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Direct resolution of chiral ‘pineno’ fused terpyridyl ligands on amylose based chiral stationary phase using long chain alcohol modifiers
Analytica Chimica Acta 534 (2005) 193–198
Direct resolution of chiral ‘pineno’ fused terpyridyl ligands on amylose based chiral stationary phase using long chain alcohol modifiers Radhakrishna Tatinia , Omowunmi Sadika,∗ , Stefan Bernhardb , Hector Abru˜nab b
a Department of Chemistry, State University of New York at Binghamton, Binghamton, NY 13902, USA Department of Chemistry and Chemical Biology, Bakers Laboratory, Cornell University, Ithaca, NY 14853, USA
Received 25 June 2004; received in revised form 10 November 2004; accepted 10 November 2004 Available online 25 December 2004
Abstract Chiral separation of novel pineno fused terpyridyl ligands have been investigated on a silica-based amylose tris(3,5-dimethylphenyl carbamate) stationary phase (Chiralpak AD), using normal phase conditions. The chromatographic parameters investigated include retention factor (k), separation factor (α), column efficiency (N) and resolution (RS ) of the analytes. In addition, the effect of alcohol modifiers on the enantioselectivity of the column has been discussed. The chiral resolution of the ligands was significantly improved by using 1-pentanol as alcohol modifier in the mobile phase. The reversal of the elution order achieved in this study allows a lower detection limit (<0.1%) of the minor enantiomer and the chromatographic system described is suitable for assessing the enantiomeric excess (ee) of the ctpy ≥ 99.9%. The method is selective and sensitive with a limit of detection (LOD) and quantification (LOQ) of 0.2 g/ml and 0.6 g/ml for ctpy, 0.6 g/ml and 1.2 g/ml for ctpy–x–ctpy, respectively. This study suggests that superior enantioseparation may be obtained for certain analytes using the same column by simply changing the type of alcohol modifier in the mobile phase instead of choosing another expensive chiral stationary phase for the same purpose. © 2004 Elsevier B.V. All rights reserved. Keywords: Chiral separation; Novel pineno fused terpyridyl ligands; Alcohol modifiers and optimization
1. Introduction There has been an increased interest in the preparation of chiral ligand systems for the coordination of transition metals due to their application in selective catalytic reactions and the interesting photophysical and chemical properties of the resulting complexes [1,2]. Von Zelewsky and co-workers first employed a strategy for controlling the helical chirality around metal centers by using optically pure ligands [3,4]. Recently, the synthesis of optically pure ‘pineno’ fused terpyridyl ligands (‘dipineno’-[4,5:4 ,5 ] fused 2,2 :6 ,2 -terpyridine, (±)-ctpy and its bridging ligand (±)[ctpy–x–ctpy], Fig. 1) has been reported and successfully employed as a promising building block for the design ∗
Corresponding author. Tel.: +1 607 777 4132; fax: +1 607 777 4478. E-mail address: [email protected] (O. Sadik).
0003-2670/$ – see front matter © 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.aca.2004.11.032
and synthesis of helical supramolecular metal coordination polymers [5,6]. Due to the chirality and electroactivity, we believe that these polymers should be able to resolve several racemic compounds of broad structural difference as chiral stationary phases (CSPs). Since these supramolecular assemblies was prepared as single enantiomers, the control of the enantiomeric purity of the above terpyridyl ligands is critical. The optically pure ctpy–x–ctpy enantiomers were prepared by using a two-fold stereo specific alkylation reaction of chiral ctpy isomers. Subsequent to synthesis, the optical purity of the ligands investigated was determined using either circular dichroism (CD) or polarimeter [5]. But it is impossible to utilize CD or polarimeter data for assessing the optical purity unless these spectroscopic parameters are initially determined using a sample with an independently measured optical purity. As a result, enantioseparation methods are needed to monitor the enantiomeric purity of
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modifiers in mobile phase to enhance the chiral resolution can provide useful and less expensive alternatives to using multiple columns during method development. Apart from the separation, it is always desirable to elute the minor enantiomer before the major one so as to avoid the difficulty in quantification caused by the tailing of the major peak, especially when the enantiomeric resolution is <2. It was found that changing the alcohol modifier in the mobile phase could reverse the elution order of the ctpy enantiomers. We are not aware of previous work in which the direct enantiomeric resolution of these novel terpyridyl ligands has been reported.
2. Experimental 2.1. Instrumentation
Fig. 1. Structures of pineno fused terpyridyl ligands.
