A Family of Single-Isomer Chiral Resolving Agentsfor Capillary Electrophoresis. 1.Heptakis(2,3-diacetyl-6-sulfato)-â-cyclodextrin

J. Bryan Vincent, Alex D. Sokolowski, Thanh V. Nguyen, and Gyula Vigh*

Department of Chemistry, Texas A&M University, College Station, Texas 77845-3255

A new, moderately hydrophobic, single-isomer chargedcyclodextrin, the sodium salt of heptakis(2,3-diacetyl-6-sulfato)-â-cyclodextrin, has been synthesized and used toseparate a variety of neutral, weak acid, strong base, weakbase, and zwitterionic racemic enantiomers in low-pH andhigh-pH background electrolytes. Separation selectivityfor the netural analytes rapidly decreases as the concen-tration of heptakis(2,3-diacetyl-6-sulfato)-â-cyclodextrinincreases. For charged analytes, selectivity can increase,decrease, or pass a maximum, depending on the numericvalues of the respective complexation constants and ionicmobilities. In addition to separation selectivity, the extentof peak resolution that can be realized strongly dependson the magnitude of the dimensionless electroosmoticflow. Heptakis(2,3-diacetyl-6-sulfato)-â-cyclodextrin provedto be a broadly applicable chiral resolving agent.

In this decade, capillary electrophoresis (CE) became firmlyestablished as a powerful method for the separation of enanti-omers,1 thanks, in great part, to the versatility of various cyclo-dextrins, the most frequently used chiral resolving agents in bothacidic and alkaline background electrolytes (BEs). Thoughspectacular separations have been achieved with native as wellas derivatized neutral cyclodextrins, as recently reviewed in ref2, the analysis of noncharged enantiomers only became possiblewhen charged cyclodextrins entered the stage.3 The chargedcyclodextrins are either weak electrolytes,3-5 which can only beused in a limited pH range (where they possess at least a fractionalcharge), or strong electrolytes,6-13 which can be used in BEs ofany pH. Most of the charged cyclodextrins used so far arecomplex mixtures which contain a large number of isomersdiffering both in their degree of substitution (the number ofcharges per cyclodextrin molecule) and the loci of substitution.The use of resolving agent mixtures is fraught with at least four

distinct problems which are of varying importance in any givenseparation. The first problem is that the number and loci ofsubstituents on the cyclodextrin can have a great effect on thechiral selectivity of the system, and, at our current level ofunderstanding, the direction and magnitude of these changescannot be predicted a priori.14 Therefore, when mixtures ofdifferent isomers of substituted cyclodextrins are used, the overallseparation selectivity of the system might be reduced (due toparasitic complexation), eliminated (due to matched, oppositebinding selectivities for the two enantiomers), or, in a few favorablecases, enhanced.15 The second problem is that kinetic bandbroadening can occur when the finite complexation rates of thedifferent cyclodextrin isomers are slightly different; this resultsin an unavoidable decrease of the separation efficiency. The thirddrawback is that fundamental molecular level studies (e.g., NMR,16

crystallographic or molecular modeling17), aimed at improving thelevel of understanding of the chiral recognition process and,perhaps, leading to a rational selection of the best chiral resolvingagent for a particular analyte, are impossible with mixtures ofresolving agent isomers. Fourth, but not least, when a criticalseparation is developed with a particular mixture of resolving agentisomers (commercial or otherwise), there is always the dangerthat the composition of the mixture will be slightly different inthe next batch, and the reproducibility of a difficult separationwill be compromised.

Therefore, a project was undertaken in our laboratory todevelop a family of pure, well-characterized, single-isomer chargedcyclodextrins which (i) are strong electrolytes and can be usedat any pH required for selectivity optimization,18 (ii) offer the sameintermolecular interactions (functional groups) on the 2- and3-positions of the glucose moieties as the well-established neutralcyclodextrins do,2 and (iii) carry the maximum number of chargedfunctional groups on the 6-position of the glucose moieties toprovide for the best possible peak resolution.18-20 This paperdescribes the synthesis, characterization, and use of the firstmember of this new, single-isomer charged cyclodextrin family,heptakis(2,3-diacetyl-6-sulfato)-â-cyclodextrin.

(1) St. Claire, R. L. Anal. Chem. 1996, 58, 569R.(2) Fanali, S. J. Chromatogr. A 1996, 735, 77.(3) Terabe, S. Trends Anal. Chem. 1989, 8, 129.(4) Fanali, S. J. Chromatogr. 1989, 474, 441.(5) Schmitt, T.; Engelhardt, H. Chromatographia 1993, 37, 475.(6) Tait, R. J.; Skanchy, D. J.; Thompson, D. O.; Chetwyn, N. C.; Dunshee, D.

