Anal. Chem. 1992, 64, 873-879 (5) Blackwell, J. A. Ph.D. Thesis, University of Minnesota, 1991. (6) M,W. Lengmuk 1989,5,96-100. (7) Bolls. V.; Mma. C.; Volante, M.; orlo, L.; Fubini, B. hngmuir 1990, 6, 695-701. (8) Marterra, C.; Giameib, E.; &io, L.; Volante, M. J . Fhys. Chem. 1990, 94. 3111-3116. (9) M e r r a , C.; Aschieri, R.; Volante, M. Meter. Chem. Fhys. 1988,20, 539-557. (IO) Rsgauoni. A. E.; Blesa, M. A.; Maroto, A. J. 0. J . CdlOM Interface SCl. 1989, 91, 560-570. (11) Regauoni, A. E.; Blesa, M. A.; Maroto. A. J. G. J . Co/bH Interface SCi. 1988, 122, 315-325. Martell, A. E. Stability Consfants of Mefal Ion Compkxes; (12) Sillen, L. 0.; The Chemical Society: London, 1984.
873
(13) Parks, G. A. Chem. Rev. 1965, 65, 177-198. (14)Rigney, M. P. Ph.D. Thesis, University of Minneosta, 1988. (15) Schafer, W. A. MS Thesis. University of Minnesota, 1990. (16) Schafer, W. A.; Funkenbusch, E. F.; Parson. K. A.; Can,P. W. J . ChrOt?lafogr. 1991, 587, 137-147. (17) Schafer, W. A.; Carr, P. W. J. Chromtogp. 1991, 587, 149-160. (18) Rigney. M. P.: Funkenbusch, E. F.; Carr, P. W. J . Chrometog. 1990, 499.291-304. (19)Pearson. R. G.J . Chem. Educ. 1968,45,581-587. (20) Pearson, R. 0.J. Chem. Educ. 1968,45, 643-648.
RECEIVED for review August 26,1991. Accepted January 21, 1992.
Multiple Enantioselective Retention Mechanisms on Derivatized Cyclodextrin Gas Chromatographic Chiral Stationary Phases Alain Berthed,' Weiyong Li) and Daniel W. Armstrong* University of Missouri-Rolla, Department of Chemistry, Rolla, Missouri 65401
2,6-DCO-pentyi-3-O-(t~acetyl) (DP-FA) derlvatired 8and ycyciodextrins are able to resolve a wlde variety of volatlie racemlc compounds. The enantiomeric recognition mechanism d these phases was Investigated. retention behavlor of homolagour series showed that iengthenlng the side alkyl chain increases the retention t h e but does not affect enantiordectlvky. The thermodynamic parameters, free energy, enthalpy, and the difference in free energy, enthalpy, and entropy between enantbmers were evaluated for 24 onantlomerlc pairs. From this data, It appoan that the compounds can be arranged in two groups. One group has high values for enthalpy, entropy, free energy, and the corm p o n c l l n g ~ ~ p a r a m e t e r s b d w e e n e nlhe a~. second roup has algnkantly bwer v a h for a i parameters. I t k shown that compounds belonging to the second group follow an enthalpy-cHltmpy compwmth regreadon (In k'vs A H ) while the group Icompounds do not. Also on a given column the mass capadty for group I 1 compounds k slgnifkantly hlgher than that for group I compounds. A mail dmerence was bund In the mass trader behavbr d the two groups of compounds. I t Is believed that there may be at least two ditferent chiral recognition mechanisms with the derlvatired cyclodextrin gas chromatographlc stationary phases. I t is postulated that one mechanism Involves cyclodextrin (CD) inclusion complex formatlon and the other doas not. Currently, more enantlomers seem to resolve through external or multiple amociatlon than through an Inckrkn process. The fact that ckrhratlzed CD drkai statbnary phases can r d v e differentenantiomen vla dtfferent mechankmr ( a d probably by combination or intennediate mecha k m r ) provkkr the practical benefit of lncreaslng the num ber and types of compounds resolved on these columns.
INTRODUCTION For approximately 25 years, gas chromatography (GC) has GC is an accurate and been used to separate *Towhom all correspondenceshould be sent.
On leave from Laboratoire des Sciences Analyti ues, UA CNRS
435, Universite de Lyon 1, 69622 Villeurbanne Cejetex, France.
