enantiomeric separation by gc on chirasil-dex: systematic study of cyclodextrin concentration,...

Enantiomeric Separation by GC on Chirasil-Dex: Systematic Study of Cyclodextrin Concentration, Polarity,

Immobilization, and Column Stability

Martin Jung and Volker Schurig* Institut fur Organische Chemie der Universitat

Auf der Morgenstelle 18, 7400 Tubingen, Germany

Abstract. Polysiloxane-anchored permethylated 6-cyclodextrin (Chirasil-Dex) was used for enantiomeric separation by GC. Four types of Chirasil-Dex, containing up to 36 % permethylated 0-cyclodextrin by weight, were synthesized and compared with 10 % permethylated P-cyclodextrin dissolved in OV-1701. The influence of the cyclodextrin weight percentage on the chiral separation factor CY and on polarity, as expressed by the Kovats indices, was studied. The chiral separation factors a did not increase significantly above a threshold of - 30 % permethylated P-cyclodextrin by weight. The plots of a vs. weight percentage depend on the complexation strength of the chiral solutes, as do the Kovats indices; for weakly complexing solutes, changes in the cyclodextrin concentration result in larger changes in a, but in less pronounced changes in the Kovats indices, as compared to strongly complexing solutes. Column performances were investigated for immobilized and nonimmobilized stationary phases. As expected, immobilization properties were found to depend on the content of Si-H groups in the polysiloxane backbone. Immobilized columns exhibited a much better long-term stability of all chromatographic variables investigated.

Key words: enantioselective gas chromatography, immobilizedpermethyl-P-cyclodextrin, Chirasil-Dex, permethylated P-cyclodextrin dissolved in OV-1701

INTRODUCTION Gas chromatography employing chiral cyclo-

dextrin stationary phases constitutes a versatile tool in modern enantiomeric analysis [ 1,2]. Enantio- selectivity of cyclodextrins is not limited to certain classes of compounds, as with complexation or hydrogen-bonding stationary phases, but is observed for an extremely wide variety of chiral solutes. Although low-melting cyclodextrin derivatives containing n-pentyl groups (e. g., per-n-pentylated or 2,6-n-penty1-3-acyl-cyclodextrins) can be used as undiluted liquid stationary phases [3], our strategy to dilute the cyclodextrin derivative in a moderately polar polysiloxane [4] proved to be more useful since this allows the use of cyclodextrin derivatives irrespective of their melting points and permits the combination of the inherent cyclodextrin enantioselectivity with the unique

*Author to whom correspondence should be addressed 1993 MicroSeparations

coating properties and excellent column efficiencies of polysiloxanes. In a recent systematic study involving more than 1.50 racemates, permethylated 0-cyclodextrin has been shown to be the most universally applicable cyclodextrin derivative currently known [5]. Columns coated with permethylated P-cyclodextrin in OV-1701 have be- come commercially available from leading column manufacturers.

A logical extension and further improvement of our approach consists in the chemical binding of the permethylated 0-cyclodextrin to a polysiloxane backbone, which was accomplished independently by Fischer et al. [6] and by our group [7] in 1990. In Chirasil-Dex the cyclodextrin is attached via a tri- [6], penta-, or octamethylene [7] spacer to a polydimethylsiloxane containing Si-H groups by

J. Microcol. Sep. 5, 11-22 (1993) 11

12 J. Microcolumn Separations, Vol. 5 , No. 1, 1993

H,PtCl,-catalyzed hydrosilylation [8,9], in analogy to the synthesis of Chirasil-Val-Nova [ 101 and Chirasil-Metal [ 113. We recently described in detail the advantages of Chirasil-Dex as compared to the original dissolved stationary phase (i. e., permethylated /3-cyclodextrin in OV-1701) for more than 100 racemates [12]. With no doubt, the most important advantage of Chirasil-Dex is the ability to immobilize it onto fused silica or glass surfaces, which has previously been observed also with Chirasil-Val [13] and a Pirkle-type chiral polysiloxane [14], thus providing the possibility of using permethylated /3- cyclodextrin as a chiral selector also in supercritical fluid chromatography [15,16] and even in capillary electrophoresis [ 171.

There have been only scattered reports on theoretical considerations and thermodynamic and mechanistic aspects of enantiomeric separation by GC on cyclodextrin stationary phases [18-211. In a previous work we investigated the influence of the cyclodextrin concentration in OV-1701 on enantioselectivity [ 191, but the reduced solubility and film stability limited the use of the dissolved phase to about 14 % permethylated cyclodextrin by weight. In the present work four types of Chirasil-Dex, containing up to 36 % permethylated /3-cyclodextrin by weight, were used. The main intention of this systematic study was to provide a better under- standing of the influence of the cyclodextrin weight percentage on enantioselectivity and to collect data on polarity, as expressed by Kovats indices, as well as data on long-term column stabilities and immobilization properties. Thus, the properties of Chir- asil-Dex can be optimized with respect to enantioselectivity, retention behavior, ease of immobilization, and long-term temperature stability of the chromatographic quantities such as chiral separation factors, capacity factors, and Kovats indices.

