Original

Chromatography, Vol.28 No.3 (2007)

Original

Enantioseparation in the Vicinity of Compensation Temperature
Masato Kazusaki＊ and Yasutaka Ohgami
Analysis Laboratories, Group 1,Technology Research and Development Center Dainippon Sumitomo Pharma Co., Ltd.
Received for review August 23, 2007, Accepted November 19, 2007.

Abstract
Extra−thermodynamic study on enantioseparation was carried out with N −carbobenzyloxy−D,L−leucine on amylose tris (3,5−dimethylbenzoate)coated on silica as the chiral stationary phase and a mixture of acetonitrile and phosphate buffer as the mobile phase at different column
temperatures. Modification of mobile phase composition had a little impact on separation factor between enantiomers. The van’t Hoff plots ofretention factor and separation factor were linear over the temperature range studied. Enthalpy−entropy compensation for enantioseparation
was formally established by the methods proposed by Krug et al . Compensation temperature for enantioseparation was calculated to be 69.4°
C, indicating nearly−constant values of the difference in Gibbs free energy change over the usable column temperature range.
Keywords : amylose tris (3,5−dimethylbenzoate), Chiralpak AD−RH, compensation temperature, enthalpy−entropy compensation.

available all over the world [5]. However, the detailed mechanism

1. Introduction
A large percentage of commercial and investigational pharma-

for enantioseparation is yet to be resolved, because the intimate

ceutical compounds are enantiomers, and many of them show sig-

structure of the analyte binding site on the chiral stationary phasenificant enantioselective differences in their pharmacokinetics andhas not be elucidated at a molecular level.

pharmacodynamics. With increasing evidence of problems related

The effect of temperature is important for understanding chro-

to stereoselectivity in drug action, enantioselective analysis bymatographic behavior of analytes and characteristics of the station-

chromatographic methods has become the focus of intensive re-ary phase [6, 7]. This is especially true for enantiomeric separation.

search of separation scientists [1−4].

Variation of column temperature has an effect on retention andGreat efforts have been devoted to the development of better

enantioselectivity of chiral analytes on a chiral stationary phase.

methodology for enantioselective chromatography, and have re-With decreasing column temperature, enantioselectivity may either

sulted in new chiral stationary phases. Chiral stationary phases

increase or decrease depending on the type of chiral discrimination

composed with polysaccharide (e.g., cellulose and amylose) could

mechanism based on the chiral stationary phase [8−10]. Adjust-separate the enantiomers by the steric fit of the molecules in thement and control of temperature are therefore useful for optimizing

chiral cavity. Cellulose−derivative chiral stationary phases andchiral separation [11].

amylose−derivative ones could separate racemic compounds in a

By varying column temperature, thermodynamic studies werecomplementary style. These columns are also acknowledged tocarried out to understand mechanisms of chiral recognition [12−separate wide range of racemic compounds, and commercially16]. Peter et al . investigated temperature effects on retention of

Correspondence : Analysis Laboratories, Group 1, Technology Research and Development Center Dainippon Sumitomo Pharma Co., Ltd. 1−5−51 Ebie, Fukushima−ku, Osaka, 553−0001, Japan
Tel : +81−6−6454−8177
Fax：+81−6−6458−3723
E−mail : masato−kazusaki@ds−pharma.co.jp

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Chromatography, Vol.28 No.3 (2007)

enantiomers on a teicoplanin and a ristcetin chiral stationary phaseat a flow−rate of 0.5 mL min−1. The column temperature was ad-[17, 18]. Oberleitner et al . studied the temperature influences onjusted at 25.0−45.0°
C, and the precision of the column temperatureenantioseparation characteristics of N −acylated amino acids on aadjustment was ± 0.1°
C. The injection volume was set at 5 µL. Du-

quinine based chiral stationary phase [19]. Weng et al. evaluatedplicate analyses were carried out at each column temperature. The

thermodynamic characteristics of amino acid derivatives on a tar-elution of N −CBZ−D−Leu and N −CBZ−L−Leu was monitored attardiamide based chiral stationary phase [20].

