Original

Chromatography, Vol.24 No.3 (2003)

Original

Enthalpy−entropy compensation for enantio−separation on cellulose andamylose tris (3, 5−dimethylphenylcarbamate) derivatives as stationary phases
Masato Kazusaki*, Toshiya Shoda and Hirofumi Kawabata
Chemical Analysis Research Group, Pharmaceutical Research & Technology Center, Dainippon Pharmaceutical Co., Ltd.
1−5−51 Ebie, Fukushima−ku, Osaka, 553−0001, Japan
Received July 17, 2003. Revised manuscript received October 1, 2003. Accepted October 3, 2003

Abstract
The thermodynamics of enantio−separation on cellulose and amylose tris (3, 5−dimethylphenylcarbamate) derivatives (CHIRALCEL OD−
RH and CHIRALPAK AD−RH) was studied in the reversed−phase mode. The differences of enthalpic and entropic changes of enantio−selective adsorption are discussed with the aim to rationalize their possible contributions to the overall chiral recognition. On the cellulose−derived
OD−RH column chiral recognition was completely enthalpy−driven with a negative entropic contribution. However, positive entropic contribution for enantio−separation was observed on an amylose−derived AD−RH column. Enthalpy−entropy compensation concerning enantio−
separation indicates that enthalpic gain/loss was substantially cancelled out by the entropic loss/gain on both columns. This relationshipleaded to constant ∆∆ G°for enantio−separation. It was also revealed that the entropic contribution caused the greater capability for enantio−
separation on an amylose−derived AD−RH column than on a cellulose−derived OD−RH column.
Keywords: CHIRALCEL OD−RH, CHIRALPAK AD−RH, enthalpy−entropy compensation, van’t Hoff plot

Introduction
The development of analytical methods that separate andquantify the enantiomers of pharmaceutical compounds continues
to play an important role in the drug development process [1].
Many chiral stationary phases have been developed and commercialized for the detection of the enantiomer as the potential impurity in the optically active drug substance. In the past, we reported
the separation of pharmaceutical compound and the corresponding
enantiomer on cellulose tris (3, 5−dimethylphenylcarbamate)(CHIRALCEL OD−RH) and amylose tris (3, 5−dimethylphenylcarbamate) (CHIRALPAK AD−RH) [2]. While the amylose−based
CHIRALPAK AD−RH had the same derivatization group (3, 5−dimethylphenylcarbamate) as its cellulose−based counter part
(CHIRALCEL OD−RH) did, the elution order of enantiomers on
the CHIRALPAK AD−RH was reversed compared to that on

Cellulose derivative

CHIRALCEL OD−RH.

Amylose derivative

Figure 1. Chemical structures of chiral selectors
Cellulose derivative: CHIRALCEL OD−RH
Amylose derivative : CHIRALPAK AD−RH

The structures of the derivatized subunits of the chiral stationary phases (CSPs) were shown in Figure 1. Okamoto’s research
Tel: 06−6454−8177, Fax: 06−6458−3723
E−mail: masato−kazusaki@dainippon−pharm.co.jp―１２１―

Chromatography, Vol.24 No.3 (2003)

group has reported numerous examples in which cellulose−based

Provided that these equations are temperature−independent, graphi-

and amylose−based columns showed different chiral recognition

cal analysis of ln α and inverse of absolute temperature gives lin-abilities [3−5]. The difference in chiral recognition ability between

ear plots (van’t Hoff plots), from which the molar differential en-

the two CSPs is attributed to the conformational difference be-thalpy (∆∆ H °) and entropy (∆∆ S °) can be extracted from thetween cellulose−derived and amylose−derived stationary phases.

slope (− ∆∆ H °/R ) and the intercept (∆∆ S °/R ), respectively.

