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J. Biochem. Biophys.

Methods 43 (2000) 59–76


www.elsevier.com / locate / jbbm

Separation of drugs by packed-column supercritical


fluid chromatography
a, b
Koji Yaku *, Fujio Morishita
a
Analytical Chemistry Department, Product & Technology Development Laboratory, Tanabe Seiyaku Co.,
Ltd., 16 -89, Kashima 3 -chome, Yodogawa-ku, Osaka 532 -8505, Japan
b
Department of Material Chemistry, Graduate School of Engineering, Kyoto University, Yoshida, Sakyo-ku,
Kyoto 606 -8501, Japan

Abstract

Packed-column supercritical fluid chromatography (pSFC) has been expected to analyze various
kinds of compounds. Many researchers have expected a new chromatographic technique that
overcomes the limitations of other techniques, HPLC and GC. In pharmaceutical development,
chromatography plays an important role in the evaluation of safety and efficacy of a new
compound. This article provides an overview of the separation of drugs by pSFC. The effects of
the chromatographic parameters were studied for the separation of steroids. In chiral separation,
the successful results were shown and compared with HPLC.  2000 Elsevier Science B.V. All
rights reserved.

Keywords: Diltiazem hydrochloride; Steroid: enantiomer separation; Pharmaceutical analysis; Thermody-


namics; SFC

1. Introduction

The applicability of supercritical fluid is still expanding in the fields of not only
chromatography but also extraction [1], chemical reaction [2] and environment [3,4].
The advantages of supercritical fluid chromatography (SFC) have recently been
recognized [5], especially packed-column supercritical fluid chromatography (pSFC),
although the chromatography with a supercritical fluid as mobile phase was suggested
more than 40 years ago. High-performance liquid chromatography (HPLC) is also
recognized as the conventional separation method for analyses of various kind of

*Corresponding author. Tel.: 181-6-6300-2632; fax: 181-6-6300-2629.


E-mail address: k-yaku@tanabe.co.jp (K. Yaku)

0165-022X / 00 / $ – see front matter  2000 Elsevier Science B.V. All rights reserved.
PII: S0165-022X( 00 )00086-5
60 K. Yaku, F. Morishita / J. Biochem. Biophys. Methods 43 (2000) 59 – 76

compounds, but long analysis times are often necessary and low column efficiency due
to the low solute diffusion in the mobile phase sometimes frustrates chromatographers.
The regions corresponding to the solid, liquid and gaseous states are clear. Above the
critical temperature, carbon dioxide does not condense any longer with increasing the
pressure, and the fluid in this state is called a supercritical fluid. We are unable to draw a
clear line between supercritical fluid and liquid or gaseous phases, as there is a
continuous transition from liquid to supercritical fluid by increasing the temperature at
constant pressure or from gas to supercritical fluid by increasing the pressure at constant
temperature. The supercritical fluid mobile phases have much greater solubilizing power
than gaseous ones and can, therefore, be used for the separation of involatile and
high-molecular-mass samples unsuited to gas chromatography (GC). The solubility or
the retention in SFC is easily controlled by changing a couple of parameters, the
temperature and the pressure. The moderate critical conditions of carbon dioxide, 318C
and 73 atm, used as the mobile phase is favorable for the analysis of the thermally
unstable compounds (1 atm 5 101 325 Pa). Although the typical solute diffusion
coefficient in supercritical fluids is intermediate between those of gas and liquid, it is
noteworthy that the diffusion coefficient is an order of magnitude greater than in liquid.
This fact has important chromatographic implications concerning separation time and
column efficiency. The typical minimum values for height equivalent to a theoretical
plate for packed-column SFC and HPLC are very similar, the most important difference,
however, is that the minimum value in SFC is achieved at linear velocities three- to
five-times greater than for HPLC [6].
Taking into account the characteristics of properties in the fluid, it can be expected
that pSFC will become a suitable technique for the separation of a chemical compound
as not only the complementary but also the alternative method. pSFC, which can use
binary or ternary mobile phases, seems to be applicable to a wide range polar
compounds [7–16]. However, there are a few problems that should be conquered for the
study on the analysis of polar compounds by pSFC. First, only a few instruments
applicable to pSFC are available commercially. Second, the guidelines for the choice of
a suitable organic modifier should be considered, since the polarity of pure carbon
dioxide fluid is poor for the elution of polar solutes. Furthermore, the features of
separation behavior in the compressible mobile phase fluid have hardly been reported in
detail. It is important to study the retention behavior of the compounds in the
compressible fluid for expanding the application of pSFC to the analysis of polar
compounds, such as drug substances.
The authors had tried to construct the pSFC instrument by modification of the HPLC
apparatus and to validate the performances. Then, the retention behavior of the polar
corticosteroids and the chiral resolution of the optical isomers in pSFC were studied in
detail. In this article, our work concerning the separations of drugs by pSFC are
reviewed briefly.