both ligands. The objective of this work is to resolve novel terpyridyl ligands to their enantiomers and then estimate the accurate enantiomeric excess of each isomer. The direct separation of enantiomers using highperformance liquid chromatography (HPLC) has rapidly advanced in the past decade leading to the development of a variety of chiral stationary phases (CSPs). Among the various commercially available CSPs, cellulose tris(3,5dimethylphenylcarbamate) (Chiralcel OD) and amylose tris(3,5-dimethylphenylcarbamate) (Chiralpak AD) CSPs have found many successful applications and these have proven to be versatile [7–17]. It has been suggested that the separation of enantiomers on the above CSPs was due to the formation of transient diastereomeric complexes between the enantiomeric solutes and the chiral cavities in the higher order structures of the CSPs [18–20]. The main chiral adsorbing sites of these CSPs are carbamate polar functional groups, which interact with the solute through hydrogen bonding using the NH and CO groups, as well as through dipole–dipole interactions using the CO moiety. In addition, the – interactions of dimethylphenyl groups of the stationary phase with aromatic groups of the solute are also equally important for chiral recognition. In the present study the direct enantioseparation of the new terpyridyl ligands on a Chiralpak AD stationary phase under normal phase conditions is discussed. In addition, the chiral stationary phase was evaluated for the influence of the kind of alcoholic modifier on the resolution of the enantiomers. The studies of Okamoto et al. [21] on cellulose based column, motivated us to use long chain alcohols as mobile phase modifier on Chiralpak AD. This study has identified that certain alcohol modifiers can significantly improve chiral resolution, which is not possible using traditional mobile phase modifiers such as 2-propanol and ethanol, recommended by column manufacturer [22]. Thus the use of different alcohol
Dionex high performance liquid chromatography (HPLC) system consisted of GP 40 gradient pump, LC 20 Chromatography Enclosure, AD 20 absorbance detector and a Rheodyne injection valve equipped with a 25 l sample loop. Chiral chromatography was performed using Chiralpak AD-H (250 mm × 4.6 mm ID and 5 m particles) column obtained from Chiral Technologies Inc., Exton, PA. HPLC was controlled and monitored using Dionex PeakNet version 5.01 software. Circular dichroism spectra (CD) were recorded in 1 cm quartz cell at 20 ◦ C on a Jobin Yvon CD 6 spectrophotometer. 2.2. Materials Hexane, 2-propanol, 1-pentanol, 1-hexanol (HPLC grades) and diethylamine were obtained from Aldrich Chemical Co., (Milwaukee, WI). Ethanol (absolute) was purchased from Pharmco Products Inc., (Brookfield, CT). Certified grade 1-propanol, 1-butanol and tert-butanol were obtained from Fisher Scientific, (Fair Lawn, NJ, USA). All the authentic enantiomers under the study were synthesized as previously reported [5]. 2.3. Chromatographic conditions Each enantiomer was first dissolved (∼3 mg) in 2propanol and then diluted with 8 ml of the mobile phase in order to obtain a final concentration of 0.3 mg/ml for the HPLC injection. The racemic samples were prepared by mixing both enantiomers at nearly equal concentrations. The injections for each experiment were made in triplicate. The mobile phase consists of various alcohols at different percentages in hexane. All solvents used were filtered and degassed in an ultrasonic bath before use. The flow rate was maintained at 1.0 ml/min throughout the study. Analysis was carried out at room temperature (∼22 ◦ C). UV detection was performed at 292 nm. The retention factors (k) were determined as k = (tR − tm )/tm . The dead time (tm ), determined with 1,3,5-tri-tert-butyl benzene, was
R. Tatini et al. / Analytica Chimica Acta 534 (2005) 193–198
2.84 min for all the eluents used, and tR is the retention time of the peak calculated. The separation factor (α) was calculated as α = k2 /k1 , where k1 and k2 are retention factors for the first and second eluting enantiomer, respectively. The resolution (RS ) between enantiomeric pairs was calculated by the following formula: RS = 2(tR2 − tR1 )/(wb1 + wb2 ), where wb1 and wb2 are the baseline peak-widths for the first and second eluting enantiomer, respectively. The column efficiency (N) was calculated using the formula 16(tR/wb )2 . In the separation of each enantiomeric pair, the elution order of the enantiomers was determined using an arbitrary mixture of the pure enantiomers. Column that was operated with the mobile phase containing diethylamine, were easily regenerated at the end of every day by washing them with ca. 100 ml of hexane-2-propanol (90:10, v/v) at a flow rate of 0.2 ml/min. In this way, no efficiency loss was observed throughout the experiment. After cleaning, the column was reconditioned with ca. 100 ml of the mobile phase.
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in the CSP caused by the alcohol modifiers of different sizes/shapes [12,21,25–30]. But these arguments were speculative until recently. Wang et al. demonstrated with spectral evidence that the modification of stereo environment of the chiral cavities in the CSP was due to the incorporation of the alcohol modifier of different sizes/shape may cause different degrees of twisting to the glucose units on the CSP helix, yielding different chiral selectivities [28,31]. Thus the influence of type and composition of the alcoholic modifier in the mobile phase on solute retention (k), elution order, selectivity (α) and resolution (RS ) were systematically studied and results obtained for both enantiomers are summarized in Tables 1 and 2. From the data shown in the tables, it was evident that using an alcohol modifier other than commonly used 2-propanol or ethanol on the column can be used to achieve superior separations. For example using 1-pentanol as mobile phase modifier, we obtained excellent separations for both ctpy and ctpy–x–ctpy enantiomeric pairs on the CSP. 3.1. Enantiomeric separation of (±)-ctpy ligands
3. Results and discussion Highly selective chiral separation was obtained with Chiralpak AD column for the terpyridyl enantiomers under normal phase conditions. Based on the chiral recognition model proposed for amylose tris (3,5-dimethylphenylcabamate) stationary phase, it was assumed that the terpyridyl ligands interact with the CSP via: (1) hydrogen bonding between the nitrogen atoms of the ligand and the amide proton of the carbamate moiety in the CSP; (2) dipole–dipole interactions between nitrogen atoms of the ligand and the carbonyl oxygen of carbamate moiety of the CSP; and (3) charge transfer (–) interactions between pyridine/phenyl moiety of the ligand and the phenyl group of the CSP. Thus, a particular orientation of the molecule is presented to the CSP to facilitate chiral recognition [8]. Initially the enantiomers of terpyridyl ligands were not resolved on the column with simple hexane and alcohol mixtures; separations were achieved only when diethylamine (DEA) at 0.2% (v/v) was added to the mobile phase. With this type of CSP, free silanol groups occur on the silica surface, resulting in secondary interactions [23,24] with the nitrogen atoms of the ligands, leading to peak asymmetry. DEA effectively masks these underivatized silanols, thus minimizing the adsorption effect and the resultant peak tailing. Further increments of this organic base did not affect retention or enantioselectivity. Optimization of the enantiomeric separation of the ligands was performed using various compositions of hexane with alcoholic modifiers such as ethanol, 1-propanol, 2-propanol, 1-butanol, tert-butyl alcohol, 1-pentanol and 1-hexanol. The change of alcohol modifier may result in different chromatographic behavior such as different chiral selectivity and, in some cases, inversion of elution order of the enantiomers. These changes in selectivity were rationalized by the authors as a result of alteration of the steric environment of the chiral cavities
The influence of the kind of alcohol modifiers on the chiral resolution of ctpy enantiomers is shown in Fig. 2. As mentioned above, the main reason for showing different separation properties is due to a changed geometry and/or size of the chiral groove of CSP, which is caused by the type of alcohol modifier. It could be seen from the data in Table 1 that only partial resolution was obtained using ethanol and 1-butanol as the mobile phase modifier. In addition, as the concentration of ethanol increases, the resolution decreased, and this became zero at hexane: ethanol: DEA (97:3:0.2) mobile phase, thus indicating no resolution. This data Table 1 Effect of alcohol modifier on chiral separation of ctpy enantiomers Modifier
Concentration (%)
k1
Elution order
α
N
RS
Ethanol
3 5 10 3 5 10 3 5 10 3 5 10 3 5 10 3 5 10 3 5 10
1.41 1.07 0.83 1.25 1.01 0.76 2.92 1.44 0.93 1.61 1.46 0.88 3.05 2.46 1.59 1.83 1.60 0.97 1.80 1.48 0.90
NSa (−)/(+) (−)/(+) (−)/(+) (−)/(+) (−)/(+) (−)/(+) (−)/(+) (−)/(+) NSa NSa (−)/(+) (−)/(+) (−)/(+) (−)/(+) (−)/(+) (−)/(+) (−)/(+) (−)/(+) (−)/(+) (−)/(+)
1.00 1.06 1.04 1.15 1.12 1.06 1.13 1.12 1.09 1.00 1.00 1.07 1.26 1.14 1.11 1.37 1.28 1.19 1.28 1.23 1.17
7250 7724 8329 7231 8049 8678 4849 6235 8452 5913 6580 7231 4582 4920 5592 7350 8622 9280 4150 4835 5670
0.0 0.9 0.6 1.3 1.1 0.6 1.9 1.3 0.9 0.0 0.0 0.4 2.0 1.8 1.6 4.1 3.4 1.8 2.2 1.9 1.4
1-Propanol
2-Propanol
1-Butanol
tert-Butanol
1-Pentanol
1-Hexanol
a
NS: no separation.
