Original

Chromatography, Vol.24 No.2 (2003)

Original

Thermodynamic Properties of Enantioseparation of Two
N −Carbobenzyloxy−D, L−Amino Acids on Cellulose
Tris (4−methylbenzoate) Chiral Stationary Phase
Masato Kazusaki*, Toshiya Shoda, Hirofumi Kawabata and Toshikazu Yamaguchi
Department of Chemical Analysis, Pharmaceutical Research Laboratories, Dainippon Pharmaceutical Co., Ltd.
1−5−51 Ebie, Fukushima−ku, Osaka, 553−0001, Japan
Received January 22, 2003 Revised manuscript received March 6, 2003 Accepted March 7, 2003

Abstract
The isocratic retention of enantiomers of N −carbobenzyloxy−D, L−tryptophan and N −carbobenzyloxy−D, L−phenylalanine was studied on cellulose tris (4−methylbenzoate) coated on silica as a chiral stationary phase at different temperatures and with different mobile phase compositions in the reversed−phase mode. The thermodynamic parameters were calculated in order to promote an understanding of the relationshipbetween enthalpy and entropy for enantioseparation in this chromatographic system. The separation was enthalpy−controlled with negative
entropic contribution in the HPLC condition studied. The enthalpy−entropy relationship indicates that enthalpic gain/loss for enantioseparation from any change of organic modifier content in the mobile phase is almost cancelled out by the entropic loss/gain.
Keywords: cellulose tris (4−methylbanzoate) chiral stationary phase, enantioseparation, enthalpy−entropy compensation

1. Introduction
The separation and analysis of biologically active chiral compounds continue to be an area of increasing interest in pharmaceutical industry since biological activities may vary dramatically between enantiomers. Through the advances of industrial technologies, direct chiral high−performance liquid chromatographic separation is now well established with many commercially available
columns containing chiral stationary phases (CSPs). In the past few
years, cellulose−based derivatives have been developed and shown
to be exceptionally useful for the separation of enantiomers of biological and pharmaceutical importance. Cellulose tris (4−methylbenzoate) is one of the most recently introduced CSPs (Figure 1).
Although there is now a considerable amount of information
regarding interaction between solutes and cellulose−based chiral
Figure 1. Chemical structure of cellulose tris (4−methylbenzoate).

stationary phases, [1−3] little is known from a view of thermodynamics, especially in the reversed−phase mode. The degree ofchiral recognition of enantiomers by a CSP depends strongly on thetures allow an evaluation of the variations in enthalpy and entropy,temperature. The separation factor usually decreases as the tem-

which govern the free energy of transfer of the analyte from theperature increases. Retention data obtained at different tempera-

mobile phase to the stationary phase. These thermodynamic pa-

TEL: 06−6454−8177, FAX: 06−6458−3723*E−mail: masato−kazusaki@dainippon−pharm.co.jp―６３―

Chromatography, Vol.24 No.2 (2003)

rameters were determined in order to better understand the thermo-

2. Experimental

dynamic driving forces affecting retention and separation of enanti-

2.1. Chemicals and reagentsomers. These effects on retention in reversed−phase high−perform-

Acetonitrile and methanol of HPLC grade, phosphoric acidance liquid chromatography have been considered for different

and sodium dihydrogen phosphate were obtained from Wako Pure

CSPs, including those based on chiral crown ethers, [4, 5] cy-

Chemical Industries Ltd. (Osaka, Japan). Water was deionized

clodextrins, [6, 7]α1−acid glycoprotein, [8] human serum albumin,prior to usage. Sodium dihydrogen phosphate was dissolved in[9] derived aromatic ring, [10−12] derived amylose [13, 14] anddeionized water and phosphoric acid was added to make a 0.02−M

cellulose, [14−19] and chiral macro cyclic antibiotics [20] by many

phosphate buffer at pH 2.0. The amino acid derivatives investi-groups.

gated were N −carbobenzyloxy−D, L−phenylalanine (CBZ−D, L−Our recent studies, from the thermodynamic point of view, re-Phe), N −carbobenzyloxy−L−phenylalanine (CBZ−L−Phe), N −car-vealed the conformation change of the CSP consisted of amylose

bobenzyloxy−D, L−tryptophan (CBZ−D, L−Trp) and N −carboben-tris (3, 5−dimethylphenylcarbamate) over the temperature range

zyloxy−L−tryptophan (CBZ−L−Trp) (Figure 2) from Tokyo Kasei

studied, [13] and unusual behavior of the enthalpy and entropy of

Kogyo Co., Ltd. (Tokyo, Japan). Derived amino acids were dis-transfer of the solute from the mobile to the CSP of cellulose tris

solved in acetonitrile at 0.01 mg/mL for chiral HPLC analysis. The(3, 5−dimethylphenylcarbamate) as a function of organic modifierL−enantiomers were employed for the identification of elution or-content in the mobile phase, [18] respectively. The aim of the pre-der of enantiomers.

sent report is to provide the information concerning the relation between enthalpy and entropy on the mechanisms involved in the2.2. Apparatus

separation of derived D, L−amino acids with cellulose tris (4−meth-HPLC measurements were performed on a Hitachi (Tokyo,ylbenzoate) coated on silica as a chiral stationary phase in the re-Japan) D−7000 System equipped with two isocratic pumps (L−versed−phase mode.

