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.J. Biochem. Biophys. Methods 48 2001 175188

www.elsevier.comrlocaterjbbm

Studies on the effect of alcohols on the chiraldiscrimination mechanisms of amylose stationary

phase on the enantioseparation of nebivololby HPLC

Hassan Y. Aboul-Enein)

, Imran Ali( )Pharmaceutical Analysis Laboratory, Biological and Medical Research Department MBC-03 , King Faisal

Specialist Hospital and Research Center, P.O. Box 3354, Riyadh-11211, Saudi Arabia

Abstract

The chiral recognition mechanism of amylose CSPs has been described by achieving the .enantiomeric resolution of " -nebivolol on Chiralpak AD and Chiralpak AD-RH columns with

methanol, ethanol, 1-propanol, 2-propanol, 1-butanol as mobile phases at different flow rates. Theenergies of interactions of methanol, ethanol, 1-propanol, 2-propanol and 1-butanol with both

.phases were calculated. The q -RRRS enantiomer eluted first when using methanol, ethanol and

1-propanol, while the elution order was reversed when using 2-propanol and 1-butanol as the

mobile phases. It has been concluded that the reversal elution order observed was due in part to

the chiral cavities on the amylose CSP which were responsible for the bondings of different

magnitude between chiral stationary phase and enantiomers, which are influenced with the type of

alcohol used as mobile phase on the conformation of the 3,5-dimethyl phenyl carbamate moiety on

the pyranose ring system of the amylose. q 2001 Elsevier Science B.V. All rights reserved.

Keywords: Chiral recognition mechanism; Resolution; Enantiomers; Nebivolol; Energy of interaction; Amy- .lose tris 3,5-dimethylphenyl carbamate ; Effect of alcohols

1. Introduction

The chiral resolution of racemates is an important area in the field of analytical

chemistry and enantioseparations. Chromatography has become the powerful tool and

)

Corresponding author. Tel.: q966-1-442-7859; fax: q966-1-442-7858. .E-mail address: [email protected] H.Y. Aboul-Enein .

0165-022Xr01r$ - see front matter q2001 Elsevier Science B.V. All rights reserved. .P I I : S 0 1 6 5 - 0 2 2 X 0 1 0 0 1 4 8 - 8

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( )H.Y. Aboul-Enein, I. Ali rJ. Biochem. Biophys. Methods 48 2001 175 188176

most practical technique of enantiomeric resolution of a wide variety of racemic

pharmaceuticals and drugs. A search of literature indicates that various chiral stationaryw xphases have been developed for this purpose 17 . The understanding of chiral

recognition mechanism at molecular level is of great importance in the field of chiral

chromatography. Further search of literature reveals that some approaches have been

done to find out the chiral recognition mechanism of different chiral stationary phases .CSPs . Attempts have been made by different workers to discuss the chiral recognition

w x w xmechanism by NMR 8 13 and computational methods 11,1422 . The chiral recogni-w x w xtion mechanisms have been developed for cyclodextrins 11,13 , Pirkle type 1417 and

w xpolysaccharides CSPs 2123 . Various rational models of interactions between CSPs

and enantiomers have been proposed. The interaction energies between CSPs and

enantiomers have been calculated by quantum mechanical calculations and the chiral

recognition mechanisms have been proposed based on these calculations and molecularw xsimulation dynamics 11,1418,20 .

Among the various CSPs, the polysaccharide-based CSPs, i.e. derivatized celluloseand amylose CSPs, are considered efficient chiral stationary phases due to their wide

w xrange of applications in the field of chiral chromatography 2430 . Although attempts

have been made to predict the chiral recognition mechanism of these stationary phases at

molecular level, the exact mechanism is still not known. NMR spectroscopy is the

. .Fig. 1. The stereochemical formulae of q -RRRS and y -SSSR enantiomers of nebivolol.

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( )H.Y. Aboul-Enein, I. Ali rJ. Biochem. Biophys. Methods 48 2001 175 188 177

powerful and main technique for revealing the chiral recognition mechanism but the

polysaccharide-based CSPs are soluble in the spectroscopic solvents such as tetrahydro-

furan, acetone and pyridine. These solvents interact with the carbamate moieties, which

is considered an essential adsorption sites for chiral recognition, of the polysaccharide-

based CSPs and hence, the chiral recognition cannot be studied using these solvents.

w xHowever, recently, Yashima, et al. 12 were able to study the chiral recognition .mechanism of cellulose tris 4-trimethyl silyl phenyl carbamate by NMR .