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Table 2 Effect of alcohol modifier on chiral separation of ctpy–x–ctpy enantiomers Modifier
Concentration (%)
k1
Elution order
α
N
RS
Ethanol
3 5 10 3 5 10 3 5 10 3 5 10 3 5 10 3 5 10 3 5 10
1.41 1.07 0.83 1.25 1.01 0.76 2.92 1.44 0.93 1.61 1.46 0.88 3.05 2.46 1.59 1.83 1.60 0.97 1.80 1.48 0.90
NSa (−)/(+) (−)/(+) (−)/(+) (−)/(+) (−)/(+) (−)/(+) (−)/(+) (−)/(+) NSa NSa (−)/(+) (−)/(+) (−)/(+) (−)/(+) (−)/(+) (−)/(+) (−)/(+) (−)/(+) (−)/(+) (−)/(+)
1.00 1.06 1.04 1.15 1.12 1.06 1.13 1.12 1.09 1.00 1.00 1.07 1.26 1.14 1.11 1.37 1.28 1.19 1.28 1.23 1.17
7250 7724 8329 7231 8049 8678 4849 6235 8452 5913 6580 7231 4582 4920 5592 7350 8622 9280 4150 4835 5670
0.0 0.9 0.6 1.3 1.1 0.6 1.9 1.3 0.9 0.0 0.0 0.4 2.0 1.8 1.6 4.1 3.4 1.8 2.2 1.9 1.4
1-Propanol
2-Propanol
1-Butanol
tert-Butanol
1-Pentanol
1-Hexanol
a
NS: no separation.
indicated that change of alcohol concentration might affect the chiral selectivity of the CSP and it confirms the results that are previously reported by Wang et al. [31]. A reasonable separation was obtained for ctpy enantiomers on the column using 2-propanol, tert-butanol and 1-hexanol as alcohol
modifiers in the mobile phase. There was a remarkable increase in the chiral resolution as the concentration of above alcohols decreased. The resolution was found to be moderately good with the mobile phases containing 1-propanol as modifier. It is interesting to find that the values of separation factor (α) and resolution (RS ) for ctpy enantiomers were highest when 1-pentanol was used as mobile-phase modifier. 1-Pentanol exhibited the most favorable effect on column efficiency, thus producing the largest plate number of any alcohol tested with this solute, resulting in a significant increase in enantiomeric resolution. We assumed that this higher plate number could result in better kinetics (mass transfer) using 1-pentanol modified mobile phase. Thus the mobile phase consisting of hexane:1-pentanol:DEA in the ratio (95:05:0.2) was found to be best suitable eluent for the chiral separation of (±)-ctpy ligand and was used in routine analysis of ctpy for the determination of enantiomeric excess. During this investigation, we also found that the elution order of the enantiomers changed according to the steric bulk of the alcohol in the mobile phase. The enantiomeric resolution was (−)/(+) with ethanol, 2-propanol, 1-propanol, and tert-butanol as the mobile phase modifiers, and the elution order was (+)/(−) with 1-butanol or 1-pentanol or 1-hexanol. The reversal of elution orders is due to different chiral selectivities of the CSP associated with the use of different alcohol modifiers that caused alterations to the steric environment of the chiral cavities in the CSP [31]. The reversal of elution orders of enantiomers on cellulose- and amylose-based CSPs upon changing the alcohol modifiers in the mobile phase have been reported by a number of research groups [12,21,25–30]. In our case, the reversal of the elution order of the ctpy enantiomers upon changing the alcohol modifier from small chain to long alkyl chain alcohols was probably due to same reason. The reversal of the elution order achieved in this current study would allow a lower detection limit (<0.1%) of the minor enantiomer and the chromatographic system described is suitable for assessing the enantiomeric excesses of the ctpy ≥99.9%. 3.2. Enantiomeric separation of (±)-ctpy–x–ctpy ligands
Fig. 2. Enantioseparations of (±)-ctpy by HPLC using Chiralpak AD column, mobile phases: n-hexane:1-pentanol:DEA, 95:5:0.2, (v/v/v) (A); nhexane:tert-butanol:DEA, 90:10:0.2, (v/v/v) (B); n-hexane:1-hexanol:DEA, 95:5:0.2, (v/v/v) (C); n-hexane:1-propanol:DEA, 93:7:0.2, (v/v/v) (D); nhexane:1-ethanol:DEA, 95:5:0.2, (v/v/v) (E); n-hexane:2-propanol:DEA, 97:3:0.2, (v/v/v) (F).