*
Current address: McNeal Pharmaceutical, McKean Road, Spring House, PA 18901. 0003-2700/92/0384-0873$03.00/0
reliable analytical method for the separation of chiral analytes that can be vaporized without decomposition. Its advantages include simplicity, speed, reproducibility, sensitivity, and ease of detection.4~~ All direct enantiomeric separations are done with chiral stationary phases (CSPs). Essentially three classes of GC-CSPs have been developed. There are: (i) amino acid derivative CSPs, such as Chirasil-Val: (ii) metal complex CSPs,Qand (iii) the derivatized cyclodextrin (CD) CSPS.~'O We used the experience gained in developing derivatized cyclodextrin CSPs for liquid chromatography"J2 to develop cyclodextrin derivative CSPs for gas chr~matography.'~J~J~ Amorphous CD derivatives, such as permethyl-0-(2hydroxypropyl)-CD~,'~J~ 2,6-di-O-pentyl-CDs,15and 2,6-diO-pentyl-3-(O-trifluoroacetyl)-CDs,10 were used to coat fused silica capillaries. Although there has been a considerable amount of speculation concerning enantioselective retention mechanisms on these CSPs, there have been few mechanistic studies. There are two factors in particular that seem to distinguish enantioseledivecyclodextrin-based GC separations from HPLC separationson cyclodextrin bonded phases. First, there seems to be far less analyte size selectivity in GC. Second, enantiomers with little functionality are easily resolved by GC but not by LC. Generally, LC separations require aromatic moieties and good hydrogen bonding groups all within reasonable proximity to the stereogeniccenter. Of course GC (unlike LC) need not account for solvent effects, which can be pronounced. The goal of this work is to investigate the enantiomeric recognition mechanism on the 2,6-di-O-penty1-3-0-(trifluoroacetyl) (DP-TFA) 8- and y-derivatized CD CSPs. The study was done in five steps: (i) The retention behavior of homologous series and the corresponding enantiomeric selectivities were studied. (ii) The thermodynamic parameters, free energy, enthalpy, and entropy of association between the enantiomers and the chiral stationary phase were measured. (iii) The thermodynamic parameters seem to indicate that there are a t least two different mechanisms. Hence these parameters were further used to make an enthalpy-entropy compensation analysis. (iv) The column capacity may be related to the retention mechanism. To investigate this possibility, column overloading experiments were performed with selected enantiomers. (v) The kinetics of the mass transfer was studied through Golay-Van Deemter plots for several enantiomeric pairs. 0 1992 American Chemical Society
874
ANALYTICAL CHEMISTRY, VOL. 64, NO. 8, APRIL 15, 1992
EXPERIMENTAL SECTION Cyclodextrin Derivative GC Phases. The nonpolar 2,6di-0-pentyl-3-(0-trifluoroacetyl) j3- and y-derivatized CD-CSPs (DP-TFA-@-CD and DP-TFA-y-CD)were prepared in two steps as previously published? Briefly, the f i t step involves formation of the dipentyl-CD preparation by a Williamson reaction. 1Bromopentane was reacted with the hydrated CD in a 3 M NaOH-DMSO solution. After purification,the dipentyl-CD was treated with trifluoroacetic anhydride in THF solution for 3 h, and the final product was purified as a clear viscous liquid. As previously shown for most derivatized cyclodextrins, the reaction product(s) is actually a mixture of homologues and isomer^.'^*'^ This was shown to be one of at least four factors that lead to "liquid-derivatized CDs" now used in GC.13J4 Gas Chromatography. Twenty-meter fused silica capillary columns (0.25-mm i.d.) were prepared by coating untreated capillary tubing obtained from Supelco (Bellefonte, PA); 0.2% w/v diethyl ether solutions of the derivatized CD were made for the coating. After filling the capillary with the ether solution, one end of the capillary was sealed and the other end was connected to a vacuum line. Ether was gently evaporated at a constant 36 "C t e m p e r a t ~ r e . ~ JThe ~ J ~derivatized CD mass content of the column is approximately 2 mg. If the derivatized CD is evenly depositated onto the tubing wall, the film thickness is approximately 0.13 bm. The efficiency of the columns was tested using dodecane. It should be higher than lo5 plates, otherwise the column was rejected. A Vtuian Model 3700 and a Shimadzu Model GC-8A gas chromatograph were used for all separations. Either flame ionization or electron capture detectors were utilized. The carrier gas, nitrogen, was used at velocities between 5 and 45 cm/s. The injector and detector were held at 200 "C. A split ratio of 100/1 was used for all columns. The injection volume was 0.5 pL. Before injection, all amines, alcohols, and diols were trifluoroacetylated by dissolving the analyte in diethyl ether and adding a large excess of trifluoroacetic anhydride. The solution was heated to a boil for several minutes, and the process was repeated until the reaction was complete.13-15 Chemicals. All chemicals were obtained from Aldrich Chemical Co. (Milwaukee,WI), Sigma Chemical Co. (St Louis, MO), or Fluka Chemical Co. (Ronkonkoma, NY). RESULTS AND DISCUSSION Several interactions between derivatized cyclodextrin CSPs and chiral analytes are possible. After derivatization, the DP-TFA-CDs are no longer good hydrogen bond donors as were the native underivatized cyclodextrins. Hydrogen bonding interactions cannot explain the variety of enantiomeric separations obtained with DP-TFA-CD CSPs. Many enantiomers resolved, e.g., hydrocarbons, halo hydrocarbons, esters, epoxide^,'^-'^ either contain no hydrogen bonding groups or contain only hydrogen bonding acceptor groups and/or have permanent dipole moments. Dipole-dipole interactions as well as other van der Waals interactions and dispersion forces, between analytes and derivatized CD molecules, are very likely important factors in chiral recognition. Inclusion complexation is another possible interaction between CSPs and solutes. Cyclodextrin inclusion complexation plays a major role in chiral recognition in LC."J2 Early on, Smolkova-Keulemansova and co-workers published GC evidence for the inclusion of nonchiral hydrocarbons in native cyclode~trins.~~J~ Retention Behavior of Homologous Series. Table I lists the retention data for three homologous series of compounds: amines, diols, both TFA derivatized, and alkyl esters of 2bromobutyric acid. For all of the compounds of these three series, there is a common structural characteristic, which is a relatively polar "head" and an apolar "tail". As is usually observed, at constant temperature, the retention times of each member of a homologous series increases exponentially with the molecular weight.19 However, the enantioselectivity factor does not change much with retention. For both CSPs and all three homologous series studied, nearly identical a values were obtained within a series regardless of the chain length or
Table I. Retention and Selectivity of Three Homologous Series on 2,6-Dipentyl-3-(trifluoroacetyl)cyclodextrinStationary Phases racemic compound" 2-aminobutane 2-amino-3,3-dimethylbutane 2-aminopentane 1,3-dimethylbutylamine 2-aminoheptane 1,5-dimethylhexylamine 1,2-propanediol 1,2-pentanediol 1,2-hexanediol 1.2-octanediol methyl 2-bromobutanoate
column temp, O C 90 90
90 90
90
90 70 70 70 70 80
80
ethyl 2-bromobutanoate
80
isopropyl 2-bromobutanoate n-butyl 2-bromobutanoate
80 80 80
n-pentyl 2-bromobutanoate
80 80
n-hexyl 2-bromobutanoate
80 80
80
k'b
a
1.55 2.10 2.40 2.80 8.15 12.15 1.61 3.50 6.64 29.3 5.07 6.71 5.25 9.93 6.57 15.5 21.5 32.3 39.5 66.4 80.0
1.14 1.22 1.22 1.22
1.22 1.22
1.49 1.23 1.23 1.23 1.56 1.57 1.29 1.16 1.08 1.16 1.09 1.16 1.08 1.16 1.09
stationary phase
B fl fl fl fl y y y Y
4 y
9, y
i
y
j3
i
y
"All amines and alcohols were resolved after trifluoroacetylation (see the Experimental Section). bFor the first eluted enantiomer.