220 nm. The chromatographic system was equilibrated by passing

The present work has carried out to determine the thermody-

the mobile phase until a stable baseline signal was obtained.

namic parameters for enantioseparation of derived D,L−leucine as a

2.3. Thermodynamic properties of liquid chromatography
The effect of temperature on the retention is related to themodel racemic compound, and to use this information to optimizechange of Gibbs free energy of the equilibrium and is expected by

the HPLC operating condition.

the van’t Hoff equation [21−24]. The retention and separation fac2. Experimental

tors, and thermodynamic parameters for the retention and separa-

2.1. Chemicals and reagentstion of two enantiomers on the amylose−derived chiral stationary

Acetonitrile of HPLC grade, potassium dihydrogen phosphate,phase is expressed by the following Eqs. (1)−(5), where the sub-potassium nitrate and uracil were obtained from Wako Purescripts L and D correspond to N −CBZ−L−Leu (first eluted enanti-

Chemical Industries Ltd. (Osaka, Japan). Water was deionizedomer) and N −CBZ−D−Leu (second eluted enantiomer), respec-prior to usage. Potassium dihydrogen phosphate was dissolved intively :deionized water, and phosphoric acid was added to make 20 mM∆∆G ＝∆GD−∆GL＝−RT ln α＝−RT ln kD/kL

phosphate buffer at pH 3.0.N −carbobenzyloxy−D,L−leucine (N −∆∆G ＝∆GD−∆GL＝(∆HD−T ∆SD)−(∆HL−T ∆SL)(1)

CBZ−D,L−Leu) and N −carbobenzyloxy−L−leucine (N −CBZ−L−＝∆∆H −T ∆∆S(2)Leu) from Tokyo Kasei Kogyo Co., Ltd. (Tokyo, Japan) were of

ln kD＝−∆HD/RT ＋∆SD/R +lnФ

(3)above 96.0% purity. Derived amino acids (Figure 1) were dis-ln kL＝−∆HL/RT ＋∆SL/R +lnФ

(4)solved in acetonitrile at 0.1 mg/mL for chiral HPLC analysis. The L

ln α＝∆∆H /RT ＋∆∆S /R(5)−enantiomer was employed for the identification of elution order ofwhere ∆G , ∆H and ∆S are the Gibbs free energy change, the mo-enantiomers. Uracil was used as a void volume marker. Acetoni-

lar enthalpy change, and molar entropy change, ∆∆G , ∆∆H andtrile was employed as organic modifier due to the poor durability∆∆S are the differences in the Gibbs free energy change, the molarof the chiral HPLC column for pressure.

enthalpy change, and molar entropy change, respectively. R is the

2.2. Apparatus

gas constant, and T represents the absolute temperature in Kelvin.

The chromatographic system used consisted of a D−7000 Sys-

The van’t Hoff plots of ln k versus 1/T (Eqs. (3) and (4)), ln α ver-tem (Hitachi, Tokyo, Japan) equipped with two isocratic pumps (Lsus 1/T (Eq.(5)) were used to evaluate the temperature influence on−7100), variable wavelength detector (L−7420), variable volume

retention and enantioseparation of amino acid derivatives on theinjector (L−7200) and column oven (L−7300) equipped with col-amylose−derived chiral stationary phase. These plots are linear if

umn cooler. The chiral HPLC column containing amylose−derived

enthalpy term and entropy term do not vary significantly within thechiral stationary phase (amylose tri−3,5−dimethylbenzoate) (4.6

temperature range studied.

mm in inside diameter and 15 cm in length with 10 µm in particle
diameter) was purchased from Daicel Chemical Industries, Ltd.