2. Instrumentation of pSFC

The constructed pSFC system is shown in Fig. 1. A HPLC system (Shimadzu, Kyoto,
Japan) was modified for SFC operation. The liquid carbon dioxide, of more than 99.9%
K. Yaku, F. Morishita / J. Biochem. Biophys. Methods 43 (2000) 59 – 76 61

Fig. 1. Schematic diagram of pSFC. 1: Carbon dioxide, 2: modifier, 3: cooling-bath, 4: LC-6A pump, 5:
LC-9A pump, 6: dynamic mixer, 7: injector, 8: oven, 9: packed column, 10 and 11: pressure monitor, 12:
detector, 13: back-pressure regulator, 14: dry-thermo unit.

purity is suitable for an analysis, is passed from the cylinder with a dip tube into the
LC-6A through the cooling-bath. To facilitate introduction of the mobile phase as a
liquid, carbon dioxide from the cylinder should be cooled by passing it, with a dip tube,
through a 1.6-mm diameter stainless steel tube in a coolant, kept at 2 108C. The pump
head of the LC-6A is also cooled to 2 108C to improve pump efficiency by circulating
the coolant. The polar modifier is fed by the LC-9A, and is mixed with the liquid carbon
dioxide in a dynamic mixer. The modifier can be mixed volumetrically with the carbon
dioxide by control of the pumping rate. This pSFC system can also be operated in
gradient elution mode by programming the flow-rate of the modifier. The sample
solution is introduced onto the column via the injector (Rheodyne, Cotati, CA, USA)
fitted with a 5-ml sample loop. Since the injection of a sample volume larger than 5 ml
(10 or 20 ml) produces a broader and / or split peak shape due to the difference of
property between the carbon dioxide fluid and the sample solvent, the injector fitted with
the 5-ml sample loop should be used. The column should be kept in the CTO-6A oven at
constant temperature. The manual back-pressure regulator (Tescom Instruments, Elk
River, MI, USA) is connected in-line after the detector and kept at ca. 408C with a
heating unit to prevent clogging with solid carbon dioxide. The mobile phase is always
fed in a constant-flow delivery mode in this system. As the pressure is controlled by the
back-pressure regulator, the inlet flow-rate can be changed independently of the
pressure. The pressure at inlet and outlet of the column is monitored using pressure
gauges between the dynamic mixer and the injector, and between the detector and the
regulator, respectively. All results are recorded with a Chromatopac C-R5A integrator.

3. Separations of drugs

pSFC has been expected to be a powerful separation technique because of the


62 K. Yaku, F. Morishita / J. Biochem. Biophys. Methods 43 (2000) 59 – 76

advantage in the mobile phase, showing a lower viscosity and higher diffusion
coefficient. To study the characteristics of the retention and separation behavior on the
analysis of drugs in pSFC, first, seven corticosteroids possessing one to four hydroxyl
groups were selected. Second, the optical isomers of diltiazem hydrochloride, a Ca-
channel blocker, were separated on a column of modified cellulose coated onto silica.
Finally, the separation of the optical isomers was studied from a thermodynamic
viewpoint.