branching of the "tail". In the amine-containing homologous series, the smallest member had a slightly smaller a value than the higher molecular weight analytes. In the other two series, the smallest members had slightly larger a values than the rest (Table I). It is reasonable to assume that for these homologous series: (a) only part of the carbon chain, one or two carbons, contributea significantly to chiral recognition because the a values of the larger homologues are independent of carbon chain length; (b) longer carbon chains affect the retention of the molecule but not the enantioseledivity; and (c) the size of the cyclodextrin can affect both the enantioselectivity (a)and retention. The functionality of the analytes may also play a role. Thermodynamic Parameters for Enantiomeric Resolution. To a first approximation, the difference in the Gibba free energy of association for an enantiomeric pair, A(AGo), can be estimated from the selectivity factor, a,by
-A(AG") = R T In a
(1)
The corresponding A(AH") and A(ASo) values can be obtained by measuring the a values of the same enantiomeric pair at different temperatures and plotting R In a versus 1/T:
R In a = -A(AH")/T
+ A(ASo)
(2)
If A(AHo) is a constant within the temperature range, a straight line should be obtained. The slope is A(AHo) and the intercept is A(ASo). The plots were always linear with regression coefficients higher than 0.995. Tables I1 and I11 list the thermodynamic parameters obtained on the two CSPs studied in decreasing order of the A(AHo) values. For selected compounds, we also listed the AHoRand AHos value corresponding to the R and S enantiomers and obtained from the In k'versus 1/T plots: -RT In k ' / 9 = AH' - TAS" (3) in which 4 is the mobile phase over stationary phase volume ratio. The differences AHoR - AHos were identical within experimental error to the A ( M 0 ) values obtained directly from eq 2. All eq 3 plots produced straight lines with regression coefficient higher than 0.998 (see Figures 1and 2). Enantiomerically pure standards are necessary to assign an R or S configuration to a compound represented by a chro-
ANALYTICAL CHEMISTRY, VOL. 84, NO. 8, APRIL 15, 1992
875
Table 11. Thermodynamic Parameters Measured by GC on DP-TFA-B-CDCSPa temp range, "C' minI max
-A(AGo),' cal/mol min max
50 70 60 60 50 70 60
100 110 100 100 90 100 100
740 730 290 290 550 210 270
80 70 2-(chloromethyl)tetrahydropyran 90 1,5-dimethylhexylamine 90 2-aminoheptane 70 2-(bromomethyl)tetrahydropyran 40 2-heptanol 70 n-hexyl2-bromobutanoate 70 n-butyl2-bromobutanoate 70 n-pentyl 2-bromobutanoate 40 2-pentanol trans-2,5-dimethoxytetrahydrofuran 40 40 2-octanol 40 2-hexanol 70 2-chlorocyclohexanone
120 120 130 130 120 90 120 120 120 70 70 90 70 100
360 220 180 180 190 150 150 140 150 180 230 150 150 52
racemic compound* methyl 2-chloropropanoate 2-chloroc yclopentanone sec-butyl2-bromobutanoate methyl 2-bromopropanoate 3-chloro-2-butanone n-hexyl2-bromopropanoate n-pentyl%-bromopropanoate 8-butyrolactone
Group I1 270 38 69 69 46 0 15 0 80 130 28 71 36
kcal/mol
kcal/mol
-A(ASO), cal/(mol.K)
TW,d "C
15.0
18.2
13.6
15.5
16.8
18.6
3.2 2.5 2.4 2.0 1.9 1.8 1.8
7.5 5.1 6.2 4.9 4.3 4.6 4.7
150 200 110 140 170 120 110
16.0 12.1 16.7 15.9
17.4 13.5 17.9 17.1
12.0 16.9 15.3 16.5
13.1 18.0 16.4 17.6
1.4 1.4 1.2 1.2 1.2 1.1 1.1 1.1 1.1 1.1 0.9 0.9 0.9 0.25
2.9 3.4 2.7 2.7 3.0 2.9 2.7 2.8 2.9 3.1 2.2 2.4 2.4 0.6
210 140 170 170 130 110 130 120 110
-AHOR,
Group I 370 530 97 110 380 70 84
0
kcal/mol
13.2
-AH"s,
- W H O ) ,
14.1
80 140 100 100 140
a 20-m wall-coated fused silica capillary columns, carrier gas nitrogen at 10-15 cm/s velocity, injection volume 0.5 p L with a 100/1 split ratio. The -AHos were used with the corresponding In k'values for enthalpy-entropy compensation studies. *All amines and alcohols were resolved after trifluoroacetylation (see the Experimental Section). cRetention and a values were measured at 10 "C intervals. Hence every van't Hoff plot contained a minimum of five data points. dTemperaturee reported to the nearest 10 O C .
