Anal. Chem.1994,66, 2893-2897
Enantiomer Separation by Capillary SFC and GC on Chirasil-Nickel: Observation of Unusual Peak Broadening Phenomena Michael Schleimer,t Markus Fluck, and Volker Schurig' Institut fur Organische Chemie der Universitat, Auf der Morgenstelle 18, 0-72076 Tubingen, Germany
Immobilized chirasil-nickel (nickel(I1) bis[3-(heptafluorobu10-methylenecamphorate]chemically bonded to tanoy1)- (1R)a poly(dimethylsi1oxane)) was employed for the separation of enantiomers by capillary supercritical fluid chromatography and gas chromatography. In SFC, the dependence of the retention behavior (k') and chiral separation factors (a)on applied pressure, density, and analysis temperature has been investigated for the two representative chiral test solute 1-phenylethanol and camphor. Even at high densities of the carbon dioxide mobile phase, the enantioselectivitiesmeasured at low temperatures under supercritical conditions were found to be similar to those expected from the extrapolation of hightemperature measurements in GC. Systematic variations in temperature or pressure in SFC resulted in unusual enantioselective peak-broadening phenomena, which were observed independently from the enantiomeric purity of the solutes. These phenomena were amplified substantially both under overload conditions and at reduced mobile-phase flow rates.
GC, the enhanced solvation strength of supercritical carbon dioxide can reduce the analysis temperature significantly.12 This reduction in temperature offers a promising alternative for increasingthe enantioselectivityof a chiral stationary phase, since the separation factor (a)increases with decreasing separation temperatures15J6 in the usually enthalpy-controlled domain of enantiomer separations.'
EXPERIMENTAL SECTION Instrumentation. For gas chromatography, a Carlo Erba Fractovap 2350 or a Mega HRGC 5300 (Fisons, Germany) with nitrogen (99.996%, Messer-Griesheim, Germany) as carrier gas was used. The flame ionization detector and injector were heated to 250 OC and the split flow was set to 100mL/minat0.5 barofnitrogen(ratioL1:lOOOatO.l MPa of nitrogen) in order not to overload the 2.0 m X 0.05 mm i.d. column and to facilitate a rapid injection. For SFC measurements, a Fisons SFC 3000 system, equipped with a syringe pump, actuated injection valve (200nL rotor), and flame ionization detector (350 "C) was used. Complexation chromatography allows the separation of Carbon dioxide (99.998 vol %, Messer-Griesheim) was passed enantiomers possessing atoms with lone-pair electrons (0,S, through an active carbon black trap before being used as mobile N, P) due to the presence of a fast and reversible coordination phase. The flow rate of the carbon dioxide was regulated equilibrium with an electronically and coordinativelyunsaturwith laboratory-made integral-type restrictors (0.5-0.6 cm/s ated nonracemic metal Thus, nickel(II), manat 10.0 MPa and 80 OC).17 The splitting ratio of the injector ganese( 11), or zinc( I I) bis [3- (heptafluorobutanoy1)-( I R)was set to 1:40 using the same conditions. The entire system camphorate] can be employed either as ~ h i r a - m e t a l s ~ - ~ was controlled by the SFC 300 software (Fisons, Mainz, dissolved in an achiral liquid matrix as stationary phase or as Germany), and data were collected either with a Chromatochirasil-metalsa-10by attaching the metal complex covalently graphic Workstation Baseline 8 10 (Millipore, Milford, MA) to a siloxane polymer. The latter can be