3.1. Steroids

The structures of the corticosteroids selected are shown in Fig. 2. The solutes were
separated on stationary phases of different polarity. The following columns were used:
Cosmosil 5NH 2 (150 3 4.6 mm I.D., Nacalai Tesque, Kyoto, Japan) modified with
aminopropyl, Ultoron VX-SIL (150 3 4.6 mm I.D., Shimadzu, Osaka, Japan), and
Zorbax phenyl (250 3 4.6 mm I.D., Shimadzu). The particle size was 5 mm. As shown in
Fig. 3, the aminopropyl column exhibited the best selectivity and peak shape with a
reasonable retention time in comparison with the others. Silica and phenyl columns
showed poor separation, especially the latter could not resolve under the operating
conditions used. A reversed elution order of compounds 5 and 6, however, was observed
between the aminopropyl and silica columns, which showed that the change of the
selectivity of retention was possible by selection of the stationary phase.
No corticosteroids were eluted from the column packed with aminopropyl support
with pure carbon dioxide as the mobile phase because of the low polarity. The effect of
modifiers with different polarities on the retention was examined. As shown in Fig. 4,
the addition of 11.8% (v / v) methanol to carbon dioxide showed the best resolution and
symmetric peak shapes within 14 min. The addition of a modifier to a mobile phase must
be considered from the viewpoint of its effect either on the stationary phase or on the
mobile phase. Berger and Deye [17] studied the effect of column and mobile phase

Fig. 2. Structures and symbols of synthetic corticosteroids.


K. Yaku, F. Morishita / J. Biochem. Biophys. Methods 43 (2000) 59 – 76 63

Fig. 3. Effect of column. (A) Cosmosil 5NH 2 , (B) Ultron VX-SIL, (C) Zorbax phenyl. Operating conditions:
mean pressure 213 kg cm 22 , flow-rate of CO 2 3 ml min 21 , flow-rate of methanol 0.4 ml min 21 , temperature
408C. Peaks as in Fig. 2.

polarity using steroids. Blilie and Greibrokk [18] mentioned that the modifiers
functioned as deactivation agents by direct interactions with residual silanol groups and
also as modifiers of the eluting power of the mobile phase. Janssen et al. [19] confirmed
that the effect of a few percent of modifier in pSFC was largely the deactivation of
residual silanol groups on the silica support, and the accessibility to the active site was
found to depend strongly on the size and structure of the modifier molecules. It was
suggested that the effect of the modifier on retention of the corticosteroids consisted in
the enhancement of the solvent strength of the mobile phase rather than the deactivation
of the active sites on the stationary phase.
The effect of pressure on the retention of seven corticosteroids was studied in the
range from 107 to 223 kg cm 22 . The theoretical plate numbers (N) of solutes were
calculated at each pressure. N values reached a maximum at 126 and 144 kg cm 22 as
shown in Fig. 5. Since the mass flow-rate was kept constant, the linear velocity varied
with pressure. The minimum plate height was obtained in this pressure range. These
results reveal that the pressure is one of the significant parameters optimizing the
operating conditions.
A modifier gradient should be effective to separate a wide range of polar cortico-
steroids. The authors attempted to analyze seven corticosteroids in modifier gradient
elution mode. All solutes were eluted within 6.5 min by increasing the methanol content
from 11.8% (v / v) to 17.0% (v / v) at 0.52% (v / v) per min, and keeping the CO 2 flow-rate
constant (Fig. 6). Good peak shapes, complete baseline separation, were obtained. The
stable baseline without drift and noise is considered dependent on the good mixing
process of the binary fluid.
The retention and the separation of the corticosteroids in pSFC using the Cosmosil
64 K. Yaku, F. Morishita / J. Biochem. Biophys. Methods 43 (2000) 59 – 76

Fig. 4. Effect of modifier. (A) Methanol, (B) ethanol (95%), (C) ethanol (99.5%), (D) 1-propanol, (E)
2-propanol. Operating conditions: column Cosmosil 5NH 2 , inlet pressure 224 kg cm 22 , outlet pressure 191 kg
cm 22 , flow-rate of CO 2 3 ml min 21 , flow-rate of modifier 0.4 ml min 21 , temperature 408C. Peaks as in Fig. 2.

