column can operate at high flow rate without a significant loss in enantioselectivity Table 2. A decrease in retention and enantioselectivity was observed, for all solutes, when the methanol fraction increased in the bulk solvent. An example was given in Table 3. This behaviour suggested that both retention and enantioselectivity mechanisms were depended on hydrophobic effects and confirmed a competition effect for the binding with the chiral selector between the enantiomer and methanol added in the mobile phase. In addition, Table 3 showed the stability of the column when the methanol fraction in the mobile phase was lower than 0.25 (v/v). Over a methanol fraction in the mobile phase equal to 0.25 (v/v) the retention decreased when near 400 injections were made. This behaviour can be explained by a possible desorption of the CS on the chromatographic support for a methanol fraction >0.25 (v/v). These results indicated that a mobile phase containing a methanol fraction lower than 0.25 (v/v) guaranteed a long lifetime and correct peak shape. 4. Conclusion This newly developed chiral stationary phase was successfully used for enantioseparations combining the chiral recognition properties of the cyclic hexapeptide molecule and the unique properties concerning the flow behaviour of silica monoliths. This work demonstrated that this CS monolithic column can operate at high flow rate without a significant loss of enantioselectivity. Consequently, faster enantioseparations can be achieved making the prepared supports of interest for high-throughput separations. References
Fig. 2. Chromatographic enantioseparation of (a) dan-nor; (b) dan-tryp; (c) flu; (d) nap. Mobile phase: phosphate buffer (M/15) (pH 5.6); flow rate 1 mL/min; T = 25 ◦ C.
[1] C. Andre, M. Thomassin, A. Umrayami, L. Ismaili, B. Refouvelet, Y.C. Guillaume, Talanta 71 (2007) 1817.
1264
Y.-C. Guillaume, C. Andr´e / Talanta 76 (2008) 1261–1264
[2] F. Gasparrini, D. Misiti, W. Still, C. Villani, H. Vennemers, J. Org. Chem. 62 (1997) 8221. [3] (a) W.H. Pirkle, T.C. Pachapsky, J. Am. Chem. Soc. 108 (1986) 352; (b) W.H. Pirkle, J.M. Finn, J. Org. Chem. 46 (1981) 2936. [4] D.S. Hage, J. Chromatogr. 3 (2002) 768. [5] T. Fornstedt, G. Gotmar, M. Andersson, G. Guiochon, J. Am. Chem. Soc. 121 (1999) 1164. [6] S.C. Jacobson, S. Golshan-Shirazi, G. Guiochon, J. Am. Chem. Soc. 112 (1990) 6492. [7] J. Hermansson, J. Chromatogr. 325 (1985) 379. [8] M. Hilton, D.W. Armstrong, J. Liq. Chromatogr. 14 (1991) 3673.
[9] G. Dotsevi, Y. Sogaah, D.J. Gram, J. Am. Chem. Soc. 97 (1975) 1259. [10] T. Kubota, C. Yamamoto, Y. Okamoto, J. Am. Chem. Soc. 122 (2000) 4056. [11] K.B. Lipkowitz, G. Pearl, B. Coner, M.A. Peterson, J. Am. Chem. Soc. 119 (1997) 600. [12] A. Berthod, S.C. Chang, D.W. Armstrong, Anal. Chem. 64 (1992) 395. ´ J. Chromatogr. A 828 [13] A. Peter, G. Torok, D.W. Armstrong, G. Toth, D. Tourve, (1998) 177. [14] D.W. Armstrong, Y. Liu, K.H. Ekborg-Ott, Chirality 7 (1995) 474. [15] D.W. Armstrong, S. Chen, Y. Zhou, C. Bagwill, J.R. Chen, Anal. Chem. 66 (1994) 1473.
Contents lists available at ScienceDirect
Talanta journal homepage: www.elsevier.com/locate/talanta
Short communication
A novel chiral column for the HPLC separation of a series of dansyl amino and arylalkanoic acids Yves-Claude Guillaume, Claire Andre´ ∗ Equipe des Sciences S´eparatives et Biopharmaceutiques (2SB)-EA 3924, Laboratoire de Chimie Analytique, Facult´e de M´edecine Pharmacie, Place Saint Jacques, 25030 Besanc¸on Cedex, France
a r t i c l e
i n f o
Article history: Received 29 October 2007 Received in revised form 12 May 2008 Accepted 14 May 2008 Available online 21 May 2008 Keywords: Separation CLHP Cyclic hexapeptide
a b s t r a c t In a previous paper [C. Andre, M. Thomassin, A. Umrayami, L. Ismaili, B. Refouvelet, Y.C. Guillaume, Talanta 71 (2007) 1817] a novel cyclic hexapeptide molecule dissolved in the mobile phase was evaluated as a chiral selector (CS) for the enantiomer separation of a series of dansyl amino and arylalkanoic acids using high performance liquid chromatography (HPLC). In this paper, this CS was immobilized to the surface of a monolithic support and the enantioselectivity and the performance of this novel column are discussed. © 2008 Elsevier B.V. All rights reserved.