EXPERIMENTAL The previously described synthesis of Chirasil-

Dex [7] was followed with some modifications. Preparation of monokis-6-(oct- 7-enyl)-P-cyclo-

dextrin. In an atmosphere of nitrogen, 18 g (16 mmol) of anhydrous P-cyclodextrin (Fluka, Buchs, Switzerland), dried in vacuo at 80°C over P40,, for 48 h, were added with stirring to 400 mL of dry dimethyl sulfoxide in a 1 L three necked flask equipped with a dropping funnel, a nitrogen inlet, and a mercury valve. To the solution, 1.9 g (47 m o l ) of powdered sodium hydroxide were added, and stirring of the yellow mixture was continued for 1 h at room temperature. A 7.58 g (40 mmol) amount of 8-bromooct-l-ene, dissolved in 50 mL of dimethyl sulfoxide, were added dropwise within

8 h. Stirring at room temperature was continued for 48 h. The reaction mixture was separated from sodium bromide and unreacted sodium hydroxide by filtration and subsequently concentrated to about 30 mL in vacuo at 0.1 torr and 60°C. The residue was diluted with 30 mL of methanol, and the product was carefully precipitated from the clear solution with 400 mL of diethyl ether. The crystal- line, slightly brownish material was separated by suction and, if necessary, again dissolved in 30 mL of dimethyl formamide and 30 mL of methanol, and precipitated with 400 mL of diethyl ether. From the crude product, the monooctenylated product was isolated by repeated column chromatography (20 cm x 5 cm i.d. column filled with 0.032-0.063 pm silica gel, ethanol/toluene 2: 1 (v/v) eluent). The column head was packed with coated silica gel obtained by dissolving the product in 60 mL of dimethyl formamide, adding 35 g of silica gel to the clear solution, and grinding after drying overnight at 0.1 torr and 60°C in a rotary evaporator. The purity was monitored using TLC (silica gel plates on a plastic support, Macherey- Nagel, Duren, Germany, detection with phenol/ concentrated sulfuric acid upon heating) and by ion-spray mass spectrometry (6 Reference [22]). R, values: 0-cyclodextrin, 0.10; monooctenylated product, 0.20; dioctenylated product, 0.27 (ethanol/toluene 2: 1 [v/v]); yield: 5.3 g (27 %) of monooctenylated product.

Preparation ofpermethylated monokis-6-(oct- 7- enyl)-fl-cyclodextrin. In an atmosphere of nitrogen, 7.0 g (290 mmol) of dry, 95% sodium hydride (Fluka, Buchs, Switzerland) were transferred into a four-necked round-bottom flask equipped with two dropping funnels, a nitrogen inlet, and a reflux condenser with a mercury valve. 7.0 g (5.6 mmol) of monokis-6-(oct-7-enyl)-~-cyclodextrin as prepared above were dissolved in 230 mL of anhydrous dimethyl formamide. Half of the solution was added via a dropping funnel to the sodium hydride, whereupon a vigorous reaction with evolution of hydrogen began. After the vigorous reaction had ceased, 13.6 mL (218 mmol) of methyl iodide were slowly added through the other dropping funnel at a bath temperature of 20°C. After stirring for 30 min, the second half of the cyclodextrin and another 13.6 mL aliquot of the methyl iodide were added to the reaction mixture. After stirring for 1 h, the reaction mixture was carefully poured into 500 mL of ice water. The aqueous phase was extracted three times with 300 mL of diethyl ether. The combined ether extracts were washed three times with 40 mL of water to remove residual dimethyl formamide and were

J . Microcolumn Separations, Vol. 5, No. 1, 1993 13

dried over anhydrous sodium sulfate. Removal of the solvent and drying in vacuo at 0.01 torr and 60°C yielded 6.9 g (80%) of the white, solid product which did not require further purifica- tion. The purity was checked by TLC (toluene/ethanol 4: 1 [v/v], R, values: 0.45 [permethyl-/3- cyclodextrin], 0.51 [permethylated mono-octenylated product], 0.53 [permethylated dioctenylated product], detection with phenol/concentrated sulfuric acid upon heating] and by ion- spray mass spectrometry (Figure 1, 6 Reference [22]). 'H-NMR (pprn, 400 MH, CDCl,): 5.78 (olefinic CH-group), 5.10 (ano- meric H), 4.97 and 4.92 (2 multiplets, olefinic CH2-group), 3.58 (singlet, OMe2), 3.44 (singlet, OMe3), 3.31 (singlet, OMe6). 13C-NMR (pprn, 400 MHz, CDC13): 139.0 (C7 of

0 1

1532 A

2 Octenyl groups

1562 1628 1 5 4 8 e & ^ .1 *

1400 1500 1600 1700

mlz

Figure 1. Zon-spray mass spectra ofpermethylated monokis-6-(oct- 7- enyl)-/3-cyclodextrin (M = 1,526) (in the presence of LiCl). (A) pure product and (B) product used in this work for the synthesis of Chirasil-Dex. The spectra show the p e a h belonging to (M + Li)' and (at low intensityl to other adducts. No undermethylated products and only small amounts of products with 0 or 2 octenyl groups are present.

octenyl group), 114.1 (C8 of octenyl group), 61.5 (OMe3), 58.9 (OMe6), 58.4 (OMe2).