The chromatograms of the separation of ctpy–x–ctpy enantiomers on Chiralpak AD column using hexane and different alcohol modifiers are shown in Fig. 3. From Table 2, no resolution occurs with 2-propanol and 1-butanol modifiers on the investigated column. Even though there was a higher α value, resolution between the isomers was somewhat lower with tert-butanol and ethanol compared to 1-propanol or 1-hexanol. This was the result of poorer column efficiency (plate number N = 774 for tert-butanol, N = 367 for ethanol) which may be caused by less favorable kinetics of the solvated stationary phase. Highest resolution and α values for ctpy–x–ctpy enantiomers was again found with 1-pentanol as mobile phase modifier, even though column efficiency was lower (N = 1373). Thus the set of conditions shown in Fig. 3
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Fig. 4. Estimation of enantiomeric excess of ‘pineno’ fused terpyridyl ligands by HPLC. Chiralpak AD column, mobile phases: n-hexane:1pentanol:DEA, 95:5:0.2, (v/v/v), UV: 292 and flow rate: 1.0 ml/min.
3.4. Limit of detection and quantification
Fig. 3. Enantioseparations of (±)-ctpy by HPLC using Chiralpak AD column, phases: n-hexane:1-pentanol:DEA, 95:5:0.2, (v/v/v) (A); n-hexane:1propanol:DEA, 93:7:0.2, (v/v/v) (B); n-hexane:tert-butanol:DEA, 90:10:0.2, (v/v/v) (C); n-hexane:ethanol:DEA, 93:7:0.2, (v/v/v) (D); n-hexane:1hexanol:DEA, 95:05:0.2, (v/v/v) (E).
using 1-pentanol on AD column provided the best separation and resolution, and were used as the method conditions for the estimation of enantiomeric excess of ctpy–x–ctpy. The elution orders of the enantiomers of ctpy–x–ctpy were found to be the same for all alcohol modifiers tested, unlike ctpy isomers. Wainer et al. have reported that the solute having aromatic functionalities could provide additional stabilizing effect to the solute–CSP complex by insertion of the aromatic portion of the solute into the chiral cavity [20]. The similar type of stabilization effect might exits with ctpy–x–ctpy due to the presence of additional phenyl ring linked between two ctpy moieties, and this is probably the reason for the above said behavior since the chiral discrimination between the enantiomers is due to the differences in their steric fit in the chiral cavities [19,20]. 3.3. Determination of enantiomeric excess The current method was applied for the estimation of enantiomeric excess (ee) using real samples of terpyridyl ligands. The representative chromatograms of each ligand are shown in Fig. 4. Interestingly, the other isomer was not detected in each of the enantiomers of ctpy. The enantiomeric excess of ctpy–x–ctpy enantiomers was found to be more than 99.5%. The precision of the method for the quantitative determination of optical purity of terpyridyl enantiomers was determined by analyzing six independent samples of each enantiomer by two analysts on two different days and two columns using different instruments and mobile phase preparations. The precision of the method was found to be less than 3.0% R.S.D.
The limit of detection (LOD) was established for both ligands by determining the concentration of a dilute enantiomer that gave a signal-to-noise ratio of 3. The limit of quantitation (LOQ) is defined as the lowest concentration that can be determined with acceptable accuracy and precision, which can be established at a signal-to-noise ratio of 10. The LOD and LOQ values were 0.2 g/ml and 0.6 g/ml for the ctpy enantiomers, and 0.6 g/ml and 1.2 g/ml for the ctpy–x–ctpy enantiomers, respectively. The possibility of potential chiral inversion of terpyridyl enantiomers in the sample preparation was also investigated. For each enantiomer of ctpy and ctpy–x–ctpy, 0.3% of other isomer was spiked and analyzed for three successive days. The results (R.S.D. < 2.5%) indicated that the terpyridyl enantiomers are chirally stable in solution for at least 3 days. Based on the precautionary statements furnished with the Chiralpak AD column instruction sheet by the manufacturer [22], we were concerned about the stability of the Chiralpak AD column when employing long chain alcohol modifiers. We compared the column performance with a fresh column and one with a history of about 500 sample injections, using a mobile phase of 5% 1-pentanol-hexane. Essentially equivalent retention and resolution for the two columns suggest good column stability in the above mobile phase.
4. Conclusions We have successfully used Chiralpak AD column for the chiral resolution of ‘pineno’ fused terpyridyl ligands under normal phase conditions. 1-Pentanol as alcohol modifier improved the enantiomeric resolution of investigated compounds in the mobile phase. It was found that long chain alcohols such as 1-pentanol and 1-hexanol reversed the elution order of ctpy enantiomers in the current study. These results suggest that using an alcohol modifier other than the commonly used 2-propanol or ethanol on the same column
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can be used to achieve better enantioseparations. This approach can provide useful and less expensive alternatives in method development of chiral resolution of certain analytes. Acknowledgement Financial support of this work by NYS Center for Advanced Technology/IEEC/NSF is gratefully acknowledged. The authors also acknowledge financial support from the USEPA Science-to-Achieve Program. References [1] F.R. Keene, Coord. Chem. Rev. 166 (1997) 121–151. [2] A. Von Zelewsky, Stereochemistry of Coordination Compounds, Wiley, New York, 1996. [3] P. Hayoz, A. Von Zelewsky, H. Stoeckli-Evans, J. Am. Chem. Soc. 115 (1993) 5111–5114. [4] H. Muerner, P. Belser, A. Von Zelewsky, J. Am. Chem. Soc. 118 (1996) 7989–7994. [5] S. Bernhard, K. Takada, D.J. Diaz, H.D. Abruna, H. Murner, J. Am. Chem. Soc. 123 (2001) 10265–10271. [6] M. Ziegler, V. Monney, H. Stoeckli-Evans, A. Von Zelewsky, I. Sasaki, G. Dupic, J.-C. Daran, G.G.A. Balavoine, J. Chem. Soc., Dalton Trans. 5 (1999) 667–675. [7] S. Lin, C.E. Engelsma, N.J. Maddox, B.K. Huckabee, D.M. Sobieray, J. Liquid Chrom. Rel. Technol. 20 (1997) 1243–1256. [8] T. Shibata, I. Okamoto, K. Ishii, J. Liquid Chromatogr. 9 (1986) 313–340. [9] P.S. Bonato, V.L. Lanchote, R. Bortocan, V.A.P. Jabor, F.O. Paias, E. Ricci-Junior, R. Carvalho, J. Liquid Chrom. Rel. Technol. 22 (1999) 1813–1827.