7100), variable wavelength detector (L−7420), variable volume injector (L−7200) and column oven (L−7300). CHIRALCEL OJ−RH(150 mm × 4.6 mm I.D., Daicel Chemical Ltd., Tokyo, Japan),kindly provided from Fuji Kagaku Yakuhin (Hyogo, Japan), wasemployed for analytical separation. The packing material of thecolumn was a silica−gel support (5 µm) coated with a polymer ofcellulose tris(4−methylbenzoate). The mobile phase consisted ofacetonitrile − 0.02 M phosphate buffer at pH 2.0 (33:67−41:59, v/v). The column temperature was adjusted at 20−45℃ at a flow−rate of 1 mL/min, and the injection volume was set at 10 µL. Duplicate injections were performed at each column temperature andmobile phase composition. The elution of amino acid derivatives
was monitored at 220 nm. The chromatographic system was equilibrated by passing the eluent until a stable baseline signal was obtained. This procedure was always followed when a new mobilephase or temperature was applied for analysis. The column void
time was determined from the first perturbation of baseline causedby methanol injection.
3. Results and discussion
Enantiomers of CBZ−D, L−Trp and CBZ−D, L−Phe were chromatographed on CHIRALCEL OJ−RH in the reversed−phasemode. Acetnitrile was used as organic modifier due to the poor durability of CHIRALCEL OJ−RH for pressure. Typicl chroma-Figure 2. Chemical structures of derived amino acids:
N −carbobenzyloxy−D−tryptophan (CBZ−D−Trp),
N −carbobenzyloxy−L−tryptophan (CBZ−L−Trp),
N −carbobenzyloxy−D−phenylalanine (CBZ−D−Phe),N −carbobenzyloxy−L−phenylalanine (CBZ−L−Phe).

tograms are shown in Figure 3. As regards the elution order ofenantiomers, the D−enantiomers always eluted before the L−enantiomers. In some cases the organic modifier in the mobile phase
plays an important role in the stereo−selectivity by binding to―６４―

Chromatography, Vol.24 No.2 (2003)

Masato Kazusaki, Toshiya Shoda, Hirofumi Kawabata and Toshikazu Yamaguchi

Figure 4. Temperature dependence of retention and enantiosepareation of derived amino acids on a CSP. Column,CHIRALCEL OJ−RH; mobile phase, acetonitrile −phosphate buffer (40:60); detection, 220 nm; flow−
rate, 1 mL/min. a) van’t Hoff plots for retention：○，CBZ−L−Trp；●，CBZ−D −Trp；□，CBZ−L −Phe；■，CBZ−D−Phe. b) van’t Hoff plot for Enantioseparation：○，CBZ−D, L−Trp；□，CBZ−D, L −Phe.

Figure 3. Chromatograms of amino acid derivatives. a) : CBZ−D,L−Trp, b) : CBZ−D, L−Phe. Column, CHIRALCEL OJ−
RH; mobile phase, acetonitrile−phosphate buffer (40:60,v/v); column temperature, 45℃; detection, 220 nm;
flow−rate, 1 mL/min.
achiral sites near or at the chiral cavities of CSP, which alters thesteric environment of these cavities. [17] However, the chiral separation on this CSP was not affected by varying acetonitrile content
in the range tested.

where k L and k D are the capacity factors of L− and D−enantiomers.

In order to calculate thermodynamic parameters for more un-∆∆ H°and ∆∆ S°represent the differences of the enthalpy and en-derstanding of the separation mechanism of enantiomers on thistropy changes of the enantiomers, respectively. ln α versus 1/T

CSP, van’t Hoff plots were constructed. The chromatographic re-(van’ Hoff plot of α) should also be linear if ∆∆ H°and ∆∆ S°are

tention, expressed by k, was expressed in terms of the following

invariant with temperature in the range tested. Linear and non−lin-van’t Hoff equation:

ear van’t Hoff plots were reported in the temperature studies in theln k = −∆ H°/RT + ∆ S°
/R + lnφ

reversed−phase mode. [13, 18, 21−24] Non−linear van’t Hoff plots(1)

In this equation, ∆ H°and ∆ S°are enthalpy and entropy changes of

may be indicative of a change in the mechanism of retention.