.Amylose tris 3,5-dimethyl phenyl carbamate is a semi synthetic polymer which .contain a polymeric chain of derivatized D- q glucose residues in a-1,4 linkage. These

chains lie side by side in a helical fashion. The three dimensional structures of cellulose-

and amylose-based CSPs were determined and compared using computational chemistryw x w x31 . Vogt and Zugenmaier 32 reported that the possible structures were 3r2 helical

.chain conformation for cellulose tris phenyl carbamate and 4r1 helical chain confor- .mation for amylose tris phenyl carbamate . The amylose CSP is more helical in nature

and has well-defined cavities, making it considerably different from the correspondingcellulose analogue, which appear to be more linear and rigid in nature.

In view of the problem associated with the experimental determination of chiral

recognition mechanism as discussed above, we have carried out some HPLC experi- .ments on amylose tris 3,5-dimethylphenyl carbamate CSPs and attempts have been

made to explain the chiral recognition mechanism by these CSPs. We have selected .nebivolol Fig. 1 to carry out this study as it is the newly developed b-adrenergic agent

w xwith superior vasodilating properties 33 36 . The purpose of this study is to explain the .chiral recognition mechanisms involved, to resolve " -nebivolol on amylose tris

.3,5-dimethylphenyl carbamate and to study the effect of alcohols on chiral discrimina-tion involved in the resolution of nebivolol enantiomers.

2. Experimental

2.1. Chemicals and reagents

. .The individual nebivolol enantiomers namely q -RRRS Product No. R85547 and . . .y -SSSR Product No. R85548 and their racemic mixture Product No. R67555 were

obtained as gifts by Janssen Research Foundation, Beerse, Belgium. Methanol, 2-pro-panol and 1-butanol of HPLC grade were purchased from Fisher Scientific Fairlawn,

. .NJ, USA . The absolute ethanol was obtained from E. Merck Darmstadt, Germany .

1-Propanol was supplied by BDH, London, UK.

2.2. Apparatus

All the HPLC experiments were performed on a HPLC system consisting of Waters . .solvent delivery pump model 510 , Waters injector model WISP 710B , Waters

. .tunable absorbance detector model 484 and Waters integrator of Waters model 740 . .The columns used were Chiralpak AD 25= 0.46 cm I.D., particle size 10 mm and

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.Chiralpak AD-RH 15= 0.46 cm. I.D., particle size, 5 mm and were obtained from

Daicel Chemical, Tokyo, Japan.

2.3. Analysis

.The stock solutions 0.1 mgrml of the racemic nebivolol and its enantiomers wereprepared in absolute ethanol. Twenty microliter of each of the solutions were injected on

to a HPLC system described above. The mobile phases used in this study were

methanol, ethanol, 1-propanol, 2-propanol and 1-butanol. The mobile phases were

filtered and degassed before use. The flow rates of the mobile phases used were 0.5, 1.0

and 1.5 mlrmin, respectively, for all the alcohols except for 1-butanol where the flow

rate was 0.1 mlrmin. The chart speed was kept at 0.1 cmrmin. The detection of

nebivolol was achieved at 220 nm. All the experiments were carried out at 23 " 18C. To .study the effect of ethanol and 2-propanol ratios on enantiomeric resolution of " -

nebivolol, different percentages of ethanol and 2-propanol were used at a flow rate of1.0 mlrmin. on Chiralpak AD-RH Chiralpak AD columns.

To determine the energy of interaction of alcohols with stationary phases, the

chromatograms of methanol, ethanol, 1-propanol, 2-propanol and 1-butanol were also .recorded using n-hexane 1.0 mlrmin. as the mobile phases on both Chiralpak AD and

Chiralpak AD-RH columns. The n-hexane was used as the mobile phase in both the

stationary phases because the polarity of the n-hexane is zero and it is supposed that

there is no interaction between hexane and the chiral stationary phases. The dead time .t of both the columns were determined by injecting 20 ml of air using n-hexane as0

the mobile phase at a flow rate of 1.0 mlrmin with detection at 220 nm. The X . .chromatographic parameters such as capacity factor k , separation factor a and

.resolution factor Rs were calculated.