'C 3
2
I
2.6
2.8
I
I
3.0
I
I 3.2
UT K ( x 1 ~ 3 ) Ffgure 2. Plots of In k'versus the reclplcol of temperature (In K) for the fkst eluted (0)and second eluted (0)enantlomers of methyl 2chloropropanoate (seeFigure 1 for experimental details).
0
10
20
M
TlflE, M I N F W e 1. Chromatograms showhrg the separatlm of racemic methyl
P-chloropropanoate at 80, 80, and 100 OC on a 20 M DP-TFA-PCD caplllary column (see the Experlmental Sectlon for further details).
matographic peak. Consequently, AHoR and AHos values could not be directly calculated for every compound in Tables I1 and 111. All A(AHo) and A ( h s o ) values were negative which means that an isoenantioselective temperature exists. The isoenantioselective temperature, Th,is the particular temperature
at which A(AGo) is nil with no enantiomer resolution. Using the Gibbs-Helmholtz equation -A(AGo) = -A(aHo) + TA(ASo) (4) the isoenantioselective temperature is expressed by Ti, = A(AH0)/A(ASo) (5) At temperaturea higher than T.,, the enantiomer elution order should be r e ~ e r s e d . ~ Tables ~ , ~ I1 and I11 list the theoretical Ti, temperatures obtained from eq 5. For most compounds T,, is far higher than the working temperature range. The disappearance of enantiomeric resolution at temperaturea close to the calculated Ti,waa observed for 2-heptanol, 2-bromobutyric acid butyl and pentyl esters on the DP-TFA-&CD phase and for 2-bromopropionic acid sec-butyl ester, 2bromobutyric acid butyl, pentyl, and hexyl eaters on the DP-TFA-7-CDphaae. However the elution reversal waa not observed. At 100 "C, on the 7-CD phaae, the two enantiomera
876
ANALYTICAL CHEMISTRY, VOL. 64, NO. 8, APRIL 15, 1992
Table 111. Thermodynamic Parameters Measured by GC on DP-TFA-r-CDCSP4
racemic compoundb
temp range, "C' min max
-A(AG"),' cal/mol min max
trans-2,5-dimethoxytetrahydrofuran 50 methyl 2-chloropropanoate 60 1,3-dibromobutane 60 styrene oxide 70 methyl 2-bromopropanoate 60 1,2-hexanediol 60 1,2-pentanediol 60 3-chloro-2-butanone 50 2-chlorocyclopentanone 80 2-hexanol 40
90 100 100 110 100 100 110 90 110 80
530 550 430 380 360 210 220 360 300 210
1,2-dibromobutane 2-pentanol 2-heptanol 2-bromoheptane 2-methylcyclohexanone j3-butyrolactone 2-chlorocyclohexanone n-butyl 2-bromobutanoate 2-octanol n-hexyl 2-bromobutanoate n-pentyl 2-bromobutanoate sec-butyl 2-bromobutanoate n-hexyl 2-bromopropanoate n-pentyl2-bromopropanoate
100 80 90 90 100
90 160 120 120 120 150 120 60 80 54 54 39 59 39
60 40 50 50 60 60 80 80 50 80 80 60 70 60
100
110 120 90 120 120 100 100
100
Group I1 30 60 35 36 36 110 72 0 30 0 0 0
-AHo& kcal/mol
12.0 14.7 14.2 15.4
15.0 17.4 16.7 17.7
16.1 15.2 12.5 15.5
17.8 16.9 14.0 16.9
13.5
14.4
12.4
13.1
14.0 15.2 16.6 15.8 13.3 16.8 16.4
14.7 15.8 17.2 16.3 13.8 17.3 16.9
- M O R ,
Group I 210 300 180 160 180 43 43 210 200 67
36 29
kcal/mol
-A(MO), -A(AS"), kcal/mol cal/ (mo1.K)
Tim? O C
3.1 2.7 2.5 2.3 1.8 1.7 1.4 1.5 1.4 1.4
8.0 6.2 6.3 5.4 4.4 4.3 4.5 3.6 3.2 3.7
110 160 120 150 140 120 100 140 160 110
0.9 0.9 0.8 0.77 0.7 0.6 0.6 0.5 0.5 0.5 0.5 0.45 0.3 0.15
2.4 2.4 2.2 2.1 1.8 1.3 1.4 1.4 1.3 1.3 1.3 1.3 0.7 0.4
100 100 90 90 120 190 160 80 110 110 110 70 160 100
a 20-m wall-coated fused silica capillary columns, carrier gas nitrogen at 10-15 cm/s velocity, injection volume 0.5 pL with a 100/1 split ratio. The -Mas were used with the corresponding In k'values for enthalpy-entropy compensation studies. bAll amines and alcohols were resolved after trifluoroacetylation. cRetention and a values were measured at 10 "C internals. Hence every van't Hoff plot contained at least five data points. dTemperatures reported to the nearest 10 "C.