thermally crossor with a Shimadzu Chromatopac CR-3A integrator (Bischoff, linked and/or immobilized onto the inner surface of fused Leonberg, Germany). silica capillaries, resulting in a nonextractable, thermally stable Preparation of Chirasil-nickel. The synthesis of nickel( 11) chiral stationary phase, which is also resistant to more dense bis[3-(heptafluorobutanoyl)-(IR)- 1O-methylenecamphormobile phases such as supercritical carbon dioxide in superate], chemically bonded to a poly(dimethylsiloxane), was critical fluid chromatography (SFC)." Compared to capillary carried out as described previously.1° Coatingand Immobilizationof Chirasil-nickel. Fused silica Present address: Marion-Merrell-Dow Research Institute, 16, rue d'Ankara, tubing of 50-pm i.d. (Chrompack International, Middelburg, 67080 Strasbourg, France. On leave to F. Hoffmann-La-Roche, PRPK, Bioanalytical Section, CH-4002Basel, Switzerland. The Netherlands) was dehydrated by purging with hydrogen (1) Schurig, V.; Biirkle, W. Angew. Chem., Int. Ed. Engl. 1978, 17, 132. at 250 OC for 4 h. The capillary columns werecoated, without (2) Schurig, V. Chromatograplria 1980, 13. 263. (3) Schurig, V.; BUrkle, W. J . Am. Chem. Soc. 1982, 104, 7573. further treatment, by the static method at room temperature (4) Schurig, V.;Weber, R. J. Chromatogr. 1984, 217, 51. with chirasil-nickel (film thickness 0.3 pm), using a carefully ( 5 ) Schurig. V. Kontakte (Darmstadt) 1986, 1, 3. (6) Schurig, V. J . Chromatogr. 1988, 441, 135. filtered 2.4% (w/v) solution of the chiral stationarv phase in (7) Schurig, V.; BIirkle, W.; Hintzer, K.;Weber, R. J . Chromatogr. 1989, 475, 23. (8) Schurig, V.; Link, R. In Chiral Separations; Stevenson, D., Wilson, I. D., Eds., Plenum Press: London, New York, 1992; p 91. (9) Schurig, V.; Schmalzing, D.; Schleimer, M. Angew. Chem., I n t . Ed. Engl. 1991, 30, 987. (10) Schleimer, M.; Schurig, V. J . Chromatogr. 1993, 638, 8 5 . (1 1) Schleimer, M.; Schurig, V. In Analysis with SupercriticalFluids; Wenclawiak, B., Ed.; Springer: Berlin, 1992; p 134. 0003-2700/94/0366-2893$04.50/0
0 1994 Amerlcan Chemical Society
(12) Bartle, K. D. In Supercritical Fluid Chromatography;Smith, R.M.,Ed., The Royal Society of Chemistry: London, 1988. (13) Mourier, F.;Sassiat, P.; Caude, M.;Rosset, R. J. Chromatogr. 1986.353,61. (14) Juvancz, Z.; Markides, K. E. LC-GClNTL 1992,5(4), 44. (15) Watabe, K.; Charles, R.; Gil-Av, E. Angew. Chem., Int. Ed. Engl. 1989, 28, 192. (16) Schurig, V.; Ossig, A.; Link, R. Angew. Chem., Int. Ed. Engl. 1989.28, 194. (17) Guthrie, E. J.; Schwarz, H. E. J . Chromatogr. Sci. 1986, 24, 85.
Analytical Chemistry, Vol. 66,No. 18, September 15, 1994 2093
Chart 1. Structure of Chlradl-nickel
CH3
L
i
5
Ink'
CHZ
in 2.3 2.4 2.5 2.6 2.7 2.8 2.9 3.0 3.1 3.2 3.3
diethyl ether. Immobilization was carried out after conditioning of the column in a GC instrument for 12 h at 160 OC and 1.0 bar of nitrogen, by heating for 24 h at 180 OC with a strongly reduced nitrogen flow. Then the column was rinsed with methanol, dichloromethane, and n-pentane (1 mL each). The degree of immobilization was found to be 85% by a comparison of the capacity factor of n-pentadecane at 120 OC before heat treatment and after rinsing of the column. The enantioselectivity factors, E % = 100[(u(after rinsing) - l / a (before heat treatment)- 11, were found tobeslightlyincreased for l-phenylethanol(l06%), 2-methyltetrahydrofuran (103%), and 2-methylcyclohexanone (108%) after the rinsing procedure.