3. Results and discussion(Osaka, Japan). The mobile phase consisted of 20 mM phosphate

The mechanism underlying enantioseparation using derivat-

buffer at pH 3.0 and acetonitrile. Acetonitrile content was set at 25

ized polysaccharide phase is a difficult process to understand due

v/v %, 30 v/v %, 35 v/v %, 40 v/v % and 45 v/v %, and delivered

to the complexity of nature of the polymeric stationary phase. In aneffort to describe some of these processes, the separation of N −CBZ−D,L−Leu was studied on a tris (3,5−dimethylbenzoate) amylose phase (Chiralpak AD−RH). Enantioseparation of N −DBZ−D,L−Leu was carried out with this column. Representative chromatogram is shown in Figure 2. Enantioseparation was evaluated byindependently varying the acetonitrile volume fraction in the mobile phase from 25 v/v % to 45 v/v % and the column temperature(25.0−45.0°
C).

Figure 1. Chemical structures of N −carbobenzyloxy−D−leucine
and N −carbobenzyloxy−L−leucine.

Influence of acetonitrile volume fraction in the mobile phase.

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Chromatography, Vol.28 No.3 (2007)

Masato Kazusaki, Yasutaka Ohgami

Figure 2. Chromatogram of N −DBZ−D,L−Leu on the amylose−derived chiral stationary phase. Column, Chiralpak AD−RH ; mobile phase, a mixture of phosphate buffer andacetonitrile (60/40 v/v%) ; detection, UV 220 nm ; column temperature, 25°
C ; flow rate,0.5 mL min−1.

Figure 4. van’t Hoff plots for (a) retention and (b) separation factor of N −CBZ−D,L−Leu. Column, Chiralpak AD−RH ;mobile phase, a mixture of phosphate buffer and acetonitrile {acetonitrile content : 25 v/v% (small square), 30
v/v % (rhomboid), 35 v/v % (circle), 40 v/v % (triangle), 45 v/v % (large square)} ; detection, UV 220 nm ;column temperature, 25−45°
C ; flow rate, 0.5 mL min−1.Retention factors of N −CBZ−D−Leu and N −CBZ−L−
Leu are expressed in the open symbol and closed symbol, respectively.
Figure 3. As shown in Figure 3(a), increment of acetonitrile content in the mobile phase exponentially reduced retention factor at agiven temperature. Interestingly, separation factor was essentiallyunchanged over the entire range of acetonitrile content at a given
temperature (Figure 3(b)).
Figure 3. Influence of volume fraction of acetonitrile on (a) retention and (b) separation of N −CBZ−D−Leu and N −CBZ
−L−Leu. Column, Chiralpak AD−RH ; mobile phase, amixture of phosphate buffer and acetonitrile ; detection,
UV 220 nm ; column temperature, 25°
C ; flow rate,
0.5 mL min−1.

Influence of temperature. The characteristics of the retention and

To study the influence of volume fraction of organic modifier on

the five set of two lines generated from enantiomeric pairs intersect

retention factors and separation factors, acetonitrile volume frac-

at a certain temperature (isoelution temperature), respectively,

tion in the mobile phase was varied between 25 v/v % and 45 v/v

where the difference of free energy change is zero and the separa-% at the column temperature of 25.0°
C. Plots of retention factor

tion of enantiomers is not observed. It is obvious from Figure 4(a)and separation factor versus acetonitrile content are presented inthat the mass transfer from the mobile phase to the stationary phaseseparation factors with the variation of column temperature can be
estimated based on the plots of the natural logarithm of the retention factor of the two enantiomers or the separation factor against
the reciprocal of absolute temperature (known as van’t Hoff plot),
respectively. These plots are depicted in Figure 4. In Figure 4(a),