1. Introduction The separation and analysis of chiral compounds is an area of increasing interest and high performance liquid chromatography columns containing immobilized chiral selector (CS) [1] have a great potential for optical resolution of such compounds. The chiral selector commonly used include amino acids [2,3], proteins [4–7], crown ethers [8–9], oligo and polysacharides [10–12] or macrocyclic antibiotics [13–15]. In a previous paper, a novel macrocyclic chiral selector synthesized in our laboratory was dissolved in the mobile phase [1]. This CS demonstrated to be effective in the separation of a great number of enantiomer pairs [1]. In this paper, this novel CS was immobilised on a monolithic support and the enantioselective properties of the resulting material was investigated using d,l-dansyl amino and arylalkanoic acids as model compounds. 2. Materials and methods 2.1. Apparatus The HPLC system for these measurements consisted of a Merck Hitachi pump L7100 (Nogent sur Marne, France), an Interchim Rheodyne injection valve model 7125 (Montluc¸on, France) fitted
∗ Corresponding author. Tel.: +33 3 81 66 55 44; fax: +33 3 81 66 56 55. E-mail address: [email protected] (Y.-C. Guillaume). 0039-9140/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.talanta.2008.05.014
with a 20 L sample loop injection, and a Merck L4500 diode array detector. The column used was an Interchim C1 monolithic (100 mm × 4.6 mm i.d.) under controlled temperature conditions (25 ◦ C) in an interchim TM 701 oven (Montluc¸on, France). The detection wavelength was fixed at 254 nm. 2.2. Solvents and samples Water was obtained from an Elgastat option I water purification system (Odil, Talant, France) fitted with a reverse osmosis cartridge. Methanol, analytical grade, was provided by Prolabo (Paris, France). Sodium hydrogen phosphate and potassium dihydrogen phosphate were obtained from prolabo (Paris, France) and the mobile phase consisted of a phosphate buffer (at pH 5.60). This phosphate buffer was prepared by mixing 10 mL of sodium hydrogen phosphate (0.06 M) and 190 mL of potassium dihydrogen phosphate. The chiral selector was synthesized as described in a previous paper [1]. Sodium nitrate was used as a dead time marker (Merck, Saint Quentin Fallavier, France). Dansyl amino acids (i.e., dansyl-alanine (dan-ala), dansyl-valine (dan-val), dansyl-norvaline (dan-nor), dansyl-leucine (dan-leu), dansyl-phenylalanine (danphe), dansyl-tryptophane (dan-try)) and arylalkanoic acids, i.e., etodolac (eto), flobuphen (flo), ibuprofen (ibu), flurbiprophen (flu), naproxen (nap), sulindac (sul), racemic standard compounds and Denantiomers were purchased from Sigma–Aldrich (Saint Louis, MO, USA). The molecular structure of the arylalkanoic acids is given in Fig. 1. Sample solutions were separately prepared at concentrations of 5 mM in the phosphate buffer. All samples and mobile phases
1262
Y.-C. Guillaume, C. Andr´e / Talanta 76 (2008) 1261–1264 Table 1 Retention factor, k, of the D-enantiomer, and enantioselectivity ˛ for the compound analysed in this study Compound
k
dan-ala dan-val dan-nor dan-leu dan-phe dan-try Eto Flo Flu Ibu Nap Sul
4.71 5.04 8.24 7.00 7.95 6.10 4.01 13.10 14.89 12.50 10.20 6.58
˛ 1.32 1.31 1.34 1.33 1.20 1.18 ≈0 1.44 1.41 1.43 1.54 ≈0
Mobile phase: phosphate buffer (pH 5.60, M/15), T = 25 ◦ C, mobile phase flow-rate 1 mL/min.
room temperature, the interchim C1 monolithic stationary phase (100 mm × 4.6 mm i.d.) was connected to the HPLC system. The chiral selector solution (2 g/L) in (M/15) phosphate buffer (pH 5.60) containing 0.5 M of sodium chloride was recycled through the column at a flow rate of 0.5 mL/min until saturation. Thereafter, the column was washed with (M/15) phosphate buffer (pH 5.60). The amount of CS bound to the column was determined from the HPLC analysis of the fractions collected at the column outlet during the percolation of the CS and the washing steps. 3. Results and discussion
Fig. 1. Arylalkanoic acid molecular structure.