of sec-butyl 2-bromopropanoate (Ti,= 73 "C) were eluted as one peak close to the dead volume. Koppenhoefer and Bayer reported results from an analogous thermodynamic study of Chira~il-Val.~Interestingly, some of the compounds in our study seemed to have relatively large values of A(AHo) and A(ASo). In particular, methyl 2-chloropropanoate(Table 11)had values approximately 1order of magnitude higher than what has been reported for analytes on 'amino acid-based" GC chiral stationary phases. The data in Tables 11and III suggested that the compounds studied fall in two groups. Group I includes the compounds with -A(AHo) energy values and -A(ASo) entropy values equal to or higher than 1.8 kcal/mol and 4.3 cal/mol-K, respectively, on the DP-TFA-&CD phase (1.4 kcal/mol and 3.2 cal/mol.K, respectively, on the DP-"FA-y-CD phase). Group I1 includes compounds with -A(AHo) values and -A(ASo) values equal to or lower than 1.4 kcal/mol and 3.4 cal/mol.K, respectively, on the 0-CD CSP (0.9 k d / m o l and 2.4 cal/mol.K on the y-CD CSP). We hypothesize that group I compounds or one of their enantiomers may be forming a dominant enantioselective inclusion complex while the group 11compounds may not. The large entropy decrease of the group I compounds may be due to the loss of degrees of freedom for the compound included in the CD cavity. The pronounced size and shape selectivities which parallel the thermodynamic parameters, e.g. 2-chlorocyclopentanonewith a -A(AHo) value of 2500 cal/mol (group I) and 2-chlorocyclohexanone with a low 250 &/mol -A(AHo) value (group 11) on the 0 phase; or truns-2,bdimethoxytetrahydrofuran with a -A(AHo) value of 3100 cd/mol and 900 cal/mol on the y-CSP (group I) and the j3-CSP (group 11),respectively, are further evidence for the formation of an enantioselective inclusion complex with group I compounds. The Tisotemperatures for all compounds separated on both CSPs were within the range 75-217 "C with no significant differences for group I and group I1 compounds. The lowest
observed Ti, temperature was 82 "C for 2-pentanol (Table 11). Enthalpy-Entropy Compensation. An extra-thermodynamic approach to the analysis of physicochemical data is known as enthalpy-entropy c o m p e n s a t i o r ~ .The ~ ~ ~enthal~~ py-entropy compensation method has been used by Horvath in studies of hydrophobic interactions and separation mechanism in reversed phase HPLC.25 Mathematically, enthalpy-entropy compensation can be expressed by the formula AHo = @ASo AGO, (6)
+
where AGO, is the Gibbs free energy of a physicochemical interaction at compensation temperature, p (j3 and AGO, are constants). AHo and ASo are the corresponding standard enthalpy and entropy, respectively. According to eq 6, when enthalpy and entropy compensation is observed with a group of compounds in a particular chemical transformation (or interaction in the case of chromatographic retention), all of the compounds have the same free energy change (AGO,) at temperature j3. For example, if enthalpy-entropy compensation is observed in LC or GC for a group of compounds, all the compounds will have the same net retention at the compensation temperature j3, although their temperature dependencies may differ. In order to express the free energy change AGOT measured at a given temperature T,the Gibbs-Helmholtz relationship can be rewritten using eq 6 as AGoT = A H O ( 1 - T/B) + (TAGo,)/@ (7) Quation 7 shows that a plot of AGOT for different compounds a t a constant temperature T,versus the corresponding AHo produces a straight line, and the compensation temperature fi can be evaluated from the slope. Rewriting eq 7 using eq 3 yields In k' = -(AHo/R)(l/T - 1/@) + AGo,/R/3 + In 4 (8)
ANALYTICAL CHEMISTRY, VOL. 64, NO. 8, APRIL 15, 1992 877 5
1.4 r
c
E
3
d
@ 3
3
1
1
13
15
I
1
17
19
K C A L I M ~ L
-AH,
Flgure 9. Plot of enthalpy-entropy compensation for the last eluted enantkmers on the DP-TFA-WD CSP at 80 "C. The enthalpy values (-AH'S) for this plot w m obtalned from Table 11. The (0)represents the three group I compounds and the (0)symbol slgnlfies the nlne group I1 compounds.
-12
-11
-9
-10 La b
l ~ OF s
-a
AVALYTE
Flgure 5. Typlcal curves of sample loadlng versus HETP on DP-TFAy-CD CSP at 80 "C. Column 20 m fused silica capillary. Tests compounds: A, tran~-2,5dlmethoxytetrahydrofuran(group I); B, O-TFA-2octand (group 11). In both cases the data Is shown for the last eluted enantiomers.
j,
3
-1
I
u
14
I
4
15
16
J
17
18
-AH, ~ c a L / i ~ o L
Flgure 4. Plot of enthalpy-entropy compensatlon for the last eluted enantiomers on DP-TFA-yCD CSP at 80 "C. The enthalpy values (-&Os) for this plot were obtalned from Table 111. The ( 0 )repregents the elght group I compounds and (0)symbols are for the nlne group
I1 compounds.