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Chromatography, Vol.28 No.3 (2007)−called “enthalpy−entropy compensation (EEC).” The slope in
Figure 5(a) is designated as compensation temperature for enantioseparation.
Enthalpy−entropy compensation. Apparent EEC concerning
enantioseparation was observed by HPLC method in this study.
However, Krug et al . criticized this procedure harshly, and proposed four methods. These methods were applied to find out
whether observed EEC was based on the substantial physico−chemical effects or results merely from a statistical compensation
due to experimental errors [27−29]. Details of these four elements
are described below.
(i) If EEC is actually present, the plot generated from enthalpy termand Gibbs free energy term at the harmonic mean of the experimental temperature (∆∆GThm) will be linear. As shown in Figure 5(b), ∆∆H and ∆∆GThm correlate well with correlation coefficient of
0.997, indicating the good linearity.
(ii) Krug et al . proposed the following equations to test the null hypothesis that Tc was equal to Thm. In this study, maximum andminimum values of Tc for enantioseparation at 95% confidence
level are calculated by the following equations.
Tc(minimum)＝Σ (∆∆H −∆∆Ĥ) (∆∆S −∆∆Ŝ) / Σ (∆∆S −∆∆Ŝ)2−t(3,0.05) V (T c)1/2
Figure 5. Linear correlation between (a) the differences of enthalpy change and entropy change and (b) the differenceof enthalpy change and that of free energy change atcompensation temperature obtained from the enantioseparation of N −CBZ−D,L−Leu.

Tc(maximum)＝Σ (∆∆H −∆∆Ĥ) (∆∆S −∆∆Ŝ) / Σ (∆∆S −∆∆Ŝ)2＋t(3,0.05) V (T c)1/2
where ∆∆Ĥ and ∆∆Ŝ are the average values of ∆∆H and ∆∆S , respectively. The t−value, t (3,0.05), is the value of the Student’s t−
test for 3 of freedom degree and a confidence level of 95%. Sample

is exothermic. It is also apparent that van’t Hoff plots of N −CBZ−

statistic, V (T c), is calculated by the following equation :D−Leu and those of N −CBZ−L−Leu gradually assemble with in-

V (T c)＝Σ (∆∆H −∆∆GTc − T c∆∆S )2 / 3 Σ (∆∆S −∆∆Ŝ)2

creasing temperature, respectively. Temperature at which someCompensation temperature (T c) was calculated 69.4°
C (343van’t Hoff plots converge is designated as compensation tempera-K), and the 95% confidence interval was 58.7−80.0°
C. Compensa-

ture. In many cases, the compensation temperature is typically welltion temperature was explicitly discriminated from the harmonic

above the temperature region normally used for liquid chromatog-

mean of the experimental temperature (34.8°
C). Very low tempera-

raphy [25, 26] and consequently the retention factors of most ana-ture for compensation was obtained for enantioseparation in the re-lytes increase as the column temperature decrease.

versed−phase mode. Miyabe et al . reported the compensation temperature of 710−1560 K with structurally−related compounds in

In a previous study, it was observed that the van’t Hoff plots
generated on the amylose−derived chiral stationary phase was non

the reversed−phase mode [21, 22, 26].

−linear with break below 20°
C. This phenomenon was concluded(iii) When enthalpy−entropy compensation is observed for a groupthat the stationary phase was underwent the conformational change

eluted with different mobile phases, all of the groups have the same[8]. In contrast, linear van’t Hoff plots of k andαwere obtained indegree of the difference in Gibbs free energy change at compensa-

this study. This result indicates that the conformational change oftion temperature. When separation is influenced by EEC, the van’t

the stationary phase was not occurred, and the mechanisms for re-

Hoff plot must intersect at a single point. As shown in Figure 4(b),tention and separation were not changed within the temperature

van’t Hoff plots obtained in this study pass through a certain point.

range studied (25−40°
C).

(iv) Probability for the intersection of the linear regression lines
was compared with that for the non−intersection on the basis of the

Figure 5(a) shows the correlation between thermodynamic parameters (∆∆H and ∆∆S ) calculated from the van’t Hoff plots instatistical data derived by ANOVA. The probability for non−inter-

Figure 4(b). It also appears that the difference of enthalpy change

section was also compared with the precision of the experimental

correlates well with that of entropy change. This phenomenon is sodata in the same manner. The value of the mean sum of squares for

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Chromatography, Vol.28 No.3 (2007)