Preparation of Chirasil-Dex by hydrosilylation (stationary phase 4). In an atmosphere of nitrogen, 0.40 g (0.262 mmol) permethylated monokisd- (oct-7-enyl)-/3-cyclodextrin, dried in vacuo at 0.05 torr and 60°C overnight, 1.02 g (ca. 0.34 mmol) dimethylpolysiloxane (MW = 3,000) containing 10% Si-H groups [23,24], and 40 mL anhydrous toluene were placed in a three-necked round-bot- tomed flask equipped with a nitrogen inlet and a reflux condenser fitted with a mercury valve. To the refluxing solution was added a solution of the hexachloroplatinic acid ( - 0.5 mg) catalyst in 1 mL of anhydrous tetrahydrofuran in several portions over 24 h. Subsequently, the solvent was removed in a rotary evaporator. Unreacted cyclodextrin was removed by filtration of a solution of the product in n-pentane. The product was washed three times with ca. 5 mL of anhydrous methanol. Centrifuga- tion was used to accelerate reprecipitation. The residue was dissolved in 15 mL of dichloromethane, and methanol was slowly added until the first droplets (rich in catalyst) formed. Then the solution was quickly decanted. This procedure was repeated two times, and the product was then filtered through a 0.5 micron filter and dried in vacuo at 60°C and 0.05 tom. The yield was 0.7 g [a]g + 37.7 (c = 0.8 [CH2C12]). 'H-NMR (ppm, 400 MH, CDC1,): in addition to the cyclodextrin

signals mentioned above (the olefinic signals disappeared), -0.15 to +0.25 (SiMe-groups).

The other described types of Chirasil-Dex were prepared analogously except that for stationary phase 5, some drops of water had to be added to the methanolic phase in order to support the precip- itation of the product which was subsequently dissolved in dichloromethane and dried over magne- sium sulfate.

Instrumentation. Carlo-Erba gas chromato- graphs (Carlo-Erba/Fisons, Milan, Italy), Frac- tovap 2150 and 2350, VEGA and MEGA, equipped with FID and suitable for operation with fused silica open tubular columns, were used. The carrier gas was hydrogen (99.999 %), and the split ratio was 1: lOO. 25 m x 0.25 mm i.d. capillaries were operated at an inlet pressure of 1.0 bar. Retention times and peak widths were determined using a Shimadzu C-R3A integrator. Net retention times were measured from the methane peak. All measurements were performed at least three times.

Preparation of open tubular columns. Fused silica tubing (25 m x 0.25 mm i.d., Chrompack, Middelburg, The Netherlands) was heated at 260°C for 2 h at a low hydrogen flow (0.2 bar inlet pressure). The columns were coated without further deactivation with carefully filtered 0.4 % solutions of Chirasil-Dex in diethyl ether by the static meth- od, yielding a film thickness of -0.25 pm. A commercial 25 m x 0.25 mm i.d. fused silica


column coated with 0.25 pm permethylated p - cyclodextrin in OV-1701 (CP-cyclodextrin-2,3,6- M-19, Chrompack) was used. (Much care should be taken not to expose the columns to air while hot or during storage.)

Immobilization. Where immobilized stationary phases were used, immobilization was accomplished thermally for 20 h at 190°C in a very slow flow of hydrogen, as described previously [25-271.

RESULTS AND DISCUSSION Five stationary phases have been used for this

work: (1) 9.1% (0.07 molal) permethylated 0- cyclodextrin in OV-1701 ("dissolved system") and (2-5) four types of Chirasil-Dex differing in the cyclodextrin weight percentage and the percentage of Si(CH3)H units contained in the polydimethylsiloxane used for the hydrosilylation (Table I). The cyclodextrin weight percentage in Chirasil-Dex can be determined by polarimetry in comparison to the pure chiral selector, i .e., permethylated monokis-6-(oct-7-enyl)-P- cyclodextrin ([a]g + 149 [CH2C12, c = 0.81). The comparison of these values to the weight percentages used for the 'reaction mixture indicates that more than 95 % of the cyclodextrin has been bonded. It should be noted that all calculated weight percentages have been corrected in order to refer to permethylated 0-cyclodextrin rather than to the octenylated derivative, i .e., 7 carbon atoms out of the octamethylene spacer are not assigned to the cyclodextrin, but to the polysiloxane backbone.