were filtered with 0.45 M syringe filter discs (Whatman, Clifton, NJ, USA) and degassed by sonification. Twenty microliters of each sample were injected in triplicate and the retention times were measured. 2.3. Chiral selector immobilisation via physical absorption The in situ process, which consists of the attachment of the chiral selector (CS) directly in pre-packed columns, was used to immobilize the CS to create the chiral column. The physical absorption was carried out on the C1 stationary phase by hydrophobic interaction. The immobilisation was carried out at a pH 5.60. At
From the retention time obtained, the retention factor (k) of all the enantiomers were determined for a phosphate buffer pH 5.6 (0.06 M). All the experiments were repeated three times. The precisions of retention times of each enantiomer was characterised by relative standard deviation (R.S.D.) values comprised between 0.10 and 0.30%. The variation coefficients of the k values were less than 1% in most cases, indicating a high reproducibility and good stability for the chromatographic system. The stability was tested by comparing the d-naproxen retention factor during the study and then after more than 4 months under the same conditions. The maximum relative difference between retention time of this compound was never more than 0.6% proving the stability of the column during an extended period of time. The chromatographic parameters – retention factor (k) selectivity (˛) – of the racemic mixtures tested are presented in Table 1. The chromatograms of dan-nor, dantryp, flu and nap are depicted in Fig. 2. For each solute molecule the L-enantiomer was more retained than the D-enantiomer. For the dansyl amino acid series, and for each enantiomer (D or L) the retention factor increased in the order: dan-ala < dan-val < dantry < dan-leu < dan-phe < dan-nor. For the arylalkanoic acids, and for each enantiomer (D or L), the retention order increased in the order: eto < sul < nap < ibu < flo < flu.
Table 2 Chromatographic parameters of naproxen (d-naproxen retention factor (k), enantioselectivity, (˛) asymmetry factor (As )) on the chiral column at different flow-rate Flow-rate (mL/min)
k
˛
As
0.5 0.8 1.0 1.2 1.5 2.0
10.19 10.21 10.22 10.20 10.21 10.23
1.54 1.53 1.55 1.56 1.53 1.53
1.20 1.19 1.22 1.21 1.20 1.19
Mobile phase: phosphate buffer (pH 5.60, M/15), T = 25 ◦ C.
Y.-C. Guillaume, C. Andr´e / Talanta 76 (2008) 1261–1264
1263
Table 3 Retention factor, k, of d-naproxen and enantioselectivity factor, ˛, for the naproxen molecule on the chiral column at different methanol fraction (v/v) in the mobile phase (phosphate buffer (pH 5.60, M/15)) and with different injection number, T = 25 ◦ C Injection number
100 200 300 400 500 600 700 800 900 1000
0.10 (v/v)
0.15 (v/v)
0.20 (v/v)
0.25 (v/v)
0.30 (v/v)
0.40 (v/v)
k
˛
k
˛
k
˛
k
˛
k
˛
k
˛
8.52 8.52 8.53 8.53 8.52 8.53 8.54 8.52 8.54 8.52
1.43 1.43 1.41 1.42 1.43 1.42 1.41 1.42 1.41 1.40
7.21 7.22 7.21 7.23 7.21 7.22 7.21 7.22 7.21 7.22
1.34 1.35 1.33 1.35 1.34 1.33 1.35 1.34 1.35 1.34
6.12 6.11 6.12 6.10 6.12 6.11 6.12 6.12 6.11 6.12
1.20 1.21 1.22 1.20 1.21 1.20 1.22 1.21 1.22 1.21
5.09 5.09 5.08 5.07 5.06 5.06 5.05 5.04 5.05 5.03
1.15 1.14 1.15 1.14 1.13 1.12 1.11 1.13 1.14 1.11
4.10 4.09 4.07 4.05 4.04 3.98 3.97 3.89 3.87 3.86
1.10 1.09 1.08 1.09 1.08 1.09 1.07 1.07 1.07 1.06
3.22 3.22 3.21 3.18 3.15 3.00 2.95 2.90 2.92 2.93
1.03 1.02 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00
Mobile phase flow-rate 1 mL/min.