RESULTS AND DISCUSSION The enantioselectivity of a chiral stationary phase in different chromatographic systems is described by parameters such as the capacity factor (k'), the chiral separation factor which (CY), and the chromatographic resolution factor (Rs), critically depend on the composition, polarity, and density of the applied mobile phase. By employing immobilized chirasil-nickel (cf. Chart 1) under supercritical mobile-phase conditions, two limiting cases can be distinguished by inspecting the plot of In k'vs inverse temperature T-l measured at different constant inlet pressures of the mobile-phasecarbondioxide (cf. Figure 1 for the second eluted enantiomer of racemic 1-phenylethanol and camphor). Under more GC-like conditions, i.e., low inlet pressure (cf. curves b at 10.0 MPa of carbon dioxide in Figure l)), the retention behavior of each of the enantiomers corresponds to that found already on achiral stationary phases.l2 Under "real" supercritical conditions, i.e., high inlet pressure (cf. curves d at 25.0 MPa of carbon dioxide in Figure 1) the overall retention of the solutes is reduced significantly, as is expected for higher density mobile phases with enhanced solvation power. However, the solute retention no longer passes through a maximum but steadily increases with a decrease in temperature, indicating that the stationary phase, particularly in the highdensity region of the mobile phase, is capable of overcompensating the strong solvation power of the supercritical carbon dioxide. This surprising phenomenon can be rationalized by a decrease in the tendency of self-association of the metal complexes in chirasil-nickel, which was found to be characteristic for closely structurally related nickel(I1) /3-diketo2894
Analytical Chemistry, Voi. 66, No. 18, September 15, 1994
3
a
0.1 MPaN,
b
10.0MPaC0,
c
15.0MPaC0,
d
25.0MPaC0,
2
SFC 1
1
1
,
1
~
1
~
1
1
1
~
1
/
1
1
1
'
2.3 2.4 2.5 2.6 2.7 2.8 2.9 3.0 3.1 3.2 3.3
Flgure 1. In k' (capaclty factor of the second eluted enantiomer) vs T-I (p = constant): (top) 1-phenylethanol, (bottom) camphor: 2.0 m X 0.05 mm 1.d. fused silica capillary, coated with immobilized chiraslinickel (fllm thickness 0.25 pm).
nates.I8J9 At high densities of the mobile phase the carbon dioxide may interact with the stationary phase either by solvating the metal complexes or by physically separating them due to extensive stationary-phase swelling. Both effects would cause an increase of the apparent concentration of available complexation sites, which then would increase the solute retention. In the region represented by curves c in Figure 1, the retention behavior of the solutes is less dependent on temperature due to the large influence of solvation on the distribution equilibrium between mobile and stationary phase. The observed enantioselectivity (CY)of a diluted chiral stationary phase is determined by the expression
where KLrepresents the apparent partition coefficient of the solute between mobile and stationary phase and R' is the retention increase (R' = Km,the product of the association constant K between selector and selectand and the molality m of the chiral selector in the liquid stationary phase). R', a chemical capacity factor, represents the normalized part of the overall retention due exclusively to the chemical (diastereomeric) interaction between solute and selector (for a (18) Bullen, J. G.; Mason, R.; Pauling, P. Inorg. Chem. 1965, 4, 546. (19) Graddon, D.P. Coord. Chem. Rev. 1969, 4, 1.
~
'
~
1
ha
sFcd41 0.6 I
I
GC
-a/ 0.0
~
'
~
'
~
'
~
c
15.0MPaC0,
d
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~
!
"'
'
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~
T-'[10-3K"] Flgwe 3. in a (separatlonfactor)v$ T-'(p = constant): 1-(2naphthylb ethanol at constant density p = 0.35 g/mL and p = 0.7 g/mL, respectively, column, 4.0 m X 0.05 mm i.d. fused silica capillary, coated with Immobilized chirasll-nlckel (film thickness 0.4 pm).
0.5 0.4 0.3 -
CHIRASIL-NICKEL
0.2 -
n
40°C 450c
C
0.1 -
I
p b
0.55
q
b
S
a 0.0""'"""""'""'" 2.3 2.4 2.5 2.6 2.7 2.8 2.9 3.0 3.1 3.2 3.3 T' (109 K') Figure 2. in a (separatlon factor) vs T-' (p = constant): (top) 1-phenyiethanol, (bottom) camphor; column, see Figure 1.