A.; Rajewsky, R. A.; Stella, V. J.; Stobaugh, J. F. J. Pharm. Biomed. Anal.1992, 10, 615.

(7) Tait, R. J.; Thompson, D. O.; Stobaugh, J. F. Anal. Chem. 1994, 66, 4013.(8) Dette, C.; Ebel, S.; Terabe, S. Electrophoresis 1994, 15, 799.(9) Wu, W. H.; Stalcup, A. M. J. Liq. Chromatogr. 1995, 18, 1289.

(10) Desiderio, C.; Fanali, S. J. Chromatogr. A 1996, 716, 183.(11) Chakvetadze, B.; Endresz, G.; Blaschke, G. Electrophoresis 1994, 15, 804.(12) Chakvetadze, B.; Endresz, G.; Blaschke, G. J. Chromatogr. A 1995, 704,

234.(13) Stalcup, A. M.; Gahm, K. H. Anal. Chem. 1996, 68, 1360.

(14) Weseloh, G.; Bartsch, H.; Konig, W. A. J. Microcolumn Sep. 1995, 7, 355.(15) Stalcup, A. M.; Gahm, K. H. Anal. Chem. 1995, 67, 19.(16) Endresz, G.; Chakvetadze, B.; Bergenthal, D.; Blaschke, G. J. Chromatogr.

A 1996, 732, 132.(17) Lipkowitz, K.; Pearl, G.; Coner, B.; Peterson, M. J. Am. Chem. Soc. 1992,

114, 1554.(18) Williams, B. A.; Vigh, Gy. J. Chromatogr. 1997, 776, 295.(19) Friedl, W.; Kenndler, E. Anal. Chem. 1993, 65, 2003.(20) Rawjee, Y. Y.; Vigh, Gy. Anal. Chem. 1994, 66, 428.

Anal. Chem. 1997, 69, 4226-4233

4226 Analytical Chemistry, Vol. 69, No. 20, October 15, 1997 S0003-2700(97)00252-7 CCC: $14.00 © 1997 American Chemical Society

EXPERIMENTAL SECTIONSynthesis of Heptakis(2,3-diacetyl-6-sulfato)-â-cyclodex-

trin. All chemicals used in this work were obtained from AldrichChemical Co. (Milwaukee, WI), except â-cyclodextrin, which wasa generous gift from CeraStar (Hammond, IN). The schematicof the synthetic procedure is shown in Figure 1. Briefly, heptakis-(2,3-diacetyl-6-sulfato)-â-cyclodextrin is produced by first reactingâ-cyclodextrin with dimethyl-tert-butylchlorosilane as describedin ref 21, purifying the intermediate by gradient elution preparativecolumn chromatography22 on silica gel using n-hexane/ethylacetate/ethanol as eluent.21 (The 50 mm i.d., 300 mm longpreparative HPLC column packed with 30 nm pore size, 10 µmirregular silica (Merck, Darmstadt, Germany) was generouslyloaned to us by Dr. Y. Y. Rawjee of SmithKline Beecham, King ofPrussia, PA.) The purified intermediate was then peracetylatedwith acetic anhydride as described in ref 21, and the product wasonce again purified by gradient elution preparative columnchromatography on silica gel using ethyl acetate/ethanol aseluent.21 The purified heptakis(2,3-diacetyl-6-dimethyl-tert-butyl-silyl)-â-cyclodextrin was then reacted with boron trifluorideetherate as described in ref 21 to remove the dimethyl-tert-butylsilyl protecting group, and the product was repurified bygradient elution preparative column chromatography on silica gelusing ethyl acetate/ethanol as eluent.21 Next, the pure heptakis-(2,3-diacetyl)-â-cyclodextrin was reacted with SO3‚pyridine in DMFas described in ref 23 to completely sulfate the primary hydroxylgroups of the cyclodextrin. Progress of the reaction was moni-tored by indirect UV detection capillary electrophoresis24 using20 mM p-toluenesulfonic acid background electrolyte, whose pHwas adjusted to 8 with tris(hydroxymethyl)aminomethane. Oncethe reaction was complete, the reaction mixture was poured intoacetone, and the semisolid material was filtered out and redis-solved in water. The aqueous solution was neutralized withNaOH, and the material was reprecipitated with ethanol. The solidmaterial was filtered out and redissolved in water, and theremaining sodium sulfate was removed by repeated addition ofethanol. Finally, the end product was dried in a vacuum oven at80 °C to obtain pure heptakis(2,3-diacetyl-6-sulfato)-â-cyclodextrin.The purity of the material was checked by indirect UV detection

capillary electrophoresis24 as above. The electropherogram of atypical heptakis(2,3-diacetyl-6-sulfato)-â-cyclodextrin sample isshown in Figure 2. The 200 MHz 1H NMR spectrum of the finalproduct is shown in Figure 3.