the transfer from the mobile to the stationary phase. R is the gas

Figure 4 illustrates the representative linear van’t Hoff plots ofconstant, and T is the absolute temperature of the chromatographic

k and α with correlation coefficients both of 0.999 for derived

system. φ means the phase ratio of the column (the volume of theamino acids employing the mobile phase, acetonitrile−phosphatestationary phase divided by the volume of the mobile phase). Thisbuffer of 40:60 (v/v), respectively. These graphs indicate that theexpression shows that the plot of ln k versus 1/T should be linear if

mechanisms for retention and separation of phenylalanine− and∆ H°, ∆ S°and φ are invariant with temperature in the range

tryptophan−derivatives on the CSP were not changed in the tem-tested. Furthermore,perature range studied.
The enthalpic and entropic contributions to the chiral separa-lnα = ln (k L / k D)= −∆∆ H°
/RT + ∆∆ S°/Rtion of D− and L−enantiomers of derived amino acids are visualized(2)―６５―Chromatography, Vol.24 No.2 (2003)

a)Thermodynamic quantity(cal/mol)

500
400
300
200
1000-100333536384033353638

40

41-200-300-400-500b)
Thermodynamic quantity(cal/mol)

500
400
300
200100Figure 6. Enthalpy−entropy compensation plots for chiral recognition of enantiomers of CBZ−D, L−Trp and CBZ−D, L−
Phe．○，CBZ−D, L−Trp；□，CBZ−D, L−Phe.

0-10041-200-300-400-500

for enantio−separation. Integration of eqution 3 gives equation 4,Figure 5. Differences in free energy, enthalpy and entropy forchiral separation of derived amino acids on cellulose
tris(4−methylbenzoate) column at 25℃ using the mobile phase of acetonitrile − phosphate buffer. The numbers on the abscissa indicate acetonitrile content in themobile phase. Filled, unfilled and shaded parts indicate−∆∆ G°, −∆∆ H°and T ∆∆ S°, respectively. a) CBZ−D,L−Phe; b) CBZ−D, L−Trp.

where T ∆∆ S0 refers to the value at ∆∆ H°= 0 (intercept) of Figure6.
T ∆∆ S°= β ∆∆ H°+ T ∆∆ S0(4)Enantiomers are solvated identically in the mobile phase, butwould release a different number of solvent molecules when they
associate with the CSP. Therefore, this contribution to entropy
would not be identical for both enantiomers. Since ∆∆ S°values arenegative, L−enantiomers have fewer degrees of freedom on thein Figure 5. On the basis of thermodynamic data, Figure 5 clearlyCSP. In other words, L−enantiomers are held at more points or areshows that the chiral separation of CBZ−D, L−Trp and CBZ−D, L−

less able to move or rotate in the CSP. Figure 6 indicates that as far

Phe on cellulose tris (4−methylbenzoate) column are exclusively

as the intercept of the ordinate is positive, that is indeed the case of

dominated by enthalpy with negative entropic contribution. With

CBZ−D, L−Phe and CBZ−D, L−Trp with this CSP under the HPLC

an increase of acetonitrile content in the mobile phase, the −∆∆ H°

condition studied, the enantioseparation can take place even in thevalues increased, reached maximum values at about 38% of aceto-absence of enthalpic gain (−∆∆ H°). This result suggests that thenitrile, and decreased. This trend was cancelled out by the T ∆∆ S°

enantioseparation without enthalpic contribution wouldvalues, resulting in the constant −∆∆ G°values. This is the reason

achieved by the difference in desolvation from each enantiomer ad-for constant separation factors for respective racemates.

sorbed through achiral contribution in the chiral cavity.

beDifferential form of Gibbs free energy function for interaction

Using these data sets for chiral separation, the differences ofentropy are plotted against the differences of enthalpy to generateof an enantiomeric pair with the CSP is the following:straight lines with correlation coefficients of more than 0.999, as∆ (∆∆ G°) = ∆ (∆∆ H°) − T ∆ (∆∆ S°)

shown in Figure 6. As the magnitude of the enthalpy for enantio−(5)

Inserting equation 3 in equation 5, equation 6 is obtained.

∆ (∆∆ G°) = (1−β) ∆ (∆∆ H°)

separation decreases, an offsetting increase in the entropy occurs. A(6)relationship of this type is referred to as enthalpy − entropy com-

Equation 6 indicates that only (1−β) proportion of the incre-pensation. [25, 26] For these straight lines the resulting change inment in enthalpy (−∆∆ H°) contributes toward raising the enantio−

entropic term is proportional to the accompanying change in en-separation (−∆∆ G°).

thalpy as described in the following equation 3:T ∆ (∆∆ S°) = β ∆ (∆∆ H°)

As can be seen from Figure 6, both linear plots give largeslopes (β: 0.98 for CBZ−D, L−Trp, 1.03 for CBZ−D, L−Phe). These(3)whereβ means the slope of this straight line. The slope (β) is aslopes (approximately 1) indicate that the enthalpic gain or loss

quantitative measure of the entropic canceling of the enthalpic gain

arising from any change caused by mobile phase composition is―６６―

Chromatography, Vol.24 No.2 (2003)

Masato Kazusaki, Toshiya Shoda, Hirofumi Kawabata and Toshikazu Yamaguchi

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