5NH 2 column were compared with those in normal- (NP) and a reversed-phase (RP)
HPLC. Under HPLC conditions, the following columns were used: Ultoron VX-SIL
(150 3 4.6 mm I.D., Shimadzu) and Inertsil ODS-2 (150 3 4.6 mm I.D., GL Science,
Osaka, Japan). The particle size was 5 mm. As shown in Fig. 7, the elution order in
pSFC was the same as that in normal-phase HPLC, as expected. It was mainly
determined by the number of hydroxyl group in solutes. Furthermore, the same range of
K. Yaku, F. Morishita / J. Biochem. Biophys. Methods 43 (2000) 59 – 76 65

Fig. 5. Relationship between theoretical plate numbers and pressure. Operating conditions: mean pressure 107,
126, 144, 162, 184, 205 and 223 kg cm 22 , other conditions as in Fig. 4. Symbols: h; hydrocortisone, ♦;
fluocinolene acetonide, s; betametthasone, n; triamcinolone.

N values was obtained in each chromatography, i.e., ca. 3600–8000 in pSFC, ca.
4800–8700 in normal-phase HPLC and ca. 2300–11 000 in reversed-phase HPLC.
These results suggests that pSFC is useful for the analysis of polar drugs, and its
applicability to a quality control and a pharmacokinetics study under the high column
efficiency can be expected.

3.2. Chiral separation

It is known that, in general, the determination of the optical impurity in the drug is
very important from the viewpoints of the efficacy and the safety. Chiral resolution using
chromatography strongly depends on the selectivity and the column efficiency. After
Mouroer et al. [20] studied the chiral separation by pSFC, many researchers have shown
the merits of chiral separation in pSFC. Especially, separation using the chiral stationary
phase (CSP) has been of interest in pSFC as well as in HPLC [21–30].
Diltiazem hydrochloride, (2S,3S)-3-acetoxy-2,3-dihydro-2-(4-methoxyphenyl)-5-(2-
dimethylaminoethyl)-1,5-benzothiazepine-4(5H)-one monohydrochloride (shown in Fig.
8), is a benzothiazepine-type Ca-antagonist developed originally by Tanabe Seiyaku
(Osaka, Japan). It is widely used over the world for the treatment of angina pectoris,
variant angina and essential hypertension, which are attributable to the Ca-antagonistic
action [31,32]. Diltiazem hydrochloride has asymmetric carbons at positions 2 and 3.
There are two isomers, cis and trans, depending on the relative positions of the
substituents. Diltiazem hydrochloride is cis-(2S,3S)-isomer.
To investigate the effect of a column on the chiral resolution of the optical isomers,
three types of CSPs with different substituents of cellulose tris(phenyl carbamate)
derivatives were used. The packings of Chiralcel OD, OC and OF columns (250 3 4.6
66 K. Yaku, F. Morishita / J. Biochem. Biophys. Methods 43 (2000) 59 – 76

Fig. 6. Gradient elution of corticosteroids by pSFC. Operating conditions: column Cosmosil 5NH 2 , flow-rate
of CO 2 3 ml min 21 , methanol gradient 11.8–17.0% (v / v) at 0.52% (v / v) per min, mean pressure 206 kg cm 22 ,
temperature 408C. Peaks as in Fig. 2.