Electrophoretic Separations Using Heptakis(2,3-diacetyl-6-sulfato)-â-cyclodextrin. All separations were carried out ona UV detector-equipped P/ACE 2100 CE unit (Beckman Instru-ments, Fullerton, CA). The detection wavelength was set at 214nm, and the cartridge coolant was thermostated at 20 °C. Theseparations were carried out in 25 µm i.d. untreated fused silicacapillaries (Polymicro Technologies, Phoenix, AZ) with a 45 cmtotal length and a 39 cm injector-to-detector length. The injectionpressure was set at 5 psi and the injection time was 1 s. Theapplied potential was varied between 12 and 20 kV in order tomaintain a power dissipation of 500-700 mW/m.

Two buffer stock solutions were prepared as required by thecharged resolving agent migration model (CHARM model) of CEenantiomer separations:18 a low-pH buffer (pH 2.5) and a high-pH buffer (pH 9.5). The low-pH buffer was prepared by adding0.0250 mol of concentrated (85% w/w) phosphoric acid to enoughdeionized water (Milli-Q, Millipore, Milford, MA) to obtain asolution of about 0.95 L. This solution was titrated to pH 2.5 witha saturated aqueous solution of LiOH using a combination glasselectrode and a precision pH meter (both of them from Corning

(21) Takeo, K.; Hisayoshi, M.; Uemura, K. Carbohydr. Res. 1989, 187, 203.(22) Vigh, Gy.; Quintero, G.; Farkas, Gy. J. Chromatogr. 1989, 484, 237.(23) Bernstein, S.; Joseph, J.; Nair, U. U.S. Patent 4,020,160, 1977.(24) Nardi, A.; Fanalli, S.; Foret, F. Electrophoresis 1990, 11, 774.

Figure 1. Synthesis scheme for heptakis(2,3-diacetyl-6-sulfato)-â-cyclodextrin.

Figure 2. Indirect UV detection electropherogram of a typicalheptakis(2,3-diacetyl-6-sulfato)-â-cyclodextrin sample. BE: 20 mMp-toluenesulfonic acid, pH 8, adjusted with TRIS. Wavelength: 214nm. Applied potential: 25 kV. Thermostat temperature: 25 °C.Capillary: 25 µm i.d., 39/45 cm effective/total length, uncoated fusedsilica.

Figure 3. 1H NMR spectrum of a typical heptakis(2,3-diacetyl-6-sulfato)-â-cyclodextrin sample. Solvent: D2O.

Analytical Chemistry, Vol. 69, No. 20, October 15, 1997 4227

Table 1. Effective Mobilities of the Less Mobile Enantiomer [µ, in 10-5 cm2/(V‚s)], Separation Selectivities (r),Measured Peak Resolution Values (Rs), Dimensionless EO Flow Values (â), and the Injector-to-Detector PotentialDrop (U, in kV) in Low-pH (pH 2.5) HDAS-âCD BEs

10 mM 15 mM 30 mM 50 mM

structure µ R Rs â U µ R Rs â U µ R Rs â U µ R Rs â U

MarkerSO3

– -28.45 ((0.07) -27.34 ((0.07) -25.93 ((0.05) -23.71 ((0.09)

Neutrals

HOCH2C

OH

H -0.6 1.44 0.7 -36.8 17.2 -0.8 1.40 0.7 -27.1 17.2 -2.7 1.19 1.7 -2.5 2.1a -3.0 1.19 3.0 -2.6 12.8

CH3CHCH2

OH -3.4 1.10 <0.5 -6.6 17.2 -3.6 1.08 <0.5 -5.8 17.2 -7.7 1.07 4.0 -0.7 12.9 -8.9 1.03 1.7 -0.7 12.9

HOCH2CH

CH3 -3.1 1.34 1.9 -7.5 17.2 -3.9 1.26 1.5 -6.0 17.2 -7.4 1.16 4.3 -0.6 2.1a -8.3 1.11 3.7 -0.7 2.1a

CHCOCH3

OH

O -0.5 1.31 0.6 -35.7 17.2 -1.3 1.21 0.9 -19.3 17.2 -3.6 1.09 1.0 -2.1 2.1a -4.2 1.09 3.0 -2.2 12.8

OO

-5.6 1.23 2.1 -4.1 17.2 -6.3 1.23 3.5 -4.0 17.2 -10.2 1.16 3.2 -0.5 2.1a -11.7 1.10 6.4 -0.8 12.8

NH

CH3

OO

-0.4 1.50 <0.5 -57.4 17.2 -1.5 1.20 1.0 -14.5 17.2 -3.9 1.07 1.0 -2.0 2.1a -5.1 1.07 4.4 -1.8 12.8