These results were in accordance with those obtained in a previous paper [1]. Indeed, the overall association of the enantiomer-CS depended, at least in part, on the overall hydrophobic interaction between the enantiomer and the methyl group of the chiral selector. In addition, the shape of the side chain R for the dansyl amino acid series for example participated in the overall association mechanism [1]. From a chiral discrimination point of view, for
the aliphatic amino acids dan-ala, dan-val, dan-nor, dan-leu) the enantioselectivity remained relatively constant around 1.31. As it was observed previously when the CS was dissolved in the mobile phase [1], for the aromatic dansyl amino acids (dan-phe, dan-try) a decrease of the enantioselectivity was observed (around 1.20). For the arylalkanoic acid derivatives, the nap molecule presented the highest separation (1.54). flo, flu and ibu presented an intermediate separation around 1.40. No separation of sul and eto enantiomers was observed in accordance with previous results obtained when the CS was dissolved in the bulk solvent [1]. An important feature of monolithic supports is their ability to operate at high flow-rate regardless of column back pressure, this is intrinsically not possible with particulate columns because by operating at flow-rate higher than 1.0 mL/min a high column back pressure would result. Focus was therefore given to the evaluation of this column with respect to speed of eluent flow. d-naproxen was used as a probe to demonstrate that this novel chiral column can operate at high flow rate without a significant loss in enantioselectivity Table 2. A decrease in retention and enantioselectivity was observed, for all solutes, when the methanol fraction increased in the bulk solvent. An example was given in Table 3. This behaviour suggested that both retention and enantioselectivity mechanisms were depended on hydrophobic effects and confirmed a competition effect for the binding with the chiral selector between the enantiomer and methanol added in the mobile phase. In addition, Table 3 showed the stability of the column when the methanol fraction in the mobile phase was lower than 0.25 (v/v). Over a methanol fraction in the mobile phase equal to 0.25 (v/v) the retention decreased when near 400 injections were made. This behaviour can be explained by a possible desorption of the CS on the chromatographic support for a methanol fraction >0.25 (v/v). These results indicated that a mobile phase containing a methanol fraction lower than 0.25 (v/v) guaranteed a long lifetime and correct peak shape. 4. Conclusion This newly developed chiral stationary phase was successfully used for enantioseparations combining the chiral recognition properties of the cyclic hexapeptide molecule and the unique properties concerning the flow behaviour of silica monoliths. This work demonstrated that this CS monolithic column can operate at high flow rate without a significant loss of enantioselectivity. Consequently, faster enantioseparations can be achieved making the prepared supports of interest for high-throughput separations. References
Fig. 2. Chromatographic enantioseparation of (a) dan-nor; (b) dan-tryp; (c) flu; (d) nap. Mobile phase: phosphate buffer (M/15) (pH 5.6); flow rate 1 mL/min; T = 25 ◦ C.
[1] C. Andre, M. Thomassin, A. Umrayami, L. Ismaili, B. Refouvelet, Y.C. Guillaume, Talanta 71 (2007) 1817.
1264
Y.-C. Guillaume, C. Andr´e / Talanta 76 (2008) 1261–1264
[2] F. Gasparrini, D. Misiti, W. Still, C. Villani, H. Vennemers, J. Org. Chem. 62 (1997) 8221. [3] (a) W.H. Pirkle, T.C. Pachapsky, J. Am. Chem. Soc. 108 (1986) 352; (b) W.H. Pirkle, J.M. Finn, J. Org. Chem. 46 (1981) 2936. [4] D.S. Hage, J. Chromatogr. 3 (2002) 768. [5] T. Fornstedt, G. Gotmar, M. Andersson, G. Guiochon, J. Am. Chem. Soc. 121 (1999) 1164. [6] S.C. Jacobson, S. Golshan-Shirazi, G. Guiochon, J. Am. Chem. Soc. 112 (1990) 6492. [7] J. Hermansson, J. Chromatogr. 325 (1985) 379. [8] M. Hilton, D.W. Armstrong, J. Liq. Chromatogr. 14 (1991) 3673.
[9] G. Dotsevi, Y. Sogaah, D.J. Gram, J. Am. Chem. Soc. 97 (1975) 1259. [10] T. Kubota, C. Yamamoto, Y. Okamoto, J. Am. Chem. Soc. 122 (2000) 4056. [11] K.B. Lipkowitz, G. Pearl, B. Coner, M.A. Peterson, J. Am. Chem. Soc. 119 (1997) 600. [12] A. Berthod, S.C. Chang, D.W. Armstrong, Anal. Chem. 64 (1992) 395. ´ J. Chromatogr. A 828 [13] A. Peter, G. Torok, D.W. Armstrong, G. Toth, D. Tourve, (1998) 177. [14] D.W. Armstrong, Y. Liu, K.H. Ekborg-Ott, Chirality 7 (1995) 474. [15] D.W. Armstrong, S. Chen, Y. Zhou, C. Bagwill, J.R. Chen, Anal. Chem. 66 (1994) 1473.