Temperature has a major impact on retention, selectivity

In this paper we explore the mechanism of enantio−separation(enantio−selectivity), resolution, and column efficiency for chro-of potential drug substance (R −2− (4−bromo−2−fluorobenzyl)−(1,matographic separation. Consequently, the variation of the column

2, 3, 4−tetrahydropyrrolo [1, 2−a] pyrazine−4−spiro−3’−pyr-operation temperature has been frequently exploited as an optimi-

rolidine)−1, 2’, 3, 5’−tetrone) (Figure 2) [20] and corresponding

zation parameter in gas and liquid chromatographic separations of(S )−enantiomer as the probe compounds on the chiral columns,

enantiomers [6−9]. Analysis based on van’t Hoff plots of retention

CHIRALCEL OD−RH and CHIRALPAK AD−RH, respectively.

and separation derived from variable−temperature operation is routinely used to access thermodynamic functions of enantio−selective

Experimental

adsorption, which may be interpreted in terms of mechanistic as-

Materials

pects of chiral recognition. Numerous studies devoted to the eluci-

R −2− (4−Bromo−2−fluorobenzyl)−(1, 2, 3, 4−tetrahydropyr-

dation of effects of temperature on the separation characteristics ofrolo [1, 2−a] pyrazine−4−spiro−3’−pyrrolidine)−1, 2’, 3, 5’−chiral selector systems, such as protein [10, 11], crown ethers [12,tetrone and corresponding S −enantiomer were synthesized in the13], cyclodextrins [14] and cellulose− and amylose−derivatives [15laboratory of Dainippon Pharmaceutical Co., Ltd. (Osaka, Japan).

−19].

Ammonium acetate was of reagent grade from Wako Pure Chemi-

Under linear chromatographic conditions the temperature de-cal Industries Ltd. (Osaka, Japan). HPLC grade acetonitrile werependence on the retention of a given analyte can be expressed withalso purchased from Wako Pure Chemical Industries Ltd. Acetoni-

the following functional thermodynamic equation:

trile was chosen for organic modifier due to the poor durability ofln k = −∆ H °/RT + ∆ S °/R + ln Φcellulose− and amylose−columns for pressure. The water was(1)

deionized prior to usage.

where k is the retention factor, R the universal gas constant, T theabsolute temperature in Kelvin, ∆ H °and ∆ S °the molar enthalpy
and molar entropy of absorption, respectively, and Φ the phase

Instrumentation

volume ratio. Combing Eq. (1) with the expression for the enantio−

The HPLC system consisted of an L−7100 intelligent pump,separation factor (α = ks/kf, with s and f arbitrarily referring thean L−7400 UV−visible spectrophotometric detector, an L−7200

second and first eluted enantiomers, respectively) and the Gibbs−

auto sampler and D−7000 interface and the chromatographic data

Helmholtz relationship (Eq (2)):

were processed using HSM 7000 chromatography data station soft-− ∆∆ G °= RT ln αware (Hitachi, Tokyo, Japan). The columns used in this study were

(2)

CHIRALCEL OD−RH and CHIRALPAK AD−RH packed withgives Eq. (3):ln α = − ∆∆ H °/RT + ∆∆ S °/R

chemically modified cellulose and amylose as the stationary phase,(3)This expression relates the temperature and the experimentally eas-

respectively. These columns were purchased from Daicel Chemical

ily accessible α value with the molar differential enthalpy

Ltd. (Tokyo, Japan).

(∆∆ H °) and entropy (∆∆ S °) of enantio−selective adsorption.
Chromatographic conditions
The mobile phase consisted of 0.01 M acetate buffer (pH 4.7)
and HPLC−grade acetonitrile that were pre−mixed before use. The
mobile phase flow−rate was 0.5 mL/min. The column temperature
was 11−45℃. UV detection was performed at 297 nm. The sample
solutions were prepared in acetonitrile at a concentration of 0.1−
0.2 mg/mL. A 10−µL volume was injected. The retention factor
was determined as k = (tR−t0) /t0. The t0 was determined by injecting methanol, noting the time of first perturbation due to methanol.

Figure 2. Chemical structure of R −2−(4−bromo−2−fluorobenzyl)−(1, 2, 3, 4−tetrahydropyrrolo [1, 2−a] pyrazine−4−
spiro−3’−pyrrolidine)−1, 2’, 3, 5’−tetrone (R −enantiomer).