3. Results and discussion

X . .The chromatographic parameters, capacity factor k , separation factor a and . . .resolution factor Rs for the resolved q -RRRS and y -SSSR nebivolol enantiomers

on normal and reversed phases are given in Tables 1 and 2, respectively. The resolvedenantiomers were identified by running the chromatograms for the individual enan-

. .tiomers, i.e. q -RRRS and y -SSSR enantiomers under the same chromatographic

conditions. Tables 1 and 2 show that the enantiomers of nebivolol have been resolved on

both Chiralpak AD and Chiralpak AD-RH columns using ethanol, 1-propanol and

2-propanol as mobile phases at different flow rates. Although the base line resolution of

racemic nebivolol was achieved using ethanol, 1-propanol and 2-propanol at all the .reported flow rates Tables 1 and 2 , the best resolution may be considered at 0.5

mlrmin. flow rate on both Chiralpak AD and Chiralpak AD-RH columns except in the

case of 2-propanol on Chiralpak AD-RH where the best resolution was achieved at 1.5 .mlrmin flow rate Fig. 2 . To optimize the resolution, a variation in the chromato-

graphic parameters was carried out. Methanol and 1-butanol were also tried but a partial

resolution could be only achieved on both of normal and reversed phases. The various

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Table 1 X . . .The chromatographic parameters, capacity factor k , separation factor a and resolution factor Rs for

.enantiomeric resolution of " -nebivolol on Chiralpak AD stationary phaseX X .Flow rate mlrmin k k a Rs1 2

MeOH

. .q -RRRS y -SSSR0.5 1.06 1.95 1.84 0.96

1.0 4.88 3.21 1.71 0.50

1.5 2.04 3.38 1.66 0.41

EtOH

. .q -RRRS y -SSSR

0.5 4.57 6.32 1.38 2.63

1.0 4.33 6.06 1.40 2.28

1.5 4.18 5.77 1.38 1.40

1-PrOH . .q -RRRS y -SSSR

0.5 4.22 5.84 1.38 1.71

1.0 2.10 3.03 1.44 1.45

1.5 1.09 1.86 1.71 1.21

2-PrOH

. .y -SSSR q -RRRS

0.5 1.53 6.32 4.13 2.45

1.0 1.36 5.96 4.38 2.36

1.5 1.40 5.85 4.18 2.53

1-BuOH

. .y -SSSR q -RRRS

0.1 3.16 3.34 1.06 0.22

ratios of the reported alcohols itself and with other solvents such as n-hexane normal. .phase mode , acetonitrile reversed phase mode in the presence of diethylamine were

tried but no improved resolution was observed. Since only partial resolution was

Table 2

X

. . .The chromatographic parameters, capacity factor k , separation factor a and resolution factor Rs for .enantiomeric resolution of " -nebivolol on Chiralpak AD-RH stationary phase

X X .Flow rate mlrmin k k a Rs1 2

EtOH

. .q -RRRS y -SSSR

0.5 5.65 7.94 1.41 1.73

1.0 3.00 4.27 1.42 1.10

1.5 2.60 3.75 1.44 1.15

1-PrOH

. .q -RRRS y -SSSR0.5 8.36 11.5 1.38 1.76

1.0 2.61 3.83 1.47 1.20

1.5 1.10 1.75 1.59 1.10

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. . Fig. 2. Chromatograms of resolved enantiomers of " -nebivolol on I: Chiralpak AD with a ethanol 0.5. . . . . .mlrmin , b 1-propanol 0.5 mlrmin and c 2-propanol 1.5 mlrmin and II: Chiralpak AD-RH with d . . .ethanol 0.5 mlrmin and e 1-propanol 1.5 mlrmin .

achieved when using methanol and 1-butanol as mobile phase, it may be concluded that

the enantiomeric resolution of nebivolol is controlled by both polarities and viscosities

of the alcohols. Accordingly, the polarities and viscosities of ethanol, 1-propanol and

2-propanol seem suitable for the resolution of enantiomers of nebivolol.