Bases

CCH2N+(CH2CH3)3

OH

H

-6.4 1.36 2.2 -4.3 17.2 -6.3 1.33 2.5 -4.2 17.2 -4.6 1.26 4.6 -0.3 2.1a -3.7 1.24 5.1 -2.5 12.8

HO

HO

CHCH2NC(CH3)3

OH H-14.9 1.13 2.6 -1.8 17.2 -14.5 1.11 2.3 -1.9 17.2 -14.2 1.05 4.6 -0.6 12.9 -13.9 1.03 5.0 -0.6 12.8

CCH2CH2N(CH3)2

Cl

OH -6.0 1.27 3.5 -4.0 17.2 -6.9 1.23 4.0 -3.7 17.1 -8.2 1.19 6.5 -0.5 2.1a -9.4 1.11 8.4 -0.7 12.9

CCOOCH2

OH

N

N

CH3

-21.5 1.15 12.7 -0.8 17.2 -18.3 1.15 8.6 -0.5 17.2 -17.5 1.15 8.7 -0.6 12.9 -15.2 1.10 13.1 -0.8 12.8

N

OCH2CH

CH2NHCH

OH

(CH3)2

-26.0 1.00 0.0 -0.7 2.8a -25.2 1.00 0.0 -0.7 17.1 -21.3 1.00 0.0 -0.5 12.9 -17.4 <1.01 <0.5 -0.7 2.1a

O

OCH2 N

-31.0 1.04 3.4 -0.6 17.2 -28.5 1.02 1.5 -0.4 17.2 -24.6 1.02 1.7 -0.5 12.9 -20.7 1.02 2.4 -0.4 12.9

Acids

Cl

SO2NH2

CNHN

OCH3 -2.4 1.10 <0.5 -9.3 17.2 -2.7 1.09 0.8 -7.9 17.2 -6.2 1.04 1.4 -0.6 2.1a -9.2 1.01 0.7 -0.7 12.9

H2C C

CH3

H

COOHHC

CH3

CH3 insoluble insoluble insoluble insoluble

O C

H

CH3

COOH

insoluble insoluble insoluble insoluble

F

C

CH3

H

COOH

insoluble insoluble insoluble insoluble

4228 Analytical Chemistry, Vol. 69, No. 20, October 15, 1997

Science Products, Corning NY). Finally, the solution was quan-titatively transferred to a 1 L volumetric flask, the volume wasbrought to the mark with deionized water, and the pH wasremeasured. The high-pH buffer was prepared similarly, exceptthat 0.0250 mol of ethanolamine was titrated to pH 9.5 with anaqueous solution of methanesulfonic acid and quantitativelytransferred to a 1 L volumetric flask, the volume was brought tothe mark with deionized water, and the pH was remeasured.

The 10, 15, 30, and 50 mM heptakis(2,3-diacetyl-6-sulfato)-â-cyclodextrin (HDAS-âCD) low-pH and high-pH BEs were preparedby weighing out the required amounts of the sodium salt of HDAS-âCD into 25 mL volumetric flasks and bringing the volumes tothe mark with the low-pH and the high-pH buffer stock solutions,respectively. Next, 0.5 mM 2-naphthalenesulfonic acid (NSA-)samples were prepared with the respective HDAS-âCD BEs. Thetrue effective mobility of NSA- (µeff

NSA-) was determined in eachHDAS-âCD BE (first line in Table 1) using the external electroos-motic (EO) flow marker method described in refs 25 and 26.Several 250 µL samples of the racemic analytes were prepared at0.5 mM concentrations in the P/ACE microvials with the respec-tive HDAS-âCD BEs. The sample solutions also contained 0.5mM NSA- so that the observed mobilities of both the NSA-

(µobsNSA-) and the enantiomers (µobs

R and µobsS) could be deter-

mined from the same runs. Since the effective mobility of NSA-

was known in each HDAS-âCD from the external EO flow markermeasurements (first line in Table 1), the electroosmotic flowmobility, µEO, could be obtained as µEO ) µobs

NSA- - µeffNSA-, from

which the effective mobilities of the enantiomers were obtainedas µeff

R ) µobsR - µEO, and the separation selectivities, R, were

calculated as µeffR/µeff

S, where subscript S refers to the less mobileenantiomer. The dimensionless electroosmotic flow values, â,were calculated as â ) µEO/µeff

S.20 The peak resolution values,Rs, were calculated as the ratio of the migration time differenceof the two enantiomers and the sum of their half peak widths atthe baseline.