detailed explanation of the concept see refs 2 and 20-22). With hydrogen-bonding (e.g., chirasil-va123)or inclusion-type (e.g., c h i r a s i l - d e ~ ~chiral ~ ~ ~stationary ~) phases, the enantioselectivity was found to decrease significantly with increases in the inlet pressure of the mobile phase. This was explained by a reduction of R',due to a blocking of the hydrophobic cavity of cyclodextrin with the nonpolar carbon dioxide or by a thermodynamic stabilization of the dissolved solute within the stationary phase. A remarkable feature of chirasil-nickel is the absence of losses in enantioselectivity when the inlet pressure is increased going from GC to SFC (cf. Figure 2). Even at high densities of the mobile-phase carbon dioxide, the enantioselectivities measured at low temperatures under supercritical conditions were found to be similar to those expected from the extrapolation of high-temperature measurements in GC. Since chirasil-nickel shows enantioselectivity usually at much higher R' than with other chiral stationary phases, eq 1 simplifies in the limiting case to 1 R'= R',Le., whenever the solute enters the stationary phase it is found to be associated to the chiral metal complex. In the achiral supercritical mobile phase both enantiomers are chemically and physically identical. The unusual increase of
+
(20) Gil-Av, E.; Herling, J. J . Phys. Chem. 1962, 66, 1208. (21) Schurig, V.; Chang, R. C.; Zlatkis, A.; Feibush, B. J . Chromatop. 1974.99, 147. (22) Jung, M.; Schmalzing, D.; Schurig, V. J . Chromafop. 1991, 552, 43. (23) Lai, G.; Nicholson, G. J.; Baycr, E. J. Chromorogr. 1992, 540, 217. (24) Schurig, V.; JuvanGz, 2.;Nicholson, G. J.; Schmalzing, D. J . High Resolut. Chromatogr. 1991, 14, 58.
(25) Schmalzing, D.; Nicholson, G. J.; Jung, M.; Schurig, V. J . MicrocolumnSep. 1992, 4, 2.
i*IuL
OH
15.0 MPa CO,
70%
10.0
17.5 20.0
JL
22.5
2s.o
co2
50 OC
Figure4. Elution profiles at varying temperaturesand pressures: (top) 1-phenylethanoiat 15 MPa of carbon dioxide, (bottom) camphor at 50 OC; column, see Figure 1.
both retention and enantioselectivity with increasing mobilephase density can thus only be ascribed to the presence of carbon dioxide in the polymeric stationary phase and its active role during the diastereomeric equilibration between the complexed and noncomplexed state of the solute. Such an influence of carbon dioxide may be attributed to changes in the properties of the selector as well as the selectand; the effect of the latter was discussed previously in a more complex system under subcritical conditions.26 Whereas a linear relationship between In a and T-1 can be derivtd12 and has, in fact, been verified,l3J4 we found significant deviation$ from linearity. The change in the slope ~~
(26) Sirct, L.; Bargmann, N.; Tambute, A,; Caudc, M. Chirality 1992, 4, 262.
Ana!vticaIChem/str~,Voi. 66, No. 18, September 15, 1994
2095
45 O C
a relatively sharp change in the slope of curve c in Figure 2 for camphor at about 70 “ C (2.93 X K-l).
1
” I 5
I
I
I
1
I
6
7
8
9
10
S
65OC
n
1
1
I
I
I
5
6
7
8
’
4
R
Time
9
(minutes)
Flgure 5. Elution proflles of dlfferent amounts of 1-phenylethanol at 45 (top) and 65 ‘C (bottom). (I) 0.25 and (11) 4.5 pmol Injected as 200 nL of a 2% (I) or 10 % (11) solution In diethyl ether (estlmated from a constant split ratio of 1:40). The lowest traces represent nonoverloaded “anaiytlcalconditions” and are Identicalto those given in Figure 4 at the corresponding temperatures: column, see Flgure 1. uo = 0.314
cm . s-’
U. = 1.335 UD
bo., (S)= 132.7 s bas (R)= 38.4 LI
. S-’
bo$ (S)= 12.7 I bas ( R ) = 12.0 I
R
S
I
I
I
0
15
30
-
0
5mkr
Flgure 6. Enantiomeric separation of racemlc camphor at different flow rates of the mobile phase (15.0 MPa of carbon dioxide), 60 ‘C: column, see Flgure 1.