Generally, the values of a for the resolved enantiomers increased with the increase

of flow rates. It is due to the fact that the retention of enantiomers decreases by

increasing the flow rates. It is of interest to note that the a values for the enantiomeric

.resolution in all cases using ethanol and 1-propanol at all flow rates are greater onreversed stationary phase than the normal stationary phase. However, the differences of

the a values for ethanol and 1-propanol at different flow rates is greater in normal

phase condition than in the reversed stationary phase which may be due that the normal

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stationary phase is more affected by the flow rate in comparison to the reversed

stationary phase. . .It was found that q -RRRS enantiomer eluted first than the y -SSSR enantiomer

when using methanol, ethanol and 1-propanol as mobile phases while the order of

elution was reversed when using 2-propanol and 1-butanol as the mobile phase on

Chiralpak AD column. However, the elution order has not been reversed on ChiralpakAD-RH column. This could be due to the fact that alcohols interact strongly with the

normal phase than the reversed phase. The poor interaction of alcohols with reversed

chiral phase stationary phase may be due to the presence of a repulsion between thealcohols alkyl chains and the alkyl chains which make the chiral stationary phase

.reversed in nature of the reversed CSP. We have used the reverse elution order

observed as the mean to explain the chiral recognition mechanism of the amylose

.Fig. 3. The relationship between the percentage of ethanol and 2-propanol and the retention times of q and .y enantiomers of nebivolol.

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stationary phases. To understand the chiral recognition mechanism on Chiralpak AD, the

resolution of nebivolol was tried with different ratios of ethanol and 2-propanol. The

resolution of nebivolol with different ratios of 1-propanol and 2-propanol was also tried

but there was a detection problem. The resolution behavior of nebivolol with different

ratios of ethanol and 2-propanol is given in Figs. 3 and 4. Fig. 3 shows that the

.q -RRRS enantiomer eluted first using ethanol and the difference of the retentiontimes between the two enantiomers decreases by increasing the volume of 2-propanol.

There was no resolution when the mobile phase consisted of ethanol:2-propanol 77:23,.vrv was used. However, by increasing the volume of 2-propanol, the resolution of

these two enantiomers occurred but with the reverse order of elution. Furthermore, the .values of resolution factors Rs were more than 1.0 using ethanol or 2-propanol, but

these values decreased by using different ratios of ethanol and 2-propanol and it became .zero at ethanol:2-propanol 77:23, vrv mobile phase indicating no resolution as shown

in Fig. 4. In order to explain this behavior, the energies of interaction of alcohols with

.Fig. 4. The relationship between the percentage of ethanol and 2-propanol and the resolution factor Rs of . .q and y enantiomers of nebivolol.

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the stationary phases were determined and shown in Table 3. The energies of thew xinteractions of alcohols were calculated by the formula described by Yuasa et al. 37 .

DDG sRT ln a

.This equation has been used to calculate the difference of the energies DDG of

interactions of the two enantiomers with chiral stationary phase but to determine the .energies of interactions G of alcohols with chiral stationary phases, we have solved

this equation as follows:

DDG sRT ln a

or

DDG sRT ln kX

rkX

2 1

or

G sRT ln kX

X

.where k is equal to t y t rt for different alcohols. The retention times of ther 0 0 alcohols were determined by using the pure n-hexane hexane has zero polarity and.supposed to be inert in both normal and reversed phases as the mobile phase with 1.0

mlrmin. as the flow rate on both normal and reversed phases. To calculate the value ofX .k , the dead times of the columns t were determined by using the same mobile phase0

and by loading 20 ml of air as the air is supposed to be inert and no interaction occurred

between the air and the stationary phases. Table 3 shows that the values of energies of

interactions of methanol, ethanol and 1-propanol were negative in magnitude with

Chiralpak AD, while the values of energy of interactions of 2-propanol and 1-butanol

were positive in magnitude and increase drastically in comparison to methanol, ethanoland 1-propanol. The values of energies of interaction of these alcohols with Chiralpak

AD-RH were of negative in magnitude indicating a very poor interaction of alcohols

with chiral reversed stationary phase. These findings clearly indicates that the alcohols

interact very strongly with the normal phase while the interaction is very poor in case of

Table 3

The energies of interactions of alcohols with Chiralpak AD and Chiralpak AD-RH stationary phasesX . .Alcohols t min k G K Calrmolr