Synthesis. We have recently reported on the advantages of Chirasil-Dex in general and of the octamethylene spacer as compared to tri- or pentamethylene spacers in particular [12]. In order to further improve the reproducibility of the Chirasil-Dex synthesis, the synthetic procedure was modified as described in the Experimental section to yield the chiral selector, i . e . , permethylated 0-cyclodextrin with only one octenyl group in the 6-position, in highly improved purity.

Table I. Stationaryphases used in this work (CJ: remarlcr in the text).

-CD% Stationary Si-H content phase (%)

1 9.1 2 5 +24.6 15.5 3 5 +38.8 24.5 4 10 +37.7 23.5 5 10 +57.6 36.3

Table II. temperatures used in this work.

Racemic solutes and corresponding oven

45oc A B C

OH OH

E F 7OoC D Trans-pinane s.Q. H O X

0

85OC G H I Cis-Pinane 6 OH rTMd OH

1 oooc K L M

13OOC R S T U DL v-Nonalactone

PhAOH G r ' h 0 phx

The ion spray mass spectrum (Figure 1B) shows the approximate purity as over 90%, compared to only ca. 40 % for the product described previously [9,15]. We found, however, no change in the properties of the resulting Chirasil-Dex stationary phases, as there seems to be no measurable contribution to the cross-linking properties of the approximately 30 % dioctenylated cyclodextrin contained in previous batches of Chirasil-Dex.

Ph = phenyl; Et = ethyl.

Dependence of the enantioselectivity on the cyclodextrin concentration. A frequently discussed issue is the optimal concentration, or weight percentage, of the cyclodextrin derivative in the stationary phase and its influence on enantioselectivity. In a previous study using the dissolved phase [22], i .e., permethylated P-cyclodextrin in OV- 1701, we found that plots of the chiral separation factors Q vs. the cyclodextrin molality m in the

J . Microcolumn Separations, Vol. 5 , No. 1, 1993 15

Figure 2. Charac- teristic chromato- grams of solute N on stationary phases 1 , 2, 4, and 5 (A, B, C, and D) exhibiting an increasing cyclodextrin weight percentage (9, 16, 24, and 36%). Conditions: (left) identical conditions and (right) increasing temperatures in order to approximately compensate increasing retention times from A to D. 25 m x 0.25 mm i.d.

fusedsilica columns, 0.25 pm film thickness, not immobilized; 1.0 bar H2 inlet pressure.

Pulegone Qo

Constant temperatures

Constant retention times

1.047

1.046

1.055

1.049

1.041

(I = 1.024

1.040

(I = 1024 Ji 4 8

Min

OI

- 110

u 7

Min

4 4 0

L 12 0

Figure 3. Char- acteristic chroma- tograms of solute S on stationary phases 1, 2, 4, and 5 (A, B, C, and D) exhibiting an increasing cyclodextrin weight percentage (9, 16, 24, and 36%). Conditions: (1 e f t ) ident ica 1 conditions and (right) increasing temperatures in order to approximately compensate increasing retention times from A to D. Conditions: 25 m x 0.25 mm i. d. fused silica columns, 0.25 pm

fZm thickness, not immobilized; 1.0 bar H2 inlet pressure.

dPh Constant Constant temperatures

1.127

1.106

/ retention times ox'o 1.

1.151 1.104 1.1 25

u = 1.065

I z 3 0 0

130 'C

OC

D

, . - ' .i ." .." . . . . . . . . . . .

i ' d ' b 0 L Min Min


Scheme I. Equilibria according to the model of the retention increase R’.

B(g) Mobile phase

Stationary B(,) + A & AB phase

. . . . . . IFLO ..................................... containing cyclodextrin A

stationary phase leveled off at higher molalities or cyclodextrin weight percentages, i. e . , an increase (e.g. , from 5 to 7 %) results in much more improvement in a than an increase from 10 to 12%. We further found that the curves are not similar for all chiral solutes, but are directly related to their complexation strength, as expressed by the retention increase, R’. These observations could be perfectly explained by the concept of R’, which is based on the assumption of two independent equilibria ( c - Scheme I): (1) a physical dissolution of the solute in the stationary phase and (2) an independent chemical interaction with the cyclodextrin. The apparent partition coefficient between the mobile and stationary phase is given by

K, = K:(1 + K m )

where m is the cyclodextrin molality and K is the thermodynamic equilibrium constant. The product (K m) is called R’ (or chemical capacity factor), accessible from relative retention data. R’ is a measure for the strength of the chemical interaction between solute and cyclodextrin. A consequence of Equation 1 is that 01 is related to the cyclodextrin molality m, or weight percentage, over R’ of the enantiomers R and S by

(2) R ’ R + 1 K R m + 1

R’, + 1 K s m + 1 - f f = -

Thus, for weakly complexing solutes (small R’) high cyclodextrin molalities or weight percentages are more important than for strongly complexing solutes, and the entire (Y vs. m curve can be pre- dicted if R’ of the enantiomers R and S is known at only one m [19].