which shows that plots of In k' versus AH" can be used to obtain the compensation temperature. A similarity in values for the compensation temperature suggests that the solutes are retained by essentially identical interaction mechanism, and thus the compensation study is a useful tool for comparing retention mechanisms in different chromatographic systems. However, the results obtained through this method must not be used alone because they can be misleading due to cumulative errors associated with the determination of enthalpy.28J7 Figures 3 and 4 show the enthalpy-entropy compensation plots of selected group I and group I1 compounds separated at 80 "C on the DP-TFA-p-CD CSP and DP-TFA-yCD CSP, respectively. The In k'and AH" values correspond to the most retained enantiomers which are the ones most likely to form inclusion complexes. Figures 3 and 4 clearly show that a linear relationship of In k' versus AH" is apparent for group I1 compounds. Group I compounds do not follow this trend; they have higher -AHo values which are randomly related to their In k'value. The group 11regression lines for Figure 3 (8-CSP) and Figure 4 (r-CSP) were In k' = -11.398 + 0.871(-AH0) r2 = 0.990
+
(9)
In k' = -11.914 0.903(-AH0) r2 = 0.975 (10) respectively. The compensation temperatures (approximately 640 "C and 700 "C on the 8- and y C D CSPs, respectively) were relatively close for the two stationary phases. Of course it is not possible to carry out any experiments at such high temperatures. If such an extra-thermodynamic approach were valid over this temperature range, it might suggest that the separation mechanism of the group I1 compounds is invarient on the two columns. As shown (Figures 3 and 4) the most
pronounced differences in entropy-enthalpy compensation are between the last eluted group I enantiomers and those of group II. Analogous plots for the first eluted enantiomer were also done.I6 Similar resulta were found in some cases, but some exceptions were noted as well. Cases where there is a major difference in the compensation behavior between enantiomers may be explained using the same inclusion vs noninclusion model. That is, one enantiomer could form an enantioselective inclusion complex while the other may not. The enthalpyentropy compensation study provides additional evidence that the separation mechanism for the two groups of analytes is different. Column Overloading Experiments. The thermodynamic measurements suggest that there is more than one mechanism of enantioselective retention. We hypothesize that the group I compounds resolve largely because they or at least one of their enantiomersform enantioseledive inclusion complexes. However, other compounds (such as group 11) resolve as a result of a "looser", possible external enantioselective association. We recognize that these may be idealized, opposite examples and that "real life" generally includes intermediate cases where there am contzibutions from both enantioselective retention mechanisms. It has been shown that native CDs form inclusion complexes with certain types of molecules in a definable host-guest molecular r a t i ~ . ~ *From * ~ ~this point of view, for group I compounds, the column capacity is determined by the number of CD molecules. For group I1 compounds that do not seem to form enantioselective inclusion complexes, the column capacity may be much higher. After all a single 8-CD molecule has 35 stereogenic centers (40centers in a y-CD molecule). Theoretically, by continually increasing the amount of analyte injected, until the separation efficiency deteriorates,two trends should be observed. The group I compounds that are thought to form inclusion complexes should not tolerate overloading to the extent of the group I1 compounds, where multiple loose enantioselective associations may occur on a single CD molecule. In the column overloading experiment, the height equivalent to a theoretical plate (HETP) was monitored as the sample loading was increased. Typical HETP versus sample loading, expressed as log injected moles, curves are shown in Figure 5. At ideally low sample concentrations, H E W is virtually independent of sample loading. After the sample loading
878
ANALYTICAL CHEMISTRY, VOL. 64, NO. 8, APRIL 15, 1992
Table IV. Column Overloading Experiment Datan racemic compoundb 2-heptanol 2-octanol 2-bromoheptane 3-chloro-2-butanone trans-2,5-dimethoxy-THF 1,3-dibromobutane methyl 2-chloropropanoate
Hmn,mol X 1Ol0
k'
A(AH"), kcal/mol
50 36 11 2.2 1.7 1.0 0.9
0.85 1.72 1.82 1.10 1.48 6.11 2.75
0.8 0.5 0.77 1.5 3.1 2.5 2.7
group
I1 I1 I1 I I I I
Temperature Effect racemic compoundb methyl 2-chloropropanoate
H w , mol x 10'0
k'
temp, "C
0.6 1.4 3.9 7.9
5.47 1.49 0.82 0.45
70 90 100 110
"Data are for the last eluted enantiomer. Column DP-TFA-7-
CD, 20 m at 80 "C unless otherwise indicated (2-chloropropionic acid methyl ester).
All alcohols were trifluoroacetylated.