Results and discussion
The chromatograms of the enantio−separation of probe enanti-

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Chromatography, Vol.24 No.3 (2003)

Masato Kazusaki, Toshiya Shoda and Hirofumi Kawabata

(a)

(b)

Figure 3. Separation of R − and S −enantiomers on (a) CHIRALCEL OD−RH and (b) CHIRALPAK AD−RH. HPLC conditions: column, CHIRALCEL OD−RH and CHIRALPAK AD−RH; column temperature, 25℃ for CHIRALCEL OD−RH and 40℃
for CHIRALPAK AD−RH; mobile phase, acetonitrile−0.01 M acetate buffer (pH 4.7) (1:1); flow rate, 0.5 mL/min; detection, 297 nm.
omeric pairs on the CHIRALCEL OD−RH and CHIRALPAK AD

1.00−RH columns in the reversed−phase mode are shown in Figure 3,
0.80

respectively. Acetonitrile concentration in the mobile phase was altered to change the magnitude of interaction between the chiral sta-

0.60

tionary phase and the enantiomers. It was noted that the separation
factors observed on the CHIRALPAK AD−RH column are always

0.40

greater than those on the CHIRALCEL OD−RH column. Figure 4
shows plots of the separation factor versus inverse temperature on

0.20
3.10

the CHIRALCEL OD−RH and CHIRALPAK AD−RH column us-

3.20

3.30

3.40

3.50

3.60

1000/T (1000/K)

ing a mixture of acetonitrile and 0.01 M acetate buffer (1:1) as the

Figure 4. van’ Hoff plots of enantio−separation on CHIRALCELOD−RH (filled circle) and CHIRALPAK AD−RH (hollow circle). HPLC conditions: column, CHIRALCELOD−RH and CHIRALPAK AD−RH; column temperature, variable; mobile phase, acetonitrile−0.01 M acetate
buffer (pH 4.7) (1:1); flow rate, 0.5 mL/min; detection,
297 nm.

mobile phase. Linear and non−linear van’t Hoff plot were observedon the CHIRALCEL OD−RH column and CHIRALPAK AD−RH,
respectively. Non−linear plot could be divided into approximately
two linear regions. Slopes of all plots observed in this study are
positive, suggesting that the differences of enthalpy change(∆∆ H °) are negative. This fact indicates that enantio−separation is

Table 1. Thermodynamic quantities of the enantio−separation on CHIRALCEL OD−RH and CHIRALPAK AD−RH
ColumnVolume fraction ofacetonitrile (%)

Q [∆∆ H °/ T∆∆ S °]−∆∆ G °(kcal/mol)−∆∆ H °(kcal/mol)T∆∆ S °(kcal/mol)

CHIRALCEL
OD−RH

50
55
57
60
65
70

1.34
1.35
1.37
1.40
1.43
1.47

0.312
0.307
0.312
0.308
0.321
0.297

1.236
1.175
1.152
1.076
1.070
0.932

−0.924−0.868−0.840−0.768−0.749−0.635HIRALPAK
AD−RH

45 a
50 a
55 a
45 b
50 b
55 b

1.69
5.12
−0.16
1.53
1.55
1.66

0.443
0.473
0.493
0.452
0.462
0.458

1.087
0.588
0.068
1.301
1.298
1.155

−0.644−0.1150.425−0.849−0.836−0.697

Mobile phase consisted of acetonitrile and 0.01 M acetate buffer, pH 4.7. ∆∆ G °, ∆∆ H °and ∆∆ S °represent the difference of free energy,
enthalpy and entropy between enantiomers concerning transfer from the mobile phase to the stationary phase. ∆∆ G °and T∆∆ S °are evaluated at 25℃. Column temperature: a) below 20℃, b) above 20℃.

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Chromatography, Vol.24 No.3 (2003)

enthalpically favorable process on these columns. Using these data

which is proportional to the difference of enthalpy change and theobtained, thermodynamic properties were calculated to understandother independent of it. Inserting Eq. (4) in the differential form of

the mechanism for enantio−separation on both chiral columns.

the Gibbs−Helmholtz Eq (6), Eq (7) is obtained.
T ∆ (∆∆ S °) = β ∆ (∆∆ H °)The thermodynamic parameters for enantio−separation and(4)enthalpy−entropy ratios Q [Q = ∆∆ H °/ T ∆∆ S °] were calculated

T (∆∆ S °) = β (∆∆ H °) + T (∆∆ S0°)

(5)

and listed in Table 1. This arbitrary factor (Q ) represents the rela-∆ (∆∆ G °) = ∆ (∆∆ H °) − T ∆ (∆∆ S °)(6)∆ (∆∆ G °) = (1−β ) ∆ (∆∆ H °)(7)tive contributions of enthalpic vs. entropic effects to ∆∆ G °at 25℃[21]. As described above, the ∆∆ H °values obtained in this study