RESULTS AND DISCUSSIONEffective Electrophoretic Mobilities of 2-Naphthalene-

sulfonate in HDAS-âCD BEs. The effective mobilities of NSA-,µeff

NSA-, were determined in each BE using the external EO flowmarker method described in ref 25 and are listed at the tops ofTables 1 and 2. (Briefly, in the external EO flow marker method,the capillary is divided into three segments: the first and the lastsegments contain the stock BE without any charged cyclodextrin,and the middle part of the capillary is filled with the BE that doescontain the charged cyclodextrin. An NSA- sample band is placedin the middle of the charged cyclodextrin-containing segment, anda neutral marker band is placed in the middle of the chargedcyclodextrin-free capillary segment. The initial distance (lini)

(25) Williams, B. A.; Vigh, Gy. Anal. Chem., in press.(26) Williams, B. A.; Vigh, Gy. Anal. Chem., in press.

Table 1 (Continued)

10 mM 15 mM 30 mM 50 mM

structure µ R Rs â U µ R Rs â U µ R Rs â U µ R Rs â U

O C

CH3

COOH

CH3

HCl

Cl insoluble insoluble insoluble insoluble

Zwitterions

OH

OH

CCH2

H2N

COOH

H

0.3 -1.9b 2.5 72.3 17.2 -0.6 2.79 5.8 -14.3 17.2 -1.8 1.70 4.9 -2.2 2.1a -1.9 1.59 14.2 -3.3 12.8

C

COOH

H2N

H CH2

-1.1 1.28 0.7 -19.5 17.2 -1.5 1.36 0.9 -13.5 17.1 -4.7 1.09 2.3 -0.6 2.1a -4.9 1.06 3.3 -1.5 12.8

N

CH2

C COOHH2N

H -6.0 1.02 <0.5 -3.7 17.2 -7.7 1.02 <0.5 -3.3 17.1 -7.9 1.02 <0.5 -0.5 2.1a -8.2 1.03 4.1 -1.3 12.8

N

CH3

CH3

S

O

O

N

H

C

H

COOH

CH2

COOH

-4.9 1.00 0.0 -4.6 17.2 -6.5 1.00 0.0 -0.6 2.8a -7.6 1.01 0.9 -0.8 2.1a -7.6 1.01 <0.5 -0.7 2.1a

N

CH3

CH3

S

O

O

N

H

C

H

COOH

CH2

CH2SCH3

-5.6 1.01 <0.5 -4.1 17.2 -6.2 1.01 1.2 -0.5 2.8a -8.6 1.01 0.8 -0.7 12.9 -8.6 1.02 0.9 -0.6 2.1a

N

CH3

CH3

S

O

O

N

H

C

H

COOH

CH2

-7.8 1.00 0.0 -3.0 17.2 -8.2 1.01 1.2 -0.7 2.8a -9.8 1.01 0.8 -0.4 12.9 -10.1 1.04 3.7 -0.7 12.9

a Analyte injected from the “short” (detector) side of the capillary. b Negative R values mean that the effective mobilities of the two enantiomerswere opposite in sign.

Analytical Chemistry, Vol. 69, No. 20, October 15, 1997 4229

Table 2. Effective Mobilities of the Less Mobile Enantiomer [µ, in 10-5 cm2/(V‚s)], Separation Selectivities (r),Measured Peak Resolution Values (Rs), Dimensionless EO Flow Values (â), and the Injector-to-Detector PotentialDrop (U, in kV) in High-pH (pH 9.5) HDAS-âCD BEs

10 mM 15 mM 30 mM 50 mM

structure µ R Rs â U µ R Rs â U µ R Rs â U µ R Rs â U

MarkerSO3

– -21.48 ((0.05) -20.94 ((0.05) -19.2 ((0.06) -17.22 ((0.09)

Neutrals

HOCH2C

OH

H -0.4 1.87 <0.5 -43.1 17.2 -0.5 1.73 <0.5 -51.1 17.1 -0.8 1.63 2.1 -14.7 12.9 -1.0 ‘.6‘ 2.2 -10.4 12.9

CH3CHCH2

OH -0.2 3.07 2.0 -78.6 17.1 -0.2 2.92 2.3 -46.3 17.1 -3.7 1.12 1.9 -2.7 12.9 -5.1 1.08 1.7 -2.8 12.9

HOCH2CH

CH3 -0.2 5.02 2.0 -86.4 17.2 -1.7 1.76 6.5 -5.7 17.1 -3.8 1.29 3.9 -2.8 12.9 -5.0 1.21 2.6 -3.2 12.9

CHCOCH3

OH

O -0.6 1.52 1.2 -31.2 17.1 -0.7 1.49 1.3 -33.5 17.1 -0.8 1.46 1.4 -17.1 12.9 -1.3 1.40 5.0 -5.7 12.9

OO

-1.4 1.87 2.8 -9.7 17.2 -2.1 1.70 2.2 -12.0 17.1 -5.6 1.29 4.2 -2.2 12.9 -7.1 1.19 1.7 -2.8 12.9