Chiralpak AD

Methanol 4.56 0.37 y0.56

Ethanol 5.60 0.70 y0.21

1-Propanol 5.70 0.73 y0.18

2-Propanol 14.23 3.33 0.71

1-Butanol 14.57 3.43 0.73

Chiralpak AD-RH

Methanol 1.96 0.026 y2.15

Ethanol 1.97 0.031 y2.04

1-Propanol 2.00 0.047 y1.802-Propanol 2.10 0.099 y1.36

1-Butanol 2.26 0.180 y1.00

t : 3.29 and 1.91 min for Chiralpak AD and Chiralpak AD-RH stationary phases respectively.0

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reversed phase and therefore no reverse order of elution was observed in case of

reversed phase. It is also interesting to observe that the interaction of methanol, ethanol

and 1-propanol with Chiralpak AD is poor in comparison to the interaction of 2-pro-

panol and 1-butanol. Therefore, 2-propanol and 1-butanol interact very strongly with

normal chiral stationary phase and create a strain on the amylose chain, which resulted

into the change of its configuration, i.e. the configuration of the pyranose ring of theamylose polysaccharide chain reversed. Besides, the long alkyl chains of these two

alcohols create a strain on amylose stationary phase and are responsible for the reverse

order of elution.w xIt has been reported 38 that, under the normal conditions, the configuration of the

glucose units of cellulose remains in the chair form with equatorial positions of hydroxyl

groups, which is supposed to be the most stable configuration. In the same way, the .amylose tris 3,5-dimethylphenyl carbamate with equatorial position of carbamate

moieties may be considered as the most stable configuration. The chiral recognition

mechanism at a molecular level on the polysaccharide-based CSPs is not knownalthough it has been reported that the chiral resolution by celluloseramylose-CSP is

`w x w xachieved through the hydrogen bonding 21 and p p interactions 39 between the

chiral stationary phase and the enantiomers. These hydrogen bondings occurred between

the carbamate moieties of the celluloseramylose-CSPs and the functional groups suchw xas hydroxyl, carbonyl, amino, etc., of enantiomers 21 . Although it is known that the

`chiral recognition occurred due to the different hydrogen and p p bondings, however,

the reason for the different magnitude of these bondings with enantiomers is not known.

Therefore, attempts have been made to explain the chiral recognition mechanism in

detail.The change in the configuration of chiral stationary phase has been explained by

.calculating a volume and energy factor F for ethanol:2-propanol mobile phase at

different ratios and it was calculated by the following equation.

Fs VE

where V and E are the volume of ethanol or 2-propanol and the energy of interaction of

ethanol or 2-propanol, respectively. The values of this factor for different ratios of .ethanol and 2-propanol are given in Table 4. The factors F for ethanol and 2-propanol

. were represented by F energy factor for ethanol and F energy factor for 2-pro-et pr.panol , respectively. Table 4 shows that the values of F are negative and the values ofet

F are positive, which corresponds to the different configurations of chiral stationaryprphase in ethanol and 2-propanol, respectively. To prove the different configuration of

the chiral stationary phases in ethanol and 2-propanol, we have calculated the sum of Fet .and F and is given in Table 4. The F q F values are either positive or negative inpr et pr

.magnitude Table 4 and the negative values correspond to the greater percentage of

ethanol while the positive values are for greater percentage of 2-propanol in ethanol and

2-propanol mixture. The negative values may be correlated with the stable configuration

of amylose stationary phase with the equatorial positions of 3,5-dimethylphenyl carba-mate moieties. On the other hand, the positive values may be due to the less stable

configuration of amylose stationary phase, i.e. with the axial positions of 3,5-dimethyl- .phenyl carbamate moieties. The values of F q F decreases as the percentage ofet pr

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Table 4 .The calculated values of F , F and F q F on Chiralpak AD stationary phaseet pr et pr

. .Volume ml F F F q Fet pr et pr

EtOH 2-PrOH

00 100 0.00 71.00 71.00

50 50 y10.50 35.50 25.00

55 45 y11.55 31.95 20.40

60 40 y12.60 28.40 15.80

65 35 y13.65 24.85 11.20

70 30 y14.70 21.30 6.35

75 25 y15.75 17.75 2.00

77 23 y16.17 16.33 0.16

80 20 y16.80 14.20 y2.60

85 15 y17.85 10.65 y7.20

90 10 y18.90 7.10 y11.80

95 05 y19.95 3.55 y16.40

100 00 y21.00 0.00 y21.00

FsVE, where F, V and E are factor, volume of alcohols and energy of interaction, respectively.