mm I.D., Daicel, Tokyo, Japan) were coated onto a silica support with polymer of
cellulose tris(3,5-dimethylphenyl carbamate), cellulose tris(phenylcarbamate) and cellu-
lose tris(4-chlorophenyl carbamate), respectively. As shown in Fig. 9, four optical
isomers were completely resolved on Chiralcel OD and OF, however, trans enantiomers
were not resolved on the Chiralcel OC column under the conditions examined. Although
the selectivity (a ) and resolution (R s ) of both trans and cis enantiomers on the Chiralcel
OD column were smaller than those on the Chiralcel OF column, the theoretical plate
numbers (N) were higher by a factor of about 3. Okamoto and co-workers [33,34]
described that the most important adsorbing sites for chiral discrimination on
phenylcarbamate derivatives of CSP were probably the polar carbamate. The p–p
interaction of phenyl groups on CSPs might be less important than the interaction of the
carbamate for the chiral recognition ability. The contribution of a steric effect of the
derivatized cellulose on the chiral separation should be also considered [35].
The effect of the alcoholic nature, methanol, ethanol and 2-propanol, on the chiral
separation of the optical isomers was studied using the Chiralcel OD column under the
other constant conditions. As shown in Table 1, although the N values obtained for
2-propanol were smaller than those on others, the a and R s of both trans and cis
K. Yaku, F. Morishita / J. Biochem. Biophys. Methods 43 (2000) 59 – 76 67

Fig. 7. Chromatograms of corticosteroids. (A) pSFC, operating conditions as in Fig. 4, (B) RP-HPLC (40%
acetonitrile), (C) RP-HPLC (55% methanol), (D) NP-HPLC (water-saturated chloroform–methanol–acetic
acid, 200:3:2). Peaks as in Fig. 2.

enantiomers were the highest. Kot et al. [23] described that more important than the
nature of the modifier was its concentrations for a chiral separation. The selectivity
decreased with the increase of 2-propanol concentrations as shown in Table 2. The
decrease in the number of stereoselective solute–CSP interactions was caused by the
preferential solvation of chiral sites by the polar modifier [36]. However, the selectivity
of (2R,3S) and (2R,3R) optical isomers was improved in the range of 2-propanol
concentration used, and the baseline resolution (R s . 1.5) was obtained in more than
68 K. Yaku, F. Morishita / J. Biochem. Biophys. Methods 43 (2000) 59 – 76

Fig. 8. Chemical structures of diltiazem optical isomers and related compounds. Diltiazem: R5COCH 3 ,
3-hydroxy form: R5H.

13.0% (v / v) 2-propanol. To improve the peak shape by the deactivation of the active sits
on the silica gel support, a low concentration of diethylamine was added to the modifier
as the basic additive.
Increasing pressure had little significant effect on the enantioselectivity of diltiazem
optical isomers. An interesting change in selectivity with pressure has been shown by an
amylose-based CSP (Chiralpak AD), it might be caused by the pressure-dependence
change of the conformation for amylose-derived CSPs [35].
To determine the three optical impurities in diltiazem hydrochloride, the precision,
accuracy, linearity and limit of detection were studied under pSFC conditions. Typical
chromatograms of diltiazem spiked with ca. 0.05 and 1.0% of the three optical impurities
are shown in Fig. 10, and a part of the three minor peaks is expanded and inserted in
Fig. 10A. The limit of detection of the impurities was 0.05% (12.5 ng at a signal-to-
noise ratio of 2). The determination of optical impurities in diltiazem hydrochloride by
the peak area percentage method exhibited acceptable precision, accuracy, linearity and
detection limit. In addition, the reproducibility of the retention times and the peak areas
was investigated by performing six replicate injections of the solution spiked with 1.0%
other isomers in diltiazem. Good reproducibility was obtained and acceptable: the
relative standard deviations of the retention times and the peak areas were 0.33–0.48%
and 1.78–3.49%, respectively.
The separations of four optical isomers on the Chiralcel OD and OF columns obtained
in pSFC were compared with those in HPLC on the same columns. As shown in Fig. 11,
K. Yaku, F. Morishita / J. Biochem. Biophys. Methods 43 (2000) 59 – 76 69

Fig. 9. Effect of columns on chiral separations of diltiazem enantiomers by SFC. SFC conditions: mobile
phase CO 2 –13% (v / v) 2-propanol containing 0.5% (v / v) diethylamine, flow-rate of CO 2 2 ml min 21 , outlet
pressure 180 kg cm 22 , temperature 508C, detection at 254 nm.