As can be seen from Eq. (7), only a proportion (1−β ) of theare negative. Therefore, positive Q values indicate that the favor-

increment in ∆∆ H °contributes toward the enantio−separation

able enthalpic contribution to enantio−separation was counterbal-(∆∆ G °). The intercept of the plot can be related to the difference

anced by unfavorable entropic contribution. The Q values obtainedof the degree of conformational change and the extent of desolva-

from CHIRALCEL OD−RH column, and CHIRALPAK AD−RH

tion induced upon enantio−separation. Eq. (5) indicates that as far

column at column temperature above 20℃ reveal that the enantio−

as the intercept (T∆∆ S0°) is positive, as is indeed the case in thisseparation under this condition is governed by enthalpy (Q ranges

study, enantio−separation can take place even in the absence of en-

between 1.34 and 1.47 on CHIRALCEL OD−RH, and 1.53 andthalpic gain (−∆∆ H °).

1.66 on CHIRALPAK AD−RH, respectively). However, on the

In our study, very large values of the slope close to unit (1.03

CHIRALPAK AD−RH column at column temperature below 20℃,

for amylose−deriven CHIRALPAK AD−RH column and 0.96 forentropy−dominated enantio−separation was observed. As is evident

cellulose−derived CHIRALCEL OD−RH column, respectively)from the data listed in Table 1, the ∆∆ G °value was kept relatively

were obtained. This fact indicates that by the modification of theconstant on each column, especially irrespective of two kind of

mobile phase composition, the enhanced enthalpic force for enantio

mechanism for enantio−separation on the CHIRALPAK AD−RH−separation is effectively cancelled out by the entropic force, re-column.

sulting in the constant enantio−separation on these columns.

The differences of the entropy changes (T ∆∆ S °) are plotted

As for the entropic contribution, larger T∆∆ S0°value of 0.49

against the differences of enthalpy changes (∆∆ H °) give straight

kcal/mol was calculated on an amylose−derived column, however,lines with correlation coefficients of 1.000 on both chiral columns,

that value for a cellulose−derived column was 0.27 kcal/mol. In-

as shown in Figure 5. As proposed previously [22], the empiricalserting Eq (5) in the Gibbs−Helmholtz Eq (8), Eq (9) is obtained.

linear relationship between ∆∆ H °and T ∆∆ S °means that the re-∆∆ G °= ∆∆ H °− T∆∆ S0°(8)

sulting change in T ∆∆ S °is proportional to the accompanying∆∆ G °= (1−β ) ∆∆ H °− T∆∆ S0°(9)change in ∆∆ H °, which leads to following Eq. (4). Integration of

In our study β is nearly equal to unit, indicating the difference

Eq. (4) gives Eq. (5), where β and T (∆∆ S0°) correspond to theof free energy is exclusively defined by the entropic term in thisslope and intercept of the plots shown in Figure 5. Eq. (5) indicatessystem. Owing to the difference of the entropic contributionthat the difference of entropy change consists of two terms one of

CHIRALCEL OD−RH column, which consists of the 3, 5−dimethyl−phenylcarbamate moiety bound to the cellulose backbone
instead of amylose, exhibited less chiral recognition ability than

Entropic quantity (kcal/mol)

0.5

CHIRALPAK AD−RH column (Table 1).
Chiral discrimination between the enantiomers is due to the0.0

differences in their steric fit in the chiral cavities [3, 4]. Many
authors discussed the importance of the higher order structure of-0.5

the polysaccharide−based CSPs in chiral recognition [23−26].
Based on the experimental data, the importance of steric fitness in-1.0

the chiral cavity is demonstrated by the comparison of entropic
-1.5
-1.5

contribution (T∆∆ S0°) on cellulose−derived CHIRALCEL OD−
-1.0

-0.5

RH column and amylose−derived CHIRALPAK AD−RH columns.