Masato Kazusaki, Yasutaka Ohgami

intersection (MSi) divided by that of squares for non−intersection(MSni) is 3.5×104 is sufficiently larger than the corresponding F −value of 10.1. This show the probability for intersection is highcompared to that for non−intersection. On the other hand, the ratioof MSni to that of squares for the residuals (MSε) is −3.4×10−4. The
negative value of MSε is probably unreasonable. However, it seems
to arise from calculation errors, which suggests that the variation
due to measurement errors is quite small. The corresponding F −value is 3.5. In conclusion, the variation due to the non−intersection is not greater than that due to the measurement errors.
On the basis of the results described above, we can state thatthe true EEC takes place for enantioseparation of N −CBZ−D,L−
Figure 6. Model van’t Hoff plots between ln α and 1/T for enantioseparation of enantiomers on a chiral stationary
phase. T c represents compensation temperature.

Leu on the amylose−derived chiral stationary phase, originatingfrom substantial physico−chemical effects at the 95% level of significance. The mechanism for enantioseparation is essentially identical under the HPLC operating condition in this study.
EEC for retention is conveniently expressed by the following
relationship [30] :∆H ＝T c ∆S ＋∆GTc(6)

where ∆GTc denotes the Gibbs free energy change of a physico−chemical interaction at compensation temperature (T c). EEC for
separation is also expressed by the following relationship :∆∆H ＝T c ∆∆S ＋∆∆GTc(7)

Eq. (7) can be rewritten in order to express the free energy change,∆GT, at a fixed temperature, T , as∆∆GT＝∆∆H (1−T / T c)＋∆∆GTc T / T c(8)
Figure 7. Differences in free energy change, enthalpy change andentropy change for chiral separation of derived aminoacids on amylose tris (3,5−dimethylbenzoate) stationary
phase at 25°
C using the mobile phase of phosphatebuffer − acetonitrile. The numbers on the abscissa indicate acetonitrile content in the mobile phase. Filled, unfilled and shaded parts indicate ∆∆G , ∆∆H , and ∆∆Stimes temperature, respectively.

Eqs. (1) and (8) give the following equation :ln αT＝−∆∆H/R (1/T −1/T c)−∆∆GTc/ R T c(9)This equation implies that in the vicinity of T c, the natural logarithm of separation factor is practically independent of temperature.
Let us suppose that EEC for enantioseparation takes place under the several HPLC operating conditions with different mobile
phases. In this instance, enantiomers are separated by the identicalmechanism. Model van’t Hoff plots of the separation factors of thetwo enantiomers on the chiral stationary phase are shown in Figuretion of N −CBZ−D,L−Leu on the amylose−derived chiral stationary

6. Needless to say, the slope of each line is related to the difference

phase is enthalpically governed process. According to Eq. (8), allof the enthalpy change (−∆∆H/R ) and the intercept is related tovalues of −∆∆G approach zero with increase of experimental tem-the difference of the entropy change (∆∆S/R ). Figure 6 indicates unperature up to compensation temperature. Figure 7 shows that with−effectiveness for improving the separation of enantiomers nearan increase of acetonitrile content in mobile phase, −∆∆H value

compensation temperature (illustrated as open symbols). As shown

gradually increases. However, according to EEC, the increment ofin Figure 3(b), separation factor is slightly increased at 25.0°
C with−∆∆H value is almost cancelled out by the increase of the value

increase of acetonitrile content in mobile phase. This experimentalof −∆∆S times temperature, resulting in the slight increment of −temperature is close to compensation temperature (69.4°
C). On the∆∆G value. This trend was observed at all experimental tempera-

other hand, it will be probably easy to achieve the better separationtures studied, and as a matter of course a small amount of incre-

in the temperature range far from compensation temperature (illus-

ment of −∆∆G value steadily observed with decreasing those tem-

trated as closed symbols).

peratures with the same mobile phase composition.

The enthalpic and entropic contribution for enanrtioseparation

The result of this study with the amylase−derived chiral sta-

at 25°C was depicted in Figure 7. Enantioselectivity for the resolu-

tionary phase indicates that separation factor of enantiomers could

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Chromatography, Vol.28 No.3 (2007)[15] Buckenmainer, S. M. C. ; McCalley, D. V. ; Euerby, M. R.

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