(2R,3S) and (2R,3R) isomers were not resolved on the Chiralcel OD column in HPLC,
though they were the diastereomeric isomer. On the other hand, in pSFC, the baseline
resolution of the four optical isomers was achieved within 8 min on the Chiralcel OD
column (Fig. 9). On the Chiralcel OF column, all isomers achieved the baseline
separations in both modes, but the elution orders of (2R,3S) and (2R,3R) isomers on
pSFC and HPLC were different.
70 K. Yaku, F. Morishita / J. Biochem. Biophys. Methods 43 (2000) 59 – 76

Table 1
Effect of alcoholic natures on chiral separation of diltiazem optical isomers by pSFC a
Modifier trans Enantiomers cis Enantiomers
k1 k2 a Rs N k1 k2 a Rs N
Methanol 3.0 3.3 1.09 1.33 7570 3.8 4.3 1.13 1.97 7280
Ethanol 3.2 3.7 1.15 2.05 6640 4.1 4.7 1.16 2.37 6830
2-Propanol 5.2 6.4 1.24 2.98 4840 6.7 8.3 1.23 2.92 4840
a
SFC conditions: column Chiralcel OD, mobile phase CO 2 –9% (v / v) modifier containing 0.5% (v / v)
diethylamine, flow-rate of CO 2 2 ml min 21 , outlet pressure 180 kg cm 22 , temperature 508C, detection at 254
nm. k 1 : Capacity factor of first-eluted enantiomer; k 2 : capacity factor of second-eluted enantiomer.

As the results demonstrate, pSFC is the effective technique for the separation of the
optical isomers in pharmaceuticals because of its ability to separate rapidly with high
column efficiency.

3.3. Thermodynamic study in chiral separation

The column temperature plays an important role for a chiral separation not only in
HPLC, GC and capillary zone electrophoresis (CZE), but also in SFC [37–46]. Smith et
al. [37] studied the temperature dependence of chiral recognition using two racemates of
very similar structures in SFC and HPLC. They described that the difference of the
chiral recognition should be ascribed to difference in the DDH 0 values for the interaction
between the analytes and a cellulose-based stationary phase. Stringham and Blackwell
[38,39] described ‘‘entropically driven’’ separation in detail. The selectivity was a
compromise between differences in enantiomeric binding enthalpy and disruptive
entropic effects. In this session, cellulose tris(4-chlorophenylcarbamate), Chiralcel OF,
was selected as the chiral stationary phase, and the temperature dependence of the
enantioselectivities on cis- and trans-diltiazem optical isomers were studied under pSFC
conditions from the thermodynamic point of view. Then, the advantage of the chiral
separation by pSFC was shown briefly.
In chromatography, the thermodynamic relationships of the capacity factor (k) and the
selectivity (a ) can be expressed by Eqs. (1) and (2), respectively:

Table 2
Effect of modifier concentrations on chiral separation of diltiazem optical isomers by SFC a
2-Propanol trans Enantiomers cis Enantiomers Geometric isomer b
(%, v/v)
k1 k2 a Rs N k1 k2 a Rs N a Rs

4.8 14.2 19.0 1.33 3.54 3330 19.0 24.5 1.29 2.81 2500 1.00 –
9.1 5.3 6.3 1.20 2.53 5100 6.9 8.4 1.22 2.75 4730 1.09 1.21
13.0 3.1 3.5 1.14 1.83 5850 3.9 4.7 1.20 2.55 6070 1.11 1.63
16.7 2.1 2.3 1.11 1.38 6260 2.6 3.1 1.17 2.22 6390 1.14 1.72
a
SFC conditions: column Chiralcel OD, mobile phase CO 2 –2-propanol containing 0.5% (v / v) diethylamine,
flow-rate of CO 2 2 ml min 21 , outlet pressure 180 kg cm 22 , temperature 508C, detection at 254 nm. k 1 :
Capacity factor of first-eluted enantiomer; k 2 : capacity factor of second-eluted enantiomer.
b
The values of separations between (2R,3S) and (2R,3R) isomers.
K. Yaku, F. Morishita / J. Biochem. Biophys. Methods 43 (2000) 59 – 76 71