Here we are extending our model to Chirasil- Dex stationary phases, where much higher cyclodextrin weight percentages can be achieved than previously possible for permethylated cyclodextrin as a chiral selector. (There have been recent reports on the use of a solution of as much as 30 % permethylated P-cyclodextrin by weight in OV-

Table DI. Chromatographic data for solutes A-U on the nonimmobilized stationary phases 1-5.

Phase

1 2 3 4 5

Separation factors aa A B C D E F G H I K L M N 0 P Q R S T U

1.009 1.044 1.023 1.020 1.033 1.025 1.059 1.021 1.015 1.085 1.000 1.138 1.025 1.032 1.025 1.032 1.048 1.066 1.014 1.017

1.015 1.054 1.035 1.035 1.036 1.034 1.073 1.027 1.017 1.122 1.029 1.200 1.042 1.045 1.032 1.047 1.066 1.104 1.031 1.028

Kovats indices Ib A B C D E F G H I K L M N 0 P Q R S T U

957 9 85

1055 1041 1048 1212 1066 1198 1254 1069 1105 1213 1366 1341 1361 1379 1318 1410 1599 1602

948 971

1033 1008 1050 1174 1067 1169 1226 1041 1056 1200 1302 1320 1338 1357 1277 1349 1470 1532

1.018 1.049 1.039 1.040 1.041 1.042 1.085 1.027 1.018 1.154 1.030 1.206 1.048 1.044 1.037 1.052 1.069 1.121 1.033 1.030

972 989

1048 1029 1072 1195 1090 1193 1246 1041 1076 1226 13 12 1345 1363 1381 13 12 1370 1497 1566

1.019 1.064 1.039 1.038 1.042 1.043 1.088 1.030 1.019 1.164 1.03 1 1.212 1.049 1.045 1.037 1.051 1.077 1.131 1.033 1.033

977 998

1049 1044 1073 1198 1093 1209 1251 1051 1089 1240 1324 1353 1373 1391 1329 1379 1505 1568

1.021 1.065 1.039 1.035 1.044 1.045 1.093 1.025 1.022 1.193 1.033 1.217 1.055 1.047 1.039 1.051 1.084 1.152 1.037 1.034

990 1010 1057 1063 1083 1211 1104 1229 1265 1055 1103 1260 1336 1373 1393 1412 1362 1399 153 1 1597

a+ 0.002. b73, for the second eluted enantiomer.

J . Microcolumn Separations, Vol. 5 , No. 1, 1993

I-. H

17

I

1

1

a 1

1

1

strong ._ M .20 - ./--.

0 10 20 30

% Permethyl-/3-CD

Figure 4. Plots of separation factors a vs. cyclodextrin weight percentage (where 7 out of 8 carbon atoms of the spacer are not counted with the cyclodextrin) f o r solutes M , K, S, and G (exhibiting strong or weak complexation, CJ: text) on stationary phases 1, 2, 4, and 5 (data from Table HI).

1.08 1 1.06

a 1.04

1.02

1 .oo 0 10 20 30

% Permethyl-P-CD

Figure 5. Plots of separation factors a vs. cyclodextrin weight percentage (where 7 out of 8 carbon atoms of the spacer are not counted with the cyclodextrin) for solutes R, B, N , Q, and E on stationary phases 1, 2, 4, and 5 (data from Table III).

1701 [28,29], but according to our experiments [30], film stabilities, coating properties, and mini- mal operating temperatures are extremely unfavor- able under such conditions.) The previously reported Chirasil-Dex [7,12] corresponds to phase 2. Unfortunately, R' cannot be determined for Chira- sil-Dex since an achiral reference column is not available. Yet, it can be assumed that the solutes exhibit a qualitatively comparable complexation behavior in the dissolved system and on Chirasil- Dex because the same chiral selector is used.

In the present study, 20 chiral solutes have been selected (Table II), belonging to different

1.04

1.03

[I

1.02

1.01

1 .oo 0 10 20 30

% Perrnethyl-/3-CD

Figure 6. Plots of separation factors a vs. cyclodextrin weight percentage (where 7 out of 8 carbon atoms of the spacer are not counted with the cyclodextrin) for solutes F, C, U, I , and A on stationary phases 1, 2, 4, and 5 (data from Table III).

1500 1-w I

llool-* 1000

Phase

Figure 7. Kovats indices on stationaryphases 1-5 (data from Table III).

classes of compounds and exhibiting separation factors ranging from poor to excellent, and both strongly and weakly complexing solutes have been included. The dependence of the chiral separation factors on the cyclodextrin weight percentage is given in Table I11 and shown in Figures 2-6. The results are in full agreement with the trends observed in our previous study [19] for the low cyclodextrin weight percentages obtainable in the dissolved system. For the strongly complexing solutes M and G (R' for the second eluted enantiomer on the dissolved system equal to 1.5 for both [19]) no further increase in a is observed above - 25 %, whereas for the very weakly complexing solutes K and S (R' = 0.1 and 0.2) a slight


increase is noticed even above 30 % . For the other solutes investigated, these different shapes of curves can also be seen. Keeping in mind that a higher cyclodextrin weight percentage also leads to longer retention times [19], percentages above 30% are generally not useful. In Figures 2 and 3, where these longer retention times are approximately com- pensated by increasingly higher analysis temperatures when going from phase 1 to 5, it can be seen that stationary phase 5 does not yield a better enantiomeric separation than phase 4 in a given time, even for the weakly complexing solutes shown.