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Direct resolution of chiral ‘pineno’ fused terpyridyl ligands on amylose based chiral stationary phase using long chain alcohol modifiers Radhakrishna Tatinia , Omowunmi Sadika,∗ , Stefan Bernhardb , Hector Abru˜nab b
a Department of Chemistry, State University of New York at Binghamton, Binghamton, NY 13902, USA Department of Chemistry and Chemical Biology, Bakers Laboratory, Cornell University, Ithaca, NY 14853, USA
Received 25 June 2004; received in revised form 10 November 2004; accepted 10 November 2004 Available online 25 December 2004
Abstract Chiral separation of novel pineno fused terpyridyl ligands have been investigated on a silica-based amylose tris(3,5-dimethylphenyl carbamate) stationary phase (Chiralpak AD), using normal phase conditions. The chromatographic parameters investigated include retention factor (k), separation factor (α), column efficiency (N) and resolution (RS ) of the analytes. In addition, the effect of alcohol modifiers on the enantioselectivity of the column has been discussed. The chiral resolution of the ligands was significantly improved by using 1-pentanol as alcohol modifier in the mobile phase. The reversal of the elution order achieved in this study allows a lower detection limit (<0.1%) of the minor enantiomer and the chromatographic system described is suitable for assessing the enantiomeric excess (ee) of the ctpy ≥ 99.9%. The method is selective and sensitive with a limit of detection (LOD) and quantification (LOQ) of 0.2 g/ml and 0.6 g/ml for ctpy, 0.6 g/ml and 1.2 g/ml for ctpy–x–ctpy, respectively. This study suggests that superior enantioseparation may be obtained for certain analytes using the same column by simply changing the type of alcohol modifier in the mobile phase instead of choosing another expensive chiral stationary phase for the same purpose. © 2004 Elsevier B.V. All rights reserved. Keywords: Chiral separation; Novel pineno fused terpyridyl ligands; Alcohol modifiers and optimization
1. Introduction There has been an increased interest in the preparation of chiral ligand systems for the coordination of transition metals due to their application in selective catalytic reactions and the interesting photophysical and chemical properties of the resulting complexes [1,2]. Von Zelewsky and co-workers first employed a strategy for controlling the helical chirality around metal centers by using optically pure ligands [3,4]. Recently, the synthesis of optically pure ‘pineno’ fused terpyridyl ligands (‘dipineno’-[4,5:4 ,5 ] fused 2,2 :6 ,2 -terpyridine, (±)-ctpy and its bridging ligand (±)[ctpy–x–ctpy], Fig. 1) has been reported and successfully employed as a promising building block for the design ∗
Corresponding author. Tel.: +1 607 777 4132; fax: +1 607 777 4478. E-mail address: [email protected] (O. Sadik).
0003-2670/$ – see front matter © 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.aca.2004.11.032
and synthesis of helical supramolecular metal coordination polymers [5,6]. Due to the chirality and electroactivity, we believe that these polymers should be able to resolve several racemic compounds of broad structural difference as chiral stationary phases (CSPs). Since these supramolecular assemblies was prepared as single enantiomers, the control of the enantiomeric purity of the above terpyridyl ligands is critical. The optically pure ctpy–x–ctpy enantiomers were prepared by using a two-fold stereo specific alkylation reaction of chiral ctpy isomers. Subsequent to synthesis, the optical purity of the ligands investigated was determined using either circular dichroism (CD) or polarimeter [5]. But it is impossible to utilize CD or polarimeter data for assessing the optical purity unless these spectroscopic parameters are initially determined using a sample with an independently measured optical purity. As a result, enantioseparation methods are needed to monitor the enantiomeric purity of
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modifiers in mobile phase to enhance the chiral resolution can provide useful and less expensive alternatives to using multiple columns during method development. Apart from the separation, it is always desirable to elute the minor enantiomer before the major one so as to avoid the difficulty in quantification caused by the tailing of the major peak, especially when the enantiomeric resolution is <2. It was found that changing the alcohol modifier in the mobile phase could reverse the elution order of the ctpy enantiomers. We are not aware of previous work in which the direct enantiomeric resolution of these novel terpyridyl ligands has been reported.
2. Experimental 2.1. Instrumentation
Fig. 1. Structures of pineno fused terpyridyl ligands.