NH

CH3

OO

-0.1 4.06 <0.5 -197 17.2 -1.1 1.40 0.9 -12.6 17.1 -1.8 1.25 1.5 -10.0 12.8 -2.3 1.13 1.9 -4.3 12.9

Bases

CCH2N+(CH2CH3)3

OH

H

-1.3 2.32 4.3 -15.1 17.2 -1.2 2.27 2.5 -23.7 17.1 -1.2 1.89 7.3 -10.9 12.9 -1.1 1.76 6.0 -12.4 12.9

HO

HO

CHCH2NC(CH3)3

OH H-13.0 1.08 4.3 -1.5 17.2 -12.0 1.11 4.2 -1.8 17.1 -9.4 1.18 6.5 -1.7 12.8 -8.3 1.13 5.9 -2.5 12.9

CCH2CH2N(CH3)2

Cl

OH -0.2 9.49 2.2 -109 17.2 -1.3 2.12 2.1 -19.8 17.1 -5.7 1.22 7.5 -2.8 12.9 -6.6 1.17 7.7 -2.4 12.9

CCOOCH2

OH

N

N

CH3

-13.3 1.27 8.8 -2.4 17.2 -12.3 1.24 20.1 -1.6 17.1 -12.0 1.16 12.9 -2.2 12.9 -10.0 1.11 8.9 -1.9 12.9

N

OCH2CH

CH2NHCH

OH

(CH3)2

-18.9 1.00 0.0 -0.6 17.2 -16.8 1.00 0.0 -0.8 17.1 -15.6 1.00 0.0 -1.4 12.8 -12.2 1.01 0.6 -1.4 12.9

O

OCH2 N

-23.3 1.03 2.4 -0.5 17.2 -20.2 1.06 3.9 -0.7 17.1 -17.7 1.05 5.8 -1.5 12.9 -14.4 1.03 3.6 -1.5 12.9

Acids

Cl

SO2NH2

CNHN

OCH3 -2.1 1.14 1.0 -8.4 17.2 -3.3 1.13 0.7 -6.5 17.1 -3.6 1.07 1.6 -3.2 12.9 -3.8 1.06 0.9 -4.5 12.9

H2C C

CH3

H

COOHHC

CH3

CH3 -15.2 1.00 0.0 -2.4 17.2 -14.7 1.00 0.0 -2.3 17.1 -13.9 1.00 0.0 -1.9 12.9 -13.2 1.01 0.7 -0.6 12.9

O C

H

CH3

COOH

-15.4 1.00 0.0 -2.6 17.2 -14.7 1.00 0.0 -2.3 17.1 -14.1 1.01 1.0 -1.6 12.8 -13.4 1.01 1.2 -1.7 12.9

F

C

CH3

H

COOH

-16.4 1.00 0.0 -2.2 17.2 -15.8 1.01 <0.5 -2.2 17.1 -15.4 1.01 0.9 -1.3 12.8 -14.3 1.01 1.0 -0.4 12.9

4230 Analytical Chemistry, Vol. 69, No. 20, October 15, 1997

between the neutral marker band and the NSA- band is deter-mined by the injection sequence. During the brief electrophoresisstep that follows the injection sequence, the distance between theNSA- band and the neutral marker band changes to lfinal as a resultof the net electrophoretic migration of NSA- in the charged

cyclodextrin-containing BE segment. The effective migrationdistance of NSA-, leff, can be calculated as leff ) lini - lfinal. Fromthis, the separation time and the effective field strength, µeff

NSA-,can be calculated.25 The µeff

NSA- values decrease as the HDAS-âCD concentration is increased, both at low pH and high pH,because the BE viscosity, the ionic strength, and the extent ofcomplexation with HDAS-âCD all increase. However, the µeff

NSA-

values in the high-pH BEs are about 30% lower than those in the

Table 2 (Continued)

10 mM 15 mM 30 mM 50 mM

structure µ R Rs â U µ R Rs â U µ R Rs â U µ R Rs â U

O C

CH3

COOH

CH3

HCl

Cl -14.4 1.00 0.0 -2.7 17.2 -13.6 1.01 0.9 -1.5 17.1 -13.5 1.01 1.0 -1.6 12.8 -12.3 1.02 1.6 -0.6 12.9

Zwitterions

OH

OH

CCH2

H2N

COOH

H

reactive reactive reactive reactive

C

COOH

H2N

H CH2

-1.08 1.00 0.0 -10.0 17.2 -2.2 1.00 0.0 -8.9 17.1 -3.6 1.00 0.0 -4.1 12.9 -4.3 1.00 0.0 -6.1 12.9