These results are not necessarily in contrast to those found by Schmarr et al. [31], who found an increase in resolution of y-lactones on "hexakis(2,6 + l-di-n-pentyl-3- acety1)-a-cyclodextrin" over the entire concentration range up to 100%. The very high molecular weight of the pentylated cyclodextrin derivatives has to be considered, i .e., the selector is already diluted by almost half of its weight by the long alkyl chains which are not very likely to participate in chiral recog- nition. In addition, R', i.e., the complexation strength, might be smaller than reported here for permethylated P-cyclodextrin. K6nig [32] recently also found that undiluted pentylated cyclodextrin derivatives do not exhibit significantly higher enantioselectivities than the 1:l mixtures with OV-1701, but the latter have much better coating properties and film stabilities.

Eflciency and resolution. No systematic decrease in column efficiency (effective theoretical plates per meter) is observed at an increasing cyclodextrin percentage, although generally the cyclodextrin stationary phases rearely exhibit more than 3,000 eflective theoretical

Table IV. Chromatographic data for solutes K-U on the nonimmobilized stationaiy phases 3 and 4, demonstrating column aging.

Phase 3 Phase 4

After After After After New 7 days 7 days New 3 days 5 days

(150°C) (220°C) (190°C) (220°C)

Separation factors aa K 1.168 1.154 L 1.032 1.030 M 1.216 1.206 N 1.048 0 1.044 P 1.037 Q 1.052 R 1.076 1.069 S 1.136 1.121 T 1.036 1.033 U 1.033 1.030

Capacity factors kb K 4.05 3.58 L 5.25 4.53 M 14.96 12.51 N 13.15 0 16.21 P 18.18 Q 20.43 R 6.34 5.35 S 8.61 7.41 T 17.64 15.04 U 26.31 22.11

Kovats indices Ic K 1042 1041 L 1079 1076 M 1230 1226 N 13 12 0 1345 P 1363 Q 1381 R 1316 1312 S 1368 1370 T 1491 1497 U 1559 1566

1.147 1.029 1.211 1.045 1.042 1.037 1.052 1.066 1.115 1.03 1 1.028

2.90 3.59 9.81

11.11 13.67 15.29 17.10 4.23 5.96

11.96 17.68

1040 1071 1217 1309 1341 1359 1376 1306 1367 1489 1558

1.164 1.031 1.212 1.049 1.045 1.037 1.051 1.077 1.131 1.033 1.033

3.15 4.08

11.66 12.12 14.65 16.62 18.77 4.63 6.19

12.95 18.71

1051 1089 1240 1324 1353 1373 1391 1329 1379 1505 1568

1.155 1.030 1.206 1.047 1.044 1.038 1.049 1.075 1.128 1.034 1.03 1

3.03 3.84

10.86 11.72 14.15 16.02 18.10 4.77 6.41

13.41 19.60

1050 1085 1239 1324 1353 1372 1392 1329 1381 1510 1576

1.144 1.029 1.192 1.044 1.043 1.035 1.054 1.066 1.117 1.03 1 1.03 1

2.44 3.10 8.42 9.16 1.02 2.36 3.91 3.57 4.88 9.89 4.38

1046 1082 1233 1321 1351 1369 1388 1323 1379 1506 1573

a & 0.002. It2 % , for the second eluted enantiomer.

plates per meter for chiral solutes which can be separated into enantiomers. However, there are fluctuations from one individual column to another. Since the a values are not affected by efficiency,

they are independent from experimental parameters such as column length, carrier gas flow rate, quality of the coating, or any peak broadening due to instrumental problems, and can thus be very

J. Microcolumn Separations, Vol. 5, No. 1, 1993 19

precisely determined. For this reason, a rather than the resolution Rs is used as a measure for enantioselectivity.

Kovats indices. Kovats indices [33] are com- monly used to characterize the polarity and retention behavior of stationary phases. For cyclodextrin stationary phases, where retention time depends on a physical and a chemical contribution [19], the Kovats indices for the stationary phases 1 to 5 (Table 111, Figure 7) are also influenced, as expected, by the two contributions. Going from phase 1 to 2 and, to a much lesser extent, from phase 4 to 3, the decrease in the polarity of the polysiloxane leads to decreases in the Kovats indices for the polar solutes, whereas for the nonpolar solutes, such as alkanes E and G , there are very small changes. The influence of the cyclodextrin can be seen when going from phase 2 to 3 and from 4 to 5. These increases in the Kovats indices are not governed by overall polarity, but by increased complexation to, or inclusion into, the cyclodextrin. Thus the weakly complexing solute K is influenced very little, whereas the indices for the strongly complexing solutes G and M increase noticeably.