Fig. 10. Chromatograms of diltiazem spiked with three optical isomers. (A) 0.05%, (B) 1.0%, SFC conditions
and peaks as in Fig. 9.

ln k 5 2 DH 0 /RT 1 DS 0 /R 1 ln b (1)

ln a 5 2 DDH 0 /RT 1 DDS 0 /R (2)


0 0
where DH and DS are the enthalpy and entropy changes in the partition process
between the mobile and stationary phases, respectively. R is the gas constant (8.314 J
mol 21 K 21 ), T is the absolute temperature, and b is the phase ratio. DDH 0 and DDS 0
are the differences in the enthalpy and entropy changes for the enantiomers, respectively.
In chiral separation, DH 0 and DS 0 can be considered as the sum of the chiral and achiral
contributions. Only stereoselective interaction with the chiral stationary selector leads to
a difference in retention, as the achiral contributions to DH 0 and DS 0 should be the same
for both enantiomeric pairs. Thus, DDH 0 and DDS 0 should be ascribed to the difference
in chiral contributions, i.e., DDH 0chiral and DDS chiral
0
, respectively. The temperature at
which the enantiomers coelute is defined as T iso , where the enthalpy and entropy terms
are equivalent or balanced, can be expressed by Eq. (3):
T iso 5 DDH 0 /DDS 0 (3)

The influence of temperature on retention and selectivity of diltiazem isomers in pSFC


72 K. Yaku, F. Morishita / J. Biochem. Biophys. Methods 43 (2000) 59 – 76

Fig. 11. Chiral separations of diltiazem optical isomers by HPLC. HPLC conditions: mobile phase n-hexane–
2-propanol (Chiralcel OD 9:1–Chiralcel OF 1:1) containing 0.1% (v / v) diethylamine, flow-rate 1 ml min 21 ,
temperature 308C, detection at 254 nm.

and HPLC were investigated using the same Chiralcel OF column. In pSFC, a mixture
of CO 2 –15% (v / v) 2-propanol containing 0.1% (v / v) diethylamine was used as the
mobile phase, and the column temperature was changed from 23 to 608C under 180 kg
cm 22 constant outlet pressure. In HPLC, a mixture of n-hexane–2-propanol (1:1)
containing 0.1% (v / v) diethylamine was also used as the mobile phase. The values of the
logarithms of k were plotted versus 1 /T, respectively. As shown in Fig. 12a, the linear
relationship could not be observed for any solute in SFC: the logarithm of k decreased,
reached the minimum value at about 358C, and increased with temperature. On the other
hand, the straight lines were obtained for any solute under HPLC conditions. Major
reasons for such curvature in SFC might be due to changes in the density of the mobile
phase and to the extent of swelling of the stationary phase with the column temperature.
In order to negate the above-mentioned temperature effects, it should be favorable to use
the separation factor a, i. e., the relative retention value for enantiomers, instead of k9.
The ln a values were also plotted versus 1 /T (Fig. 12b) and the obtained thermo-
dynamic parameters in pSFC and HPLC are summarized in Table 3. The plots of ln a
versus 1 /T yielded straight lines for both cis- and trans-enantiomers in pSFC and
HPLC, as expected. In pSFC, the chiral separation of cis-enantiomers was improved by
a decrease in temperature, whereas that of trans-enantiomers was improved by an
increase in temperature. In HPLC, the chiral separations of cis- and trans-enantiomers
K. Yaku, F. Morishita / J. Biochem. Biophys. Methods 43 (2000) 59 – 76 73

Fig. 12. Temperature dependence of retention and selectivity in pSFC and HPLC. pSFC and HPLC conditions
as in Table 3. Symbols: d; pSFC, s; HPLC.