N

CH2

C COOHH2N

H -2.2 1.33 2.2 -8.1 17.2 -2.3 1.32 1.7 -8.9 17.1 -4.1 1.22 4.6 -3.7 12.9 -4.8 1.17 4.0 -2.8 12.9

N

CH3

CH3

S

O

O

N

H

C

H

COOH

CH2

COOH

-23.1 1.00 0.0 -0.5 17.2 -21.7 1.00 0.0 -0.7 17.1 -19.1 1.00 0.0 -1.2 12.8 -16.7 1.00 0.0 -0.4 12.9

N

CH3

CH3

S

O

O

N

H

C

H

COOH

CH2

CH2SCH3

-12.3 1.00 0.0 -2.9 17.2 -11.5 1.00 0.0 -2.9 17.1 -11.1 1.01 0.8 -1.5 12.8 -10.2 1.01 0.9 -0.8 12.9

N

CH3

CH3

S

O

O

N

H

C

H

COOH

CH2

-12.1 1.00 0.0 -3.1 17.2 -11.5 1.00 0.0 -2.8 17.1 -11.2 1.01 0.7 -2.1 12.8 -10.5 1.01 0.9 -0.6 12.9

Figure 4. Typical electropherograms of noncharged analytes inhigh-pH BEs. The numbers next to the structures indicate the HDAS-âCD concentrations (mM). Applied effective potential: 17 kV for the10 and 15 mM HDAS-âCD BEs, 12 kV for the 30 and 50 mM HDAS-âCD BEs. Capillary: 25 µm i.d., 39/45 cm effective/total length,uncoated fused silica. Other conditions: see Experimental Section.

Figure 5. Typical electropherograms of noncharged analytes inhigh-pH BEs. Conditions as in Figure 4.

Analytical Chemistry, Vol. 69, No. 20, October 15, 1997 4231

low-pH BEs. Since in the respective low-pH and high-pH BE pairsthe ionic strength is identical and the viscosities (measured byusing the P/ACE unit as a capillary viscosimeter27,28) agree within2%, the lower µeff

NSA- values in the high-pH BEs may be explainedby considering that the high-pH BE constituents (ethanolamine/ethanolammonium and methanesulfonate) interact more stronglywith HDAS-âCD than the low-pH BE constituents (phosphoricacid/dihydrogen phosphate and lithium ion) and/or that thehydrated ion radii of the highly charged cyclodextrins change asthe ionic environment is changed.

Separation of Enantiomers in HDAS-âCD BEs. A seriesof nonionic, strong base, weak base, weak acid, and zwitterionicenantiomers were separated with both low-pH and high-pH BEs.Tables 1 and 2 list the effective mobilities of the less mobileenantiomers (µ), the separation selectivities (R), the measuredpeak resolution values (Rs), the corresponding dimensionless EOflow values (â), and the injector-to-detector potential drop (U).

Since the effective mobilities of the enantiomers are influencedby both the extent of their complexation and the ionic strengthof the BE, the µ values in Tables 1 and 2 cannot (and should not)be interpreted quantitatively, because the ionic strength in theBEs increases very rapidly as the concentration of HDAS-âCD(seven permanent negative charges!) is increased. A few qualita-tive conclusions can be drawn, though, as follows. (i) Fornoncharged analytes, both at low pH and at high pH, the effective(anionic) mobility increases as the concentration of HDAS-âCDis increased, indicating more extensive complexation, whichovercomes the mobility reducing effects of higher ionic strength.(ii) For weak bases, both at low pH and at high pH, the effective(anionic) mobility decreases as the concentration of HDAS-âCDis increased, indicating that complexation is strong, even initially,and the rapidly increasing ionic strength effects reduce theeffective (anionic) mobility of the complex. Chlophedianol (Tables1 and 2) is an exception, both at low pH and at high pH, indicating,possibly, that two HDAS-âCDs are involved in the complexation,and thus the complexation effects can outpace the ionic strengtheffects. (iii) For weak acids, reliable data could not be collectedin the low-pH BEs, because the acids studied are not sufficientlysoluble at low pH. At high pH, when the effective mobility of the

noncomplexed anion is higher than that of the fully complexedanion, both the complexation and the ionic strength effects act inconcert and lead to lower effective mobilities as the concentrationof HDAS-âCD is increased. (iv) Zwitterionics, at low pH, behavelike chlophedianol: the effective mobility becomes increasinglyanionic as the concentration of HDAS-âCD is increased. At highpH, the behavior depends on their respective pI values: the dansylderivatives studied behave as most bases and weak acids, whiletryptophan and phenylalanine behave like chlophedianol.