0.0

Enthalpic quantity (kcal/mol)

Dalgliesh established 3−point theory for chiral discrimination attributed by the differences of interactions between the chiral ab-

Figure 5. Enthalpy−entropy compensation plot of the enantio−
separation on cellulose−derived CHIRALCEL OD−RH(filled circle) and amylose−derived CHIRALPAK AD−
RH (hollow circle).

sorption sites and enantiomers [27]. This theory is valid well in thenormal−phase mode. We suggest that the participation of the entropic contribution for chiral discrimination (Table 1) and the pos-

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Chromatography, Vol.24 No.3 (2003)

Masato Kazusaki, Toshiya Shoda and Hirofumi Kawabata[13] Machida, Y.; Nishi, H.; Nakamura, K.; Nakai, H.; Sato, T. J.

sibility of enantio−separation without the differences of the molar
enthalpic change (∆∆ H °= 0) (Figure 5) in the reversed−phase

Chromatogr. A 1998, 805, 85−92.
[14] N. Morin, N.; Guiaume, Y. C.; Peirin, E.; Rouland J. −C. J.

mode.

Chromatogr. A 1998, 808, 51−60..
[15] Yashima, E.; Sahavattanapong, P.; Okamoto, Y. Chirality

References[1]

1996, 8, 446−451.

ICH, International Conference on Harmonization Guideline.

[16] Smith, R. J.; Taylor, D. R.; Wilkins, S. M. J. Chromatogr. A

Specifications: Test procedures and Acceptance Criteria for

1995, 697, 591−596.

Drug Substances and Drug Products: Chemical Substances.

[17] O’Brien, T.; Crocker, L.; Thompson, R.; Torma P. H.; Con-

http://www.nhi.go.jp/dig/ich/quality/q 6 a/q 6 a−e.pdf[2]

Kazusaki, M.; Kawabata, H.; Matsukura, H. J. Liq. Chrom.

[3]

Okamoto, Y; Yashima, E, Angew Chem. Int. Ed. 37 (1998)[4]

Okamoto, Y.; Kaida, Y. J. Chromatogr. A 1994, 666, 403−

lon D. A.; Feibush, B.; Moeder, C.; Bicker G.; Grinberg, N.
Anal. Chem. 1997, 69, 1999−2007..

& Rel. Tech. 2000, 23, 2819 − 2828.

[18] Kazusaki, M.; Shoda, T.; Kawabata, H.; Matsukura, H. J.
Liq. Chorom. & Rel. Technol. 2001, 24, 141−151.

1020.

[19] Kazusaki, M.; Kawabata, H.; Matsukura, H. J. Liq. Chrom.
& Rel. Technol. 2000, 23, 2937−2946.

419.
[5]

[20] Negoro, T; Murata, M.; Ueda, S.; Fujitani, B.; Ono, Y.;

I. W. Wainer, R. M. Stiffin, T. Shibata, J. Chromatogr. 411

Kuriyama, A.; Komiya, M.; Suzuki, K.; Matsumoto, J. J.

(1987) 65.
[6]

Schurig, V. J. Chromatogr. A 2001, 906, 275−299.

[7]Cabrena, K.; Lubda, D. J. Chromatogr. A 1994, 666, 433−

Med. Chem. 1998, 41, 4118−4129.
[21] Oberleitner, W. R.; Maier, N. M.; Lindner, W. J. Chromatogr. A 2002, 960, 97−108.

438.
[8]

[22] Kazusaki, M.; Shoda, T.; Kawabata, H.; Yamaguchi, T.

Kusters, E.; Loux, V.; Schmidt, E.; Floersheim, P. J. Chro-

Chromatography 2003, 24, 13−19.

matogr. A 1994, 666, 421−432.
[9]Kirkland, K. M.; McCombs, D. A. J. Chromatogr. A 1994,[23] Hesse, G.; Hagel, R. Chromatographia, 1973, 6, 277−280.

666, 211−219.

[24] Ichida, A.; Shibata, T.; Okamoto, I.; Yuki, Y.; Namikoshi,
H.; Toga, Y. Chromatographia 1984, 19, 280−284.

[10] Fornstedt, T.; Sajonz, P.; Guiochon, G. J. Am. Chem. Soc.

[25] Shibata, T.; Sei, T.; Nishimura, H.; Degichi, K. Chroma-

1997, 119, 1254−1264.

tographia 1987, 24, 552−554.

[11] Karlsson, A.; Charron, C. J. Chromatogr. A 1996, 732, 245[26] Francotte, E.;Zhang, T. J. Chromatogr. A 1995, 718, 257−

− 253.

266.

[12] Naemura, K; Fuji, J.; Ogashara K.; Hirose, K.; Tobe, Y.

[27] Dalgliesh, C. E. J. Chem. Soc. 1952, 3940−3942.

Chem. Commun. 1996, 2749−2750.

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