reaches a large enough amount, the column efficiency deteriorates. For some compounds, the change was gradual whereas for other compounds the change could be dramatic (Figure 5). The sample loading at which 50% of the column efficiency is lost is recorded and defined as the value. The HW%values are gathered in Table IV. The data were measured on the same column at the same column temperature (80"C), same splitter ratio (60/1), and same injection volume (0.5 pL)in order to obtain comparable results. In a comparison such as this, one must set the conditions so that the compounds compared are rapidly and completely volatized in the injector or have approximately the same volatility so that split injection does not give rise to significant fractionation. Also compounds of approximately the same retention (k? should be compared 80 as to minimize retention-dependent loading differences. The highest column capacity was obtained with trifluoroacetylated 2-heptanol and the lowest with methyl-2-chloropropanoate with a 50-fold difference in the capacities of these two compounds. In general, there was a higher column capacity for the group I1 compounds than the group I compounds. However, it must be noted that column capacity is very temperature dependent (Table IV, bottom). Although carrying out an enantiomeric separation at higher temperature decreases the separation factor (a),it significantly increases the column capacity. Both are a consequence of the increase in the average amount of analyte in the gas phase and its corresponding decrease in the stationary phase. In Figure 5 we compare two compounds that have similar retention but very different capacities. Since each compound could have a different temperature capacity profile, this data should not be taken as absolute proof of mechanism. However, when taken with other evidence (vide supra), it appears to support the existence of at least two separation mechanisms. This data also suggests that a few chiral compounds could be resolved via one mechanism at low temperatures and another at high temperatures. Mass-Transfer Kinetics. Mass-transfer kinetics are linked to column efficiency and could give more information on the chromatographic separation mechanism. Inclusion complex formation may be slower than simple external CD interaction near a stereogenic center. The mass transfer kinetics in wall-coated capillary GC can be easily studied with the Van Deemter-Golay equation HETP = B / u
+ Cu
where B is the constant for longitudinal diffusion or the solute diffusion coefficient in the gas phase, C is the mass-transfer constant in both the mobile and the stationary phase, and u is the carrier gas linear velocity. For a thin stationary phase film, the maas transfer in the gas phase is the ratedetermining process. Solutes with similar molecular weights (similar diffusion coefficients) should produce similar C values in Van D e e m t e e l a y plots. If there is inclusion complexation which could be a slow process, the mass transfer in the stationary phase may not be neglected even though a thin film column is used. The Van Deemter-Golay plots of methyl 2-chloropropanoate, 2-chloro-2-butanone,2-bromoheptane, and 0TFA-2-heptanolwere done on DP-TFA-yCD column, at 80 OC. The C values were respectively 1.72, 1.97, 1.52, and 1.50. The first two analytes are group I compounds (inclusion) and the second two are group I1 compounds (noninclusion). One might expect strongly included compounds to have higher maas-transfer coefficients. The group I compounds did seem to have slightly higher C values (between 20 and 50%) than the group I1 compounds. However, comparisons such as this may not provide direct mechanistic evidence in this case. It must be remembered that we are grouping compounds according to their enantioselective retention only and not on total retention behavior. Most frequently, nonenantiwlective retention factors dominate the separation of chiral molecules. Hence, the a values for most GC enantiomeric separations are small. The difference in these mass transfer constants is fairly small. It is likely that, for a thin-film (0.13-pm) capillary column, the mass transfer is not as significantly affected by inclusion complex formation. It may be possible, however, to observe more significant effects with thick-film columns coated with CD-based stationary phases.
CONCLUSION It appears that at least two different enantioselective retention mechanism exist for the derivatized cyclodextrin GC stationary phases in this study. We postulate that one involves a classic inclusion complex formation while the other is a loose, probably external, multiple aasociation with the cyclodextrin (top, side, and/or bottom). Compounds can be identified that appear to resolve by one or the other mechanism. Group I compounds are mainly resolved through enantioselective inclusion complex formation. They show high thermodynamic values for AHo, A(AH"), and A(AS") and a relatively low column capacity. Group I1 compounds show lower AH", A(AH"), and A(ASo) values. They obey an entropy-enthalpy compensation scheme with a compensation temperature in the 650 "C range on both CSPs studied. Undoubtedly, there are numerous compounds that show intermediate behavior between these two ideal models. In fact, the enantioselective retention mechanism of some compounds may be temperature dependent (Le., more inclusion-like at low temperature but not at higher temperaturea). There are at least two practical aspects to the multimechanistic behavior of these CSPs. One is a better understanding of how derivatized cycldextrin GC phases work. The other is that a large variety of enantiomers can be resolved on these CSPs (via different mechanisms), thereby greatly increasing their utility. ACKNOWLEDGMENT Support of this work by the Department of Energy, Office of Basic Sciences (Grant DE FG02 88ER138191, is gratefully acknowledged. REFERENCES ( 1 ) GiCAv, E.; Feibush, B.; Chsrles-Sgk, R. Tetrahedon Left. 1966, 1004. (2) Kiinig, W. A.; Nicholson, G. J. Anal. Chem. 1975. 4 7 , 951
Anal. Chem. 1002, 64,879-888 (3) KGnig, W. A. The Recti98 of Enantlomw Separetbn by Capllary Oes Chrometogrephy;Hvthi: Heidelbecg, 1987. (4) Schurlg, V. J . Ckomtogr. 1988,441, 135. (5) Frank, H.; Nicholson, G. J.; Bayer, E. J . Chromatogr. Scl. 1977, 75, 174. (6) Smdkova-Keulemansova, E. J . Chrometogr. 1982,257, 17. (7) Koscielskl, T.; Sybllska, D.; Jurczak, J. J . Chrometogr. 1983,280, 131. (8) Khig, W. A.; Lutz, S.; Wenz, G.; von der Bey, E. H@h Res. Chromatogr. Chfomogr. Commun. 1988, 7 7 . 508. (9) SchuriQ, V.; Nowotny. H. P. Angew. Chem., Int. Ed. Engl. 1990,29, 939. (10) Berthod, A.; Ll. W. Y.; Armstrong, D. W. carbahydr. Res. 1990,207, 175. (11) Hen. S. H.; Armstrong, D. W. I n Chkel Separetions by H R C ; Krstulovic, A. M., Ed.: Ellis Hotwood Limlted: Chlchester. 1989; ChaDter 10, pp 208-284. (12) Armstrong, D. W. Anal. Chem. 1987,59, 84A. (13) Armstrong, D. W.; LI, W. Y.; Chang, C. D.; Pltha, J. Anal. Chem. 1990. ... 62. .- 914. . (14) Ll, W. Y.; Jln. H.-L.; Armstrong, D. W. J . Chromatogr. 1990, 509, 303. (15) Armstrong, D. W.; LI, W. Y.; Stalcup, A. M.; Secor, H. V.; Izac. R. R.; Seeman, J. I. Anal. Chlm. Acta 1990,234, 385. (16) Ll, W. Ph.D. Dkrsertatlon, Unhrerslty of Mlssouri-Rolla, 1990. (17) Kosclelskl, T.; Sybllska. D.; Felt, L.; Smolkova-Keulemansova,E. J. J . Chromatogr. 1984,286, 23.