Separation selectivity can be readily interpreted with the helpof the CHARM model.18 For noncharged analytes, separationselectivity is predicted to decrease as the concentration of HDAS-âCD is increased. This is, indeed, the case at both low and highpH, e.g., selectivity drops from R ) 1.5 at 10 mM to R ) 1.07 at50 mM at low pH (R-methyl-R-phenylsuccinimide) or from R ) 5to R ) 1.2 at high pH (2-phenyl-1-propanol). The selectivity valuesfor noncharged analytes are less distorted by ionic strength effects,because selectivity for the noncharged analytes is a single-termexpression,18 and both the numerator and the denominator areinfluenced similarly. For weak acids, weak bases, and zwitteri-onics, R can increase (flurbiprofen at high pH, pindolol at lowpH), pass a maximum (terbutaline at high pH), or decrease(chlophedianol) with the HDAS-âCD concentration over the rangestudied. According to the CHARM model,18 for these analytes,

(27) Williams, B. A.; Vigh, Gy. Anal. Chem. 1996, 68, 1174.(28) Bello, M. S.; Rezzonico, R.; Righetti, P. G. J. Chromatogr. 1994, 659, 199.

Figure 6. Typical electropherograms of weak base analytes in high-pH BEs. Conditions as in Figure 4.

Figure 7. Typical electropherograms of weak base analytes in high-pH BEs. Conditions as in Figure 4.

Figure 8. Typical electropherograms of weak base analytes in high-pH BEs. Conditions as in Figure 4.

4232 Analytical Chemistry, Vol. 69, No. 20, October 15, 1997

the selectivity trends cannot be predicted a priori without the exactknowledge of the complexation constants and the respective ionicmobilities.

The peak resolution values depend not only on the selectivitiesbut also, very sensitively, on the â values and the magnitude ofthe potential drop between the injector and the detector. There-fore, general conclusions cannot (and should not) be drawn fromthe resolution values: as long as R * 1, manipulation of thedimensionless electroosmotic flow or bulk flow can furnish anydesired peak resolution value (naturally, at the expense ofseparation time29,30). Since, in some cases, the â value was veryclose to -1, the separations were carried out from the “short side”of the capillary to decrease the required separation time.

Finally, a few typical separations, some of them not studied assystematically as those in Tables 1 and 2, are shown in Figures4-10. All BEs were made with the high-pH stock buffer, exceptfor the zwitterionics in Figure 10. The numbers next to theelectropherograms indicate the concentration of HDAS-âCD used(in millimolar). Figures 4 and 5 show the separations of non-charged analytes, Figures 6-8 those of the basic analytes, Figure9 that of the weak acid analytes, and Figure 10 that of thezwitterionics. In general, the peak resolution values are quitelarge, permitting the injection of much larger sample quantitiesfor the accurate quantitation of minor enantiomers or the reductionof the separation times by changing to shorter capillaries.

CONCLUSIONSA new, moderately hydrophobic, single-isomer charged cyclo-

dextrin, the sodium salt of heptakis(2,3-diacetyl-6-sulfato)-â-cyclo-dextrin, has been synthesized and used to separate a variety ofnoncharged, weak acid, strong base, weak base, and zwitterionicracemic enantiomers in low-pH and high-pH BEs. In agreementwith the predictions of the CHARM model of CE enantiomerseparations,18 separation selectivity for the neutral analytesdecreases as the concentration of HDAS-âCD increases. Forcharged analytes, R can increase, decrease, or pass a maximum,depending on the numeric values of the complexation constantsand ionic mobilities. The magnitude of the electroosmotic flow,characterized by the â value, plays a major role in determiningthe success or failure of any particular separation. HDAS-ΒCDproved to be a broadly applicable chiral resolving agent.

ACKNOWLEDGMENTPartial financial support of this project by the Texas Coordina-

tion Board of Higher Education ARP program (Project No. 010366-016) and Beckman Instruments (Fullerton, CA) is gratefullyacknowledged. We are also indebted to CeraStar (Hammond, IN)for the â-cyclodextrin sample used in the synthesis of HDAS-âCD,and Professor D. Romo of Texas A&M University for valuablediscussions.

Received for review March 6, 1997. Accepted July 21,1997.X

AC970252R(29) Culbertson, C. T.; Jorgenson, J. W. Anal. Chem. 1994, 66, 955.(30) Williams, B. A.; Vigh, Gy. Enantiomer 1996, 1, 183. X Abstract published in Advance ACS Abstracts, September 1, 1997.

Figure 9. Typical electropherograms of weak acid analytes in high-pH BEs. Conditions as in Figure 4.

Figure 10. Typical electropherograms of zwitterionic analytes inlow-pH BEs. Conditions as in Figure 4.

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