were improved by a decrease in temperature. Considering the T iso of the enantiomers,


the separation of cis-enantiomers was enthalpy-controlled, whereas the separation of
trans-enantiomers was entropy-controlled in pSFC in the temperature range examined.
In HPLC, both cis- and trans-enantiomers showed enthalpy-controlled separation.
Recently, Wada et al. [47] reported the local excess density about substituted benzene
compounds in supercritical CO 2 based on Fourier transform infrared (FT-IR) spec-
troscopy. They found that the local density of CO 2 about substituents on the aromatic
ring was different from that around the aromatic ring itself and also strongly dependent
on the kind of substituents. The steric environment around a functional group should
have some influence on the local density of solvents around it. The environments around
the chiral active sites including the phenyl groups of diltiazem isomers and the
phenylcarbamate group of the CSP in pSFC may be different from those in HPLC.
The advantages of a chromatography using the compressible fluid as the mobile phase
are the separation time and column efficiency because of the lower viscosity and the
higher diffusivity in the mobile phase. The simultaneous chiral separations of diltiazem
and 3-hydroxy optical isomers (shown in Fig. 8) were investigated by pSFC and HPLC.

Table 3
Thermodynamic parameters for diltiazem enantiomers by HPLC and pSFC
trans Enantiomers cis Enantiomers
0 0
DDH DDS T iso DDH 0 DDS 0 T iso
(J mol 21 ) (J mol 21 K 21 ) (8C) (J mol 21 ) (J mol 21 K 21 ) (8C)
HPLC a 28000 221 114 28200 221 111
pSFC b 4700 16 24 29500 225 104
a
HPLC conditions: column, Chiralcel OF; mobile phase n-hexane–2-propanol (1:1) containing 0.1% (v / v)
diethylamine; flow-rate 1 ml min 21 ; detection at 254 nm.
b
pSFC conditions: column Chiral OF, mobile phase CO 2 –15% (v / v) 2-propanol containing 0.1% (v / v)
diethylamine, flow-rate of CO 2 2 ml min 21 , outlet pressure 180 kg cm 22 , detection at 254 nm.
74 K. Yaku, F. Morishita / J. Biochem. Biophys. Methods 43 (2000) 59 – 76

Since the 3-hydroxy form of diltiazem hydrochloride is the precursor, the main
degradation product and the metabolite, it is very important to study the simultaneous
separation from the viewpoints of safety and efficacy. The results of the separation are
shown in Fig. 13. The peaks in HPLC are broader than those in SFC possibly due to
lower diffusivity and higher viscosity of liquid than compressible fluid; all of the optical
isomers could not be separated simultaneously in HPLC. These results reveal that pSFC
is superior to HPLC as a chiral separation technique.

4. Conclusions

pSFC must have the potential for the analysis of a drug. Especially, in chiral
separation, the improvements of selectivity, resolution and efficiency can be expected
rather than in HPLC. Increasing number of reports on pSFC should be necessary in order
confirm it as a complementary or alternative analytical technique.

Fig. 13. Chromatograms of diltiazem and 3-hydroxy form isomers by (a) pSFC and (b) HPLC. pSFC
conditions: column Chiralcel OF, mobile phase CO 2 –22.5% (v / v) 2-propanol containing 0.1% (v / v)
diethylamine, flow-rate of CO 2 2 ml min 21 , temperature 608C, pressure 180 kg cm 22 , detection at 254 nm.
HPLC conditions: column Chiralcel OF, mobile phase n-hexane–2-propanol (3:7) containing 0.1% (v / v)
diethylamine, flow-rate 1 ml min 21 , temperature 308C, detection at 254 nm. Peaks: 15(2S,3R)-diltiazem,
25(2R,3S)-diltiazem, 35(2R,3R)-diltiazem, 45(2S,3S)-diltiazem, 55(2R,3R)-3-OH, 65(2S,3S)-3-OH, 75
(2S,3R)3-OH, 85(2R,3S)-3-OH.
K. Yaku, F. Morishita / J. Biochem. Biophys. Methods 43 (2000) 59 – 76 75

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