both ligands. The objective of this work is to resolve novel terpyridyl ligands to their enantiomers and then estimate the accurate enantiomeric excess of each isomer. The direct separation of enantiomers using highperformance liquid chromatography (HPLC) has rapidly advanced in the past decade leading to the development of a variety of chiral stationary phases (CSPs). Among the various commercially available CSPs, cellulose tris(3,5dimethylphenylcarbamate) (Chiralcel OD) and amylose tris(3,5-dimethylphenylcarbamate) (Chiralpak AD) CSPs have found many successful applications and these have proven to be versatile [7–17]. It has been suggested that the separation of enantiomers on the above CSPs was due to the formation of transient diastereomeric complexes between the enantiomeric solutes and the chiral cavities in the higher order structures of the CSPs [18–20]. The main chiral adsorbing sites of these CSPs are carbamate polar functional groups, which interact with the solute through hydrogen bonding using the NH and CO groups, as well as through dipole–dipole interactions using the CO moiety. In addition, the – interactions of dimethylphenyl groups of the stationary phase with aromatic groups of the solute are also equally important for chiral recognition. In the present study the direct enantioseparation of the new terpyridyl ligands on a Chiralpak AD stationary phase under normal phase conditions is discussed. In addition, the chiral stationary phase was evaluated for the influence of the kind of alcoholic modifier on the resolution of the enantiomers. The studies of Okamoto et al. [21] on cellulose based column, motivated us to use long chain alcohols as mobile phase modifier on Chiralpak AD. This study has identified that certain alcohol modifiers can significantly improve chiral resolution, which is not possible using traditional mobile phase modifiers such as 2-propanol and ethanol, recommended by column manufacturer [22]. Thus the use of different alcohol
Dionex high performance liquid chromatography (HPLC) system consisted of GP 40 gradient pump, LC 20 Chromatography Enclosure, AD 20 absorbance detector and a Rheodyne injection valve equipped with a 25 l sample loop. Chiral chromatography was performed using Chiralpak AD-H (250 mm × 4.6 mm ID and 5 m particles) column obtained from Chiral Technologies Inc., Exton, PA. HPLC was controlled and monitored using Dionex PeakNet version 5.01 software. Circular dichroism spectra (CD) were recorded in 1 cm quartz cell at 20 ◦ C on a Jobin Yvon CD 6 spectrophotometer. 2.2. Materials Hexane, 2-propanol, 1-pentanol, 1-hexanol (HPLC grades) and diethylamine were obtained from Aldrich Chemical Co., (Milwaukee, WI). Ethanol (absolute) was purchased from Pharmco Products Inc., (Brookfield, CT). Certified grade 1-propanol, 1-butanol and tert-butanol were obtained from Fisher Scientific, (Fair Lawn, NJ, USA). All the authentic enantiomers under the study were synthesized as previously reported [5]. 2.3. Chromatographic conditions Each enantiomer was first dissolved (∼3 mg) in 2propanol and then diluted with 8 ml of the mobile phase in order to obtain a final concentration of 0.3 mg/ml for the HPLC injection. The racemic samples were prepared by mixing both enantiomers at nearly equal concentrations. The injections for each experiment were made in triplicate. The mobile phase consists of various alcohols at different percentages in hexane. All solvents used were filtered and degassed in an ultrasonic bath before use. The flow rate was maintained at 1.0 ml/min throughout the study. Analysis was carried out at room temperature (∼22 ◦ C). UV detection was performed at 292 nm. The retention factors (k) were determined as k = (tR − tm )/tm . The dead time (tm ), determined with 1,3,5-tri-tert-butyl benzene, was
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2.84 min for all the eluents used, and tR is the retention time of the peak calculated. The separation factor (α) was calculated as α = k2 /k1 , where k1 and k2 are retention factors for the first and second eluting enantiomer, respectively. The resolution (RS ) between enantiomeric pairs was calculated by the following formula: RS = 2(tR2 − tR1 )/(wb1 + wb2 ), where wb1 and wb2 are the baseline peak-widths for the first and second eluting enantiomer, respectively. The column efficiency (N) was calculated using the formula 16(tR/wb )2 . In the separation of each enantiomeric pair, the elution order of the enantiomers was determined using an arbitrary mixture of the pure enantiomers. Column that was operated with the mobile phase containing diethylamine, were easily regenerated at the end of every day by washing them with ca. 100 ml of hexane-2-propanol (90:10, v/v) at a flow rate of 0.2 ml/min. In this way, no efficiency loss was observed throughout the experiment. After cleaning, the column was reconditioned with ca. 100 ml of the mobile phase.
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in the CSP caused by the alcohol modifiers of different sizes/shapes [12,21,25–30]. But these arguments were speculative until recently. Wang et al. demonstrated with spectral evidence that the modification of stereo environment of the chiral cavities in the CSP was due to the incorporation of the alcohol modifier of different sizes/shape may cause different degrees of twisting to the glucose units on the CSP helix, yielding different chiral selectivities [28,31]. Thus the influence of type and composition of the alcoholic modifier in the mobile phase on solute retention (k), elution order, selectivity (α) and resolution (RS ) were systematically studied and results obtained for both enantiomers are summarized in Tables 1 and 2. From the data shown in the tables, it was evident that using an alcohol modifier other than commonly used 2-propanol or ethanol on the column can be used to achieve superior separations. For example using 1-pentanol as mobile phase modifier, we obtained excellent separations for both ctpy and ctpy–x–ctpy enantiomeric pairs on the CSP. 3.1. Enantiomeric separation of (±)-ctpy ligands
3. Results and discussion Highly selective chiral separation was obtained with Chiralpak AD column for the terpyridyl enantiomers under normal phase conditions. Based on the chiral recognition model proposed for amylose tris (3,5-dimethylphenylcabamate) stationary phase, it was assumed that the terpyridyl ligands interact with the CSP via: (1) hydrogen bonding between the nitrogen atoms of the ligand and the amide proton of the carbamate moiety in the CSP; (2) dipole–dipole interactions between nitrogen atoms of the ligand and the carbonyl oxygen of carbamate moiety of the CSP; and (3) charge transfer (–) interactions between pyridine/phenyl moiety of the ligand and the phenyl group of the CSP. Thus, a particular orientation of the molecule is presented to the CSP to facilitate chiral recognition [8]. Initially the enantiomers of terpyridyl ligands were not resolved on the column with simple hexane and alcohol mixtures; separations were achieved only when diethylamine (DEA) at 0.2% (v/v) was added to the mobile phase. With this type of CSP, free silanol groups occur on the silica surface, resulting in secondary interactions [23,24] with the nitrogen atoms of the ligands, leading to peak asymmetry. DEA effectively masks these underivatized silanols, thus minimizing the adsorption effect and the resultant peak tailing. Further increments of this organic base did not affect retention or enantioselectivity. Optimization of the enantiomeric separation of the ligands was performed using various compositions of hexane with alcoholic modifiers such as ethanol, 1-propanol, 2-propanol, 1-butanol, tert-butyl alcohol, 1-pentanol and 1-hexanol. The change of alcohol modifier may result in different chromatographic behavior such as different chiral selectivity and, in some cases, inversion of elution order of the enantiomers. These changes in selectivity were rationalized by the authors as a result of alteration of the steric environment of the chiral cavities
The influence of the kind of alcohol modifiers on the chiral resolution of ctpy enantiomers is shown in Fig. 2. As mentioned above, the main reason for showing different separation properties is due to a changed geometry and/or size of the chiral groove of CSP, which is caused by the type of alcohol modifier. It could be seen from the data in Table 1 that only partial resolution was obtained using ethanol and 1-butanol as the mobile phase modifier. In addition, as the concentration of ethanol increases, the resolution decreased, and this became zero at hexane: ethanol: DEA (97:3:0.2) mobile phase, thus indicating no resolution. This data Table 1 Effect of alcohol modifier on chiral separation of ctpy enantiomers Modifier
Concentration (%)
k1
Elution order
α
N
RS
Ethanol
3 5 10 3 5 10 3 5 10 3 5 10 3 5 10 3 5 10 3 5 10
1.41 1.07 0.83 1.25 1.01 0.76 2.92 1.44 0.93 1.61 1.46 0.88 3.05 2.46 1.59 1.83 1.60 0.97 1.80 1.48 0.90
NSa (−)/(+) (−)/(+) (−)/(+) (−)/(+) (−)/(+) (−)/(+) (−)/(+) (−)/(+) NSa NSa (−)/(+) (−)/(+) (−)/(+) (−)/(+) (−)/(+) (−)/(+) (−)/(+) (−)/(+) (−)/(+) (−)/(+)
1.00 1.06 1.04 1.15 1.12 1.06 1.13 1.12 1.09 1.00 1.00 1.07 1.26 1.14 1.11 1.37 1.28 1.19 1.28 1.23 1.17
7250 7724 8329 7231 8049 8678 4849 6235 8452 5913 6580 7231 4582 4920 5592 7350 8622 9280 4150 4835 5670
0.0 0.9 0.6 1.3 1.1 0.6 1.9 1.3 0.9 0.0 0.0 0.4 2.0 1.8 1.6 4.1 3.4 1.8 2.2 1.9 1.4
1-Propanol
2-Propanol
1-Butanol
tert-Butanol
1-Pentanol
1-Hexanol
a
NS: no separation.