of the high-temperature branch of curves b-d in Figure 2 (constant pressure) and the unusual shape of curve b in Figure 3 (constant density) may indicate the presence of two (or more) competing mechanisms for enantiomer discrimination which, dependent on temperature, differ in their contribution to the observed enantioselectivity. A similar behavior under constant pressure conditions in G C has been discussed by Gil-Av et al.15 for the enantiomeric separation of racemic amino acids on diamide phases. The assumption of overlayed mechanisms for enantiomer discrimination is augmented by 2898
Analytical Chemistty, Vol. 66, No. 18, September 15, 1994
An unusual enantioselective peak broadening of the quantitatively separated enantiomers of racemic l-phenylethanol and camphor was observed, which was studied by systematically varying the column temperature and/or the carbon dioxide inlet pressure (cf. Figure 4). Following the isobaric curve c for racemic 1-phenylethanol (cf. Figures 1 and 2), it can be seen in Figure 4 that the peak of the first eluted S enantiomer becomes very broad between 45 and 5 5 OC although there is no deviation of the In CY vs T-l curve from linearity. The fact that the first eluted enantiomer is broader in peak shape than the second eluted enantiomer is quite uncommon in chromatography of enantiomers and special attention in regard to its unknown origin is warranted. Related, though less pronounced, peak-broadening phenomena have previously been observed in complexation gas chromatograp h ~ , inclusion ~ ~ * ~gas~ c h r o m a t ~ g r a p h y , and ~ ~ , ~ligand ~ exchange c h r ~ m a t o g r a p h y . ~ ~ A different behavior was found for racemic camphor, as is evident from the isothermal elution profiles obtained at varying inlet pressures ( T = 50 OC corresponds to a vertical K-1 in Figures 1 and 2). In this case, the line at 3.09 X first eluted peak became broader between 11 and 16 MPa of carbon dioxide pressure while the second eluted R enantiomer showed peak broadening above 16 MPa of applied pressure. It should be noted that the areas of the broadened vs unbroadened elution curves show the correct 1:1 ratio expected for a racemic solute. Recently, the unusual peak-broadening phenomenon in inclusion G C has been related to overload conditions in the gas which areeasily induced in short, narrow, and thin film c a p i l l a r i e ~similar , ~ ~ to the column used for the experiments shown in Figure 1-3, while the presence of different interaction sites with different kinetic requirements has tentatively been discussed in complexation GC.27 In order to determine column overload as a possible source of the observed peak broadening, varying amounts of racemic and highly enriched ( S ) -1-phenylethanol were injected. Peak broadening already occured under nonoverloaded analytical conditions, as clearly demonstrated in Figure 5, and the injection of larger amounts resulted in distorted peak shapes frequently encountered in overloaded preparative liquid ~hromatography.~~ Peak-broadening phenomena may likewise be attributed to slow kinetics of diastereomeric equilibration as suggested p r e v i o ~ s l ybecause , ~ ~ ~ ~they ~ ~were ~ also affected by the linear velocity of the mobile phase (cf. Figure 6 ) . At low carbon dioxide flow rates, peak broadening of the first eluted enantiomer was strongly amplified, which was alsore(27) Schurig, V.; Betschinger, F. Chem. Rev. 1992, 92, 873. (28) KBnig, W. A.; Icheln, D.; Hardt, I. J . High Resolur. Chromotogr. 1991, 14, 694. (29) JuvanGz, Z.; Grolimund. K.; Schurig, V. J . High Resolut. Chromarogr. 1993, 16, 202. (30) Kurganov, A.; Davankov, A.; Unger, K.; Eisenbeiss, F.; Kinkel, F. J . Chromatogr. 1994, 666, 99. (31) Yabaumuto,K.;Ingraham,D. F.;Jennings.W.J.J . HighReso/ut.Chromorogr. Chromarogr. Commun. 1980, 3, 248. (32) Guiochon, G.; Golshan-Shirazi, S.; Jaulmes, A. AMI. Chem. 1988,60, 1856. (33) Jacobson, S.C.; Seidel-Morgenstern, A.; Guiochon, G. J . Chromatogr. 1993, 637, 13.
flected in the effective plate numbers found (S,305 N/m, k’ = 0.83;R, 5226 N/m, k’ = 1.21; a = 1.46). As the linearvelocity of the mobile phase was increased, the expected peak heights of the separated enantiomers were found while peak widths and plate numbers (S, 1700 N/m, k’ = 0.81;R, 281 1 N/m, k’ = 1.19;a = 1.47)were still reduced for the first eluted enantiomer.
ACKNOWLEDGMENT This work was supported by Deutsche Forschungsgemeinschaft and Fonds der chemischen Industrie. for rev’ew
1994a
*&
1994.’ *Abstract published in Aduance ACS Absrrucrs. July 15, 1994.
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