F and F are factors for ethanol and 2-propanol respectively.et pr

.ethanol increase in ethanol and 2-propanol mobile phase. The values of F q Fet prchange from positive to negative through a value of 0.16, which is the smallest values

and equal to zero. Therefore, it may be concluded that the chiral configuration of

amylose phase changed by replacing ethanol by 2-propanol but at the mixture of

.ethanol:2-propanol 77:23, vrv the environment of amylose phase is 50% with .equatorial and 50% with axial positions of the 3,5-dimethylphenyl carbamate moieties.

`Therefore, at this mobile phase composition, the magnitude of hydrogen and p p

bondings are the same for both of the enantiomers and hence, no resolution occurred .Figs. 3 and 4 .

The cavities on the amylose CSPs provide the suitable site for a particular enan- . .tiomer, i.e. either for q -RRRS or y -SSSR. One of the two enantiomers fits more

`closely with greater chances of the bondings by hydrogen and p p interactions. It may

be assumed that the configuration of amylose chiral stationary phase remains in the form

of chair with equatorial position of 3,5-dimethylphenyl carbamate moieties in presence .of ethanol and the chiral cavities provide the suitable site for y -SSSR enantiomer of

. .nebivolol, i.e. the y -SSSR enantiomer fit more closely than the q -RRRS enan-`tiomer. Therefore, the chances of the hydrogen and p p interactions in case of

. .y -SSSR enantiomer is greater than the q -RRRS enantiomer. Accordingly, the . .y -SSSR enantiomer retained more and eluted after q -RRRS enantiomer. On the

contrary, in the presence of 2-propanol or 1-butanol, the configuration of the amylose

stationary phase reversed, i.e. the 3,5-dimethylphenyl carbamate moieties are changed .from equatorial to axial position. This configuration provides the better site for q -

. .RRRS enantiomer than y -SSSR enantiomers and hence, q -RRRS enantiomer .retained more and eluted after y -SSSR enantiomer. The change of cellulose configu-

ration through equatorial and axial positions in different mobile phases has beenw xreported by Yuasa et al. 38 .

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Attempts have also been made to find out the different hydrogen bonding interactions

between chiral stationary phase and nebivolol enantiomers. It has been observed that the .magnitude of hydrogen bonding between y -SSSR and chiral stationary phase with

.equatorial position was greater in comparison to q -RRRS enantiomer. On the otherhand, the hydrogen bondings were greater between chiral stationary phase with axial

. .positions of 3,5-dimethylphenyl carbamate moieties and q -RRRS enantiomer in .comparison to y -SSSR enantiomer. Besides, we have also observed that the differ-

ence of the magnitude of the hydrogen bondings of the two enantiomers was greater in

case of amylose chiral stationary phase with axial positions of 3,5-dimethylphenyl

carbamate moities. This explains the greater difference of retention times of the two

enantiomers on the chiral stationary phase with the axial positions of carbamate

moieties.

The reverse elution order on cellulose chiral stationary phases has also been reported

by other workers. The high percentage of 2-propanol has reversed the order of elution of

w . x w xb-blockers on Chiracel OD column cellulose tris 3,5-dimethyl phenyl carbamate 40 .The different enantioselectivity by different alcohols has been observed by Tang et al.w x w x w x25 , Wainer et al. 41 and Wainer and Stiffin 42 . Therefore, it may be concluded that

the chiral recognition mechanism of celluloseramylose phases are more or less similar

in nature.

In summary, it may be concluded that the configuration of the chiral cavities in the .amylose tris 3,5-dimethylphenyl carbamate is determined by the composition of mobile

phase in normal phase mode while the configuration of reversed phase mode remains

unchanged. The chiral recognition is not simply due only to hydrogen bondings and

`

p p interactions but also due to the chiral cavities with specific configuration on thestationary phases that are responsible for bondings of different magnitude between the

stationary phase and enantiomers.

Acknowledgements

The authors are thankful to JANSSEN Research Foundation, Beerse, Belgium for

providing the nebivolol enantiomers and the racemic form used in this study. The

authors would also like to thank the King Faisal Specialist Hospital and Research Centre

administration for their support for the Pharmaceutical Analysis Laboratory Research

Programs.

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