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Table 2 Effect of alcohol modifier on chiral separation of ctpy–x–ctpy enantiomers Modifier
Concentration (%)
k1
Elution order
α
N
RS
Ethanol
3 5 10 3 5 10 3 5 10 3 5 10 3 5 10 3 5 10 3 5 10
1.41 1.07 0.83 1.25 1.01 0.76 2.92 1.44 0.93 1.61 1.46 0.88 3.05 2.46 1.59 1.83 1.60 0.97 1.80 1.48 0.90
NSa (−)/(+) (−)/(+) (−)/(+) (−)/(+) (−)/(+) (−)/(+) (−)/(+) (−)/(+) NSa NSa (−)/(+) (−)/(+) (−)/(+) (−)/(+) (−)/(+) (−)/(+) (−)/(+) (−)/(+) (−)/(+) (−)/(+)
1.00 1.06 1.04 1.15 1.12 1.06 1.13 1.12 1.09 1.00 1.00 1.07 1.26 1.14 1.11 1.37 1.28 1.19 1.28 1.23 1.17
7250 7724 8329 7231 8049 8678 4849 6235 8452 5913 6580 7231 4582 4920 5592 7350 8622 9280 4150 4835 5670
0.0 0.9 0.6 1.3 1.1 0.6 1.9 1.3 0.9 0.0 0.0 0.4 2.0 1.8 1.6 4.1 3.4 1.8 2.2 1.9 1.4
1-Propanol
2-Propanol
1-Butanol
tert-Butanol
1-Pentanol
1-Hexanol
a
NS: no separation.
indicated that change of alcohol concentration might affect the chiral selectivity of the CSP and it confirms the results that are previously reported by Wang et al. [31]. A reasonable separation was obtained for ctpy enantiomers on the column using 2-propanol, tert-butanol and 1-hexanol as alcohol
modifiers in the mobile phase. There was a remarkable increase in the chiral resolution as the concentration of above alcohols decreased. The resolution was found to be moderately good with the mobile phases containing 1-propanol as modifier. It is interesting to find that the values of separation factor (α) and resolution (RS ) for ctpy enantiomers were highest when 1-pentanol was used as mobile-phase modifier. 1-Pentanol exhibited the most favorable effect on column efficiency, thus producing the largest plate number of any alcohol tested with this solute, resulting in a significant increase in enantiomeric resolution. We assumed that this higher plate number could result in better kinetics (mass transfer) using 1-pentanol modified mobile phase. Thus the mobile phase consisting of hexane:1-pentanol:DEA in the ratio (95:05:0.2) was found to be best suitable eluent for the chiral separation of (±)-ctpy ligand and was used in routine analysis of ctpy for the determination of enantiomeric excess. During this investigation, we also found that the elution order of the enantiomers changed according to the steric bulk of the alcohol in the mobile phase. The enantiomeric resolution was (−)/(+) with ethanol, 2-propanol, 1-propanol, and tert-butanol as the mobile phase modifiers, and the elution order was (+)/(−) with 1-butanol or 1-pentanol or 1-hexanol. The reversal of elution orders is due to different chiral selectivities of the CSP associated with the use of different alcohol modifiers that caused alterations to the steric environment of the chiral cavities in the CSP [31]. The reversal of elution orders of enantiomers on cellulose- and amylose-based CSPs upon changing the alcohol modifiers in the mobile phase have been reported by a number of research groups [12,21,25–30]. In our case, the reversal of the elution order of the ctpy enantiomers upon changing the alcohol modifier from small chain to long alkyl chain alcohols was probably due to same reason. The reversal of the elution order achieved in this current study would allow a lower detection limit (<0.1%) of the minor enantiomer and the chromatographic system described is suitable for assessing the enantiomeric excesses of the ctpy ≥99.9%. 3.2. Enantiomeric separation of (±)-ctpy–x–ctpy ligands
Fig. 2. Enantioseparations of (±)-ctpy by HPLC using Chiralpak AD column, mobile phases: n-hexane:1-pentanol:DEA, 95:5:0.2, (v/v/v) (A); nhexane:tert-butanol:DEA, 90:10:0.2, (v/v/v) (B); n-hexane:1-hexanol:DEA, 95:5:0.2, (v/v/v) (C); n-hexane:1-propanol:DEA, 93:7:0.2, (v/v/v) (D); nhexane:1-ethanol:DEA, 95:5:0.2, (v/v/v) (E); n-hexane:2-propanol:DEA, 97:3:0.2, (v/v/v) (F).