It has to be recalled that the chromatographic behaviors of the n-alkanes are also influenced by an increasing cyclodextrin concentration [ 191. Thus the capacity factors k of the n-alkanes continuously increase by 10 to 30% from phase 2 to phase 5. Obviously, even for n-alkanes the complexation and inclusion properties of the cyclodextrin are much more important than any overall polarity increase of the stationary phase produced by an increasing cyclodextrin percentage. Otherwise the k values of the n-alkanes and the Kovats indices of the alkane solutes E and G should decrease rather than increase.

Column aging. Nonimmobilized Chirasil-Dex capillary columns are subject to considerable deterioration when kept at elevated temperatures for extended periods of time (Table IV, Figure 8). In addition to the decreases in k and the a, the column efficiency decreases by about 10 to 40% when the column is kept for 1 week at 220°C. The Kovats indices also decrease slightly.

Immobilization. Immobilization does not only allow the rinsing of a column in case of contamination, but also greatly improves the long-term stability of the retention behavior and column performance of the Chirasil-Dex stationary phases (Tables V-VI, Figure 9). The use of a better quality carrier gas (oxygen-free) might further improve the long-term stability at high temperatures. Immobilization is accomplished thermally

Table V. Chromatographic data for solutes K-U on stationary phase 3 during and after immobilization.

Phase 3

Before After After After After immob. irnmob. rinsing 3 days 3 days

(19OOC) (220°C)

Separation factors ora K 1.159 1.161 L 1.032 1.033 M 1.206 1.208 N 1.048 1.049 0 1.047 1.045 P 1.040 1.038 Q 1.052 1.056 R 1.069 1.071 S 1.122 1.125 T 1.033 1.035 U 1.033 1.031

Capacity factors kb K 3.99 4.03 L 5.08 5.14 M 14.12 14.39 N 14.63 14.83 0 18.33 18.53 P 20.47 20.76 Q 22.99 23.39 R 5.57 5.69 S 7.66 7.77 T 15.10 15.52 U 22.47 23.04

Kovats indices I' K 1038 1040 L 1074 1076 M 1224 1228 N 1309 1312 0 1344 1347 P 1362 1365 Q 1380 1383 R 1311 1313 S 1367 1367 T 1487 1489 U 1558 1559