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(18) Andera, L.; Smolkova-Keulemansova, E. J. J . Inclusion &nom. 1987,5,397. (19) Ettre, L. S. The Kovats Retention Index System. Anal. Chem. 1964, 36. - - , 31A. - .. .. (20) Watabe, K.; Charles, R.; OICAv, E. Angew. Chem., Int. Ed. €ngl. lgag. 28. 192. -(21) Schurig. V.; Osslg, J.; Link, R. Angew. Chem.. Int. Ed. Engl. 1989, 28, 194. (22) Koppenhoefer, B.; Bayer, E. Chromatographia 1984, 79, 123. (23) Lumty. R.; Rajender, S. Bispolvmes 1970. 9 , 1125. (24) Leffbr, J.; Grunwald, E. R8t8S end Equlllbrle or Orgenlc RaecHons; Wiley: New York, 1963. (25) Melander. W.; Campbell, D. E.; Horvath, C. J . Chromatogr. 1978. 758, 215. (26) Krug, R. R.; Hunter, W. G.; m r , R. A. J . phvs. Chem. 1978,80, 2335. (27) Krug, R. R.; Hunter, W. G.; Grbger, R. A. J . Fhys. Chem. 1978,80, 2341. (28) Armstrong, D. W.; Nome. F.; Spino, L. A.; Golden, T. D. J . Am. Chem. Soc.1986, 708, 1410. (29) Smolkova-Keulemansova, E. J.; SoJak, L. ACS Symposium Ser. 1987, 342.247.
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RECEIVED for review September 12,1991. Accepted January 22, 1992.
Peptide Mapping of Complex Proteins at the Low-Picomole Level with Capillary Electrophoretic Separations Kelly A. Cobbt and Milos V. Novotny*
Department of Chemistry, Indiana University, Bloomington, Indiana 47405
A varlety of dyfereni pepWe-mapplng schemes are plesented,
wlth emphark on the development of procedures whlch can be done wlth llmlted quantltks (Le. 5 pmol) of proteln. Resub are OMaIned hwn model proteins whkh contah dzsuMde bonds, whlch must be broken prlor to fragmentatlon of the proteln. A reactlon lnvolvlng the dmultaneous use of trC butylphosphlna and Smethylazlrldlne to reduce and alkylate the dkufflde bonds Is employed, due to favorable attrlbutes of these reagents for the rcaleddown procedure. The tradltlonal performlc acld oxldatlon reactlon to cleave cystlne groupr k ako successfully u w d wlth lowglcomole quantltles of proteln. Three dmerent proteh d@stbn reagents are u w k trypeln, chymotrypsin, and cyanogen bromkle. Each reagent produces a unlque mlxture of peptldes. Caplllary electre phorerk Is used to separate the peptMer, offerlng hlgh sep aratlon efflclencles, short analydr times, and compatlblllty wlth small sample slzer. I n addttlon to the conventional use of UV detectlon for underlvatlzed peptldes, laser-Induced fluorescence detectlon Is employed In conjunctlon wlth an arglnlne-solecllve derlvatlzatlon reactlon. Thls latter procedure for derlvatlzatlon and detectlon offers an alternatlve peptklamapplngmode, In whlch only the arglnlnacontalnlng peptides are detected, and is useful In sknplltylng the peptlde maps of large protelns.
INTRODUCTION The study of proteins frequently involves the use of peptide mapping, a powerful and efficient means of protein charac*To whom all corres ondence should be addressed. Present address: Tfe Dow Chemical Co., 1897 Building, Midland, MI 48667.
terization or identification. The basic premise of peptide mapping is to enzymatically or chemically cleave a protein into a number or smaller peptide fragmenta and then separate the resulting peptides, either chromatographically or electrophoretically, to yield a characteristic peak profile or “map” of that particular protein. Peptide mapping is primarily a qualitative, comparative technique, and ita popularity stems from the ability to ascertain very subtle differences between proteins, such as amino acid substitutions or posttranslational
modification^.'-^ Over the years, a number of sample treatment procedures have been developed for the purpose of peptide mapping. Most often, these procedures have been designed for micromole or nanomole quantities of protein.&l0 However, there is a growing need for the ability to obtain reproducible and informative peptide maps from much smaller amounts of protein, down to the picomole (or nanogram) level. This need arises from the fact that many proteins of interest to the medical or bioanalytical community, such as those occurring in physiological fluids, cell surface receptors, or growth factors,llare typically isolated in extremely s m d quantities. To be able to obtain needed information from such proteins, it is necessary that reliable procedures be developed for reduced-scale sample treatment. We have previously addressed some of the needs pertaining to high-sensitivity peptide mapping by demonstrating the use of immobilized trypsin for the digeation of as little as 50 ng of protein, followed by peptide separation using either capillary electrophoresis (CE)or microcolumn liquid chromatography.12 However, our previous studies were performed on proteins with relatively simple tertiary structures, with no disulfide bridges. Because many proteins of interest contain disulfide linkages, which tend to complicate the sample treatment procedure for peptide mapping, it is imperative that the methods be expanded to
0003-2700/92/0364-0879$03.00/0 0 1992 American Chemical Society