The chromatograms of the separation of ctpy–x–ctpy enantiomers on Chiralpak AD column using hexane and different alcohol modifiers are shown in Fig. 3. From Table 2, no resolution occurs with 2-propanol and 1-butanol modifiers on the investigated column. Even though there was a higher α value, resolution between the isomers was somewhat lower with tert-butanol and ethanol compared to 1-propanol or 1-hexanol. This was the result of poorer column efficiency (plate number N = 774 for tert-butanol, N = 367 for ethanol) which may be caused by less favorable kinetics of the solvated stationary phase. Highest resolution and α values for ctpy–x–ctpy enantiomers was again found with 1-pentanol as mobile phase modifier, even though column efficiency was lower (N = 1373). Thus the set of conditions shown in Fig. 3
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Fig. 4. Estimation of enantiomeric excess of ‘pineno’ fused terpyridyl ligands by HPLC. Chiralpak AD column, mobile phases: n-hexane:1pentanol:DEA, 95:5:0.2, (v/v/v), UV: 292 and flow rate: 1.0 ml/min.
3.4. Limit of detection and quantification
Fig. 3. Enantioseparations of (±)-ctpy by HPLC using Chiralpak AD column, phases: n-hexane:1-pentanol:DEA, 95:5:0.2, (v/v/v) (A); n-hexane:1propanol:DEA, 93:7:0.2, (v/v/v) (B); n-hexane:tert-butanol:DEA, 90:10:0.2, (v/v/v) (C); n-hexane:ethanol:DEA, 93:7:0.2, (v/v/v) (D); n-hexane:1hexanol:DEA, 95:05:0.2, (v/v/v) (E).
using 1-pentanol on AD column provided the best separation and resolution, and were used as the method conditions for the estimation of enantiomeric excess of ctpy–x–ctpy. The elution orders of the enantiomers of ctpy–x–ctpy were found to be the same for all alcohol modifiers tested, unlike ctpy isomers. Wainer et al. have reported that the solute having aromatic functionalities could provide additional stabilizing effect to the solute–CSP complex by insertion of the aromatic portion of the solute into the chiral cavity [20]. The similar type of stabilization effect might exits with ctpy–x–ctpy due to the presence of additional phenyl ring linked between two ctpy moieties, and this is probably the reason for the above said behavior since the chiral discrimination between the enantiomers is due to the differences in their steric fit in the chiral cavities [19,20]. 3.3. Determination of enantiomeric excess The current method was applied for the estimation of enantiomeric excess (ee) using real samples of terpyridyl ligands. The representative chromatograms of each ligand are shown in Fig. 4. Interestingly, the other isomer was not detected in each of the enantiomers of ctpy. The enantiomeric excess of ctpy–x–ctpy enantiomers was found to be more than 99.5%. The precision of the method for the quantitative determination of optical purity of terpyridyl enantiomers was determined by analyzing six independent samples of each enantiomer by two analysts on two different days and two columns using different instruments and mobile phase preparations. The precision of the method was found to be less than 3.0% R.S.D.
The limit of detection (LOD) was established for both ligands by determining the concentration of a dilute enantiomer that gave a signal-to-noise ratio of 3. The limit of quantitation (LOQ) is defined as the lowest concentration that can be determined with acceptable accuracy and precision, which can be established at a signal-to-noise ratio of 10. The LOD and LOQ values were 0.2 g/ml and 0.6 g/ml for the ctpy enantiomers, and 0.6 g/ml and 1.2 g/ml for the ctpy–x–ctpy enantiomers, respectively. The possibility of potential chiral inversion of terpyridyl enantiomers in the sample preparation was also investigated. For each enantiomer of ctpy and ctpy–x–ctpy, 0.3% of other isomer was spiked and analyzed for three successive days. The results (R.S.D. < 2.5%) indicated that the terpyridyl enantiomers are chirally stable in solution for at least 3 days. Based on the precautionary statements furnished with the Chiralpak AD column instruction sheet by the manufacturer [22], we were concerned about the stability of the Chiralpak AD column when employing long chain alcohol modifiers. We compared the column performance with a fresh column and one with a history of about 500 sample injections, using a mobile phase of 5% 1-pentanol-hexane. Essentially equivalent retention and resolution for the two columns suggest good column stability in the above mobile phase.
4. Conclusions We have successfully used Chiralpak AD column for the chiral resolution of ‘pineno’ fused terpyridyl ligands under normal phase conditions. 1-Pentanol as alcohol modifier improved the enantiomeric resolution of investigated compounds in the mobile phase. It was found that long chain alcohols such as 1-pentanol and 1-hexanol reversed the elution order of ctpy enantiomers in the current study. These results suggest that using an alcohol modifier other than the commonly used 2-propanol or ethanol on the same column
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can be used to achieve better enantioseparations. This approach can provide useful and less expensive alternatives in method development of chiral resolution of certain analytes. Acknowledgement Financial support of this work by NYS Center for Advanced Technology/IEEC/NSF is gratefully acknowledged. The authors also acknowledge financial support from the USEPA Science-to-Achieve Program. References [1] F.R. Keene, Coord. Chem. Rev. 166 (1997) 121–151. [2] A. Von Zelewsky, Stereochemistry of Coordination Compounds, Wiley, New York, 1996. [3] P. Hayoz, A. Von Zelewsky, H. Stoeckli-Evans, J. Am. Chem. Soc. 115 (1993) 5111–5114. [4] H. Muerner, P. Belser, A. Von Zelewsky, J. Am. Chem. Soc. 118 (1996) 7989–7994. [5] S. Bernhard, K. Takada, D.J. Diaz, H.D. Abruna, H. Murner, J. Am. Chem. Soc. 123 (2001) 10265–10271. [6] M. Ziegler, V. Monney, H. Stoeckli-Evans, A. Von Zelewsky, I. Sasaki, G. Dupic, J.-C. Daran, G.G.A. Balavoine, J. Chem. Soc., Dalton Trans. 5 (1999) 667–675. [7] S. Lin, C.E. Engelsma, N.J. Maddox, B.K. Huckabee, D.M. Sobieray, J. Liquid Chrom. Rel. Technol. 20 (1997) 1243–1256. [8] T. Shibata, I. Okamoto, K. Ishii, J. Liquid Chromatogr. 9 (1986) 313–340. [9] P.S. Bonato, V.L. Lanchote, R. Bortocan, V.A.P. Jabor, F.O. Paias, E. Ricci-Junior, R. Carvalho, J. Liquid Chrom. Rel. Technol. 22 (1999) 1813–1827.
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