1.178 1.033 1.216 1.052 1.047 1.040 1.057 1.076 1.137 1.036 1.034

2.53 3.38 9.66 9.51 12.14 13.65 15.41 3.69 4.86 9.90 14.71

1043 1085 1240 1318 1356 1374 1393 1328 1376 1501 1570

1.188 1.033 1.220 1.054 1.049 1.041 1.058 1.076 1.142 1.037 1.035

2.49 3.40 9.84 9.64 12.45 13.99 15.79 3.76 4.89 9.98 14.88

1044 1090 1248 1322 1362 1380 1399 1334 1380 1506 1576

1.195 1.034 1.217 1.056 1.049 1.041 1.060 1.076 1.143 1.036 1.035

2.41 3.32 9.53 9.32 12.02 13.51 15.28 3.63 4.70 9.67 14.36

1043 1090 1245 1329 1359 1377 1396 1335 1381 1508 1577

~~~~

a+ 0.002. bk2%, for the second eluted enantiomer. '+3, for the second eluted enantiorner.

since radical immobilization thus far resulted in an almost complete loss of enantioselectivity [25,34]. This is monitored by measuring the k values of some solutes during and after the immobilization process. The degree of immobilization, as given by

20

5-

0:

J. Microcolumn Separations, VoI. 5 , No. 1 , 1993

S I R I

L K I

9 I

I -4 1 1 - + '

a I

Figure 8. Column aging with respect to separation factors 01

and capacity factors k for solutes K-U on the nonimmobilized stationary phases 3 and 4 (data from Table ZV).

a

k

Figure 9. Capacity factors k for

r)

R

i solutes K-U on stationary phases 3 and 4 during and after immobilization (data from Tables V and W). (The separation factors 01 remain constant or increase slightly.)

15

10

before after I after after 3 immobili- immobili- I rinsing days at zation zation I 190 o c

rinsing

the k values before and after rinsing, was measured as 64 % for phase 3 (75 % in another experiment) and 89 % for phase 4. As expected and reported by Schomburg et al. [14] for other chiral polysiloxanes, immobilization is easier when the polysiloxane used for the synthesis contains more Si-H groups. However, care has to be taken not to "over-immobilize" the stationary phase since in that case cross-linking continues even after the rinsing of the column, and the k values further decrease.

after 3 days at 220 oc

Although immobilization seems to be supported by a stoichiometric reaction with the remaining hydrosilylation catalyst H,PtCl,, we use less catalyst than described by Schomburg et al. [ 171 in order to avoid the resulting metal contamination and the excessively high viscosity of the stationary phase. (A high level of platinum may lead to decomposi- tion phenomena for, e.g. , cY,P-unsaturated ke- tones.) During and after immobilization, the column efficiencies do not significantly decrease

J. Microcolumn Separations, Vol. 5, No. 1, 1993 21

Table VI. Chromatographic data for solutes K-U on stationary phase 4 during and after immobilization.

Phase 4

Before After After After After immob. immob. rinsing 3 days 3 days

(19OOC) (220°C)

Separation factors aa K 1.168 1.169 L 1.031 1.033 M 1.208 1.210 N 1.048 1.051 0 1.046 1.045 P 1.039 1.040 Q 1.052 1.052 R 1.076 1.074 S 1.135 1.132 T 1.035 1.042 U 1.033 1.033

Capacity factors kb K 3.50 3.35 L 4.61 4.40 M 13.14 12.59 N 13.29 12.87 0 16.73 16.34 P 18.85 18.35 Q 21.25 20.78 R 5.26 5.09 S 7.03 6.75 T 14.35 13.75 U 21.24 20.35

Kovats indices I' K 1044 1044 L 1084 1084 M 1237 1238 N 1318 1318 0 1354 1355 P 1372 1373 Q 1391 1393 R 1326 1327 S 1376 1377 T 1500 1502 U 1568 1571

1.170 1.032 1.213 1.052 1.046 1.039 1.054 1.075 1.134 1.036 1.033

3.09 4.11

11.67 11.83 14.83 16.71 18.82 4.50 6.01

12.24 18.04

1043 1085 1238 1320 1355 1374 1392 1327 1377 1502 1570

1.179 1.034 1.215 1.052 1.047 1.041 1.057 1.075 1.137 1.036 1.033

2.97 4.00

11.35 11.37 14.40 16.21 18.29 4.34 5.76

11.77 17.40

1043 1086 1238 1319 1355 1374 1392 1328 1377 1502 1570

1.178 1.033 1.214 1.052 1.047 1.040 1.057 1.074 1.136 1.035 1.033

2.82 3.80

10.67 10.81 13.71 15.41 17.37 4.09 5.44

11.10 16.40

1043 1087 1238 1320 1357 1375 1393 1328 1378 1502 1570

Table VII. Chromatographic data for solutes R-U on a blended stationaryphase (g. text) corresponding to stationaryphase 4 during and after immobilization.

"Pseudo-phase 4"

Before After After After immob. immob. rinsing 5 days

(220°C)

Separation factors ola

R 1.077 1.078 1.077 1.078 S 1.137 1.140 1.144 1.144 T 1.036 1.036 1.036 1.037 U 1.033 1.034 1.035 1.035

Capacity factors kb R 5.84 5.88 4.76 4.58 S 7.81 7.80 6.25 5.96 T 16.07 15.97 12.83 12.16 U 23.86 23.92 19.09 18.26

Kovats indices R 1325 1329 1334 1336 S 1375 1378 1380 1382 T 1500 1503 1504 1505 U 1568 1573 1573 1576

a+ 0.002. b+2%, - for the second eluted enantiomer. '+3, for the second eluted enantiomer.

deterioration process described above for the nonimmobilized columns.

Mixed stationary phases. The synthesis of several batches of Chirasil-Dex demonstrated that the reproducibility is quite good with respect to optical rotation [a]k0 (+1) and content of Si-H groups in the polysiloxane backbone used for the hydrosilylation. Still it may sometimes be desirable to modify these properties after synthesis, which can be accomplished by blending of polysiloxanes. Thus a stationary phase corresponding to phase 4 has been prepared by mixing appropriate amounts of polysiloxanes with optical rotations of +25 and +57 which were themselves prepared from blends of polydimethylsiloxanes containing 8 % and 20 % Si-H groups. The resulting stationary phase (Table VII) exhibits very similar qualities as the pure stationary phase 4 described above, including column efficiency and immobilization properties.

As a consequence of the presented data, we consider immobilized Chirasil-Dex containing 25- 30 % permethylated P-cyclodextrin and prepared from a polydimethylsiloxanewith 10 % Si-H groups

a+ 0.002. b+2%, for the second eluted enantiomer. 'f3, for the second eluted enantiomer.

(around 10 %). Generally, immobilization does not measurably affect the polarity of the stationary phase, and the Kovats indices on immobilized and nonimmobilized phases are very similar. The Kovats indices and CY values increase slightly during and after immobilization, in contrast to the * - - -


the most useful type of Chirasil-Dex in terms of enantioselectivity, immobilization properties, and long-term stability.

ACKNOWLEDGMENTS This work has been supported by Deutsche

Forschungsgemeinschaft and by Fonds der Chemis- chen Industrie. We wish to thank Dr. J. Metzger for recording the ion-spray mass spectra.

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Received: August 17, 1992 Accepted: November 4, 1992

enantiomeric separation by gc on chirasil-dex: systematic study of cyclodextrin concentration,...

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