form of polyphenols may be responsible for some of this
therapeutic effect; however, studies on related compounds (biofla-
vonoids)19,20 have shown that excessive consumption by expectant
women could elevate the risk for leukemia in their infants.
In general, polyphenols are encountered in various biological
samples21 as complex mixtures of homologues and isomers with
differing degrees and sites of polymerization (see Figure 1) and
thus represent a truly challenging separation problem. Most of
the methods currently employed to separate polyphenols use high
performance liquid chromatography (HPLC)22-24 and, to a lesser
extent, gas chromatography.25 More recently, capillary electro-
phoresis (CE) has been used for the analysis of polyphenols;26-30
however, none of the current methods can provide complete
resolution of all of the constituents in these very complicated
samples.
In some CE studies, alkylammonium salts have been used as
electroosmotic flow (EOF) modifiers.31-33. Recently, however,
Yanes and co-workers34 reported the development of a fairly robust
capillary electrophoretic method for the separation of polyphenols
found in grape seed extracts that uses tetraethylammonium
tetrafluoroborate (TEA-TFB) as the only electrolyte in the
background electrolyte. In this study, the cation not only acted
as an EOF modifier but also played an active role through
association with the polyphenols. The excellent reproducibility that
was achieved was attributed to the coating of the capillary wall
by the tetraalkylammonium cations with a permanent charge
group not subject to pH-induced variations in ionization.
The purpose of the present study is to investigate the potential
application of ionic liquids to the capillary electrophoretic separa-
tion of polyphenols. Currently, the majority of studies pertaining
to ionic liquids focus on their characterization and their potential
use in industrial processes. However, given the similarity of
structure and properties of the TEA-TFB salt to the so-called
“green chemistry” solvents of the ionic liquids, the extension of
the previous results that were obtained using TEA-TFB in the
separation of polyphenols to ionic liquids seems a natural progres-
sion. The present work describes a simple and reproducible
electrophoretic method for the analysis of phenolic compounds
found in grape seed extracts. The method primarily involves the
use of 1-alkyl-3-methylimidazolium-based ionic liquids as the
running electrolyte solutions.
EXPERIMENTAL SECTION
Materials. The (-)-catechin, (-)-epicatechin, (-)-catechin
gallate, (-)-gallocatechin gallate, (-)-epicatechin gallate, gallic
acid, and resveratrol were purchased from Sigma Chemical Co.
(St, Louis, MO). The 1E-3MI-TFB, 1E-3MI-HFP, 1-ethyl-3-methyl-
imidazolium nitrate (1E-3MI-NT) and 1-ethyl-3-methylimidazolium
trifluoromethanesulfonate (1E-3MI-TFMS) were purchased from
Fluka Chemical Corp. (Ronkondoma, NY). Sodium hydroxide,
hydrochloric acid, and nitromethane were purchased from Fisher
Scientific (Fair Lawn, NJ). All other chemicals were purchased
from either Aldrich Chemical Co. or Fisher Scientific and used
as is unless otherwise specified in the procedures. Filters (0.2
µm) were obtained from Nalge Nunc International Corporation
(Rochester, NY). The fused-silica capillaries were obtained from
Bio-Rad Laboratories, Inc. (Hercules, CA).
The 1B-3MI-TFB and 1B-3MI-HFP were prepared from 1-butyl-
3-methylimidazolium chloride (1B-3MI-Cl) according to the fol-
lowing procedures.
1B-3MI-Cl. The 1B-3MI-Cl was prepared by a slight modifica-
tion of a literature procedure.35 A 50-mL aliquot of 1-methylimi-
dazole (dried over MgSO4) was combined with 250 mL of distilled
1-chlorobutane and allowed to reflux for 24 h. The excess
1-chlorobutane was removed by rotary evaporation, leaving a clear,
yellow, viscous oil. This oil was heated (100 °C) under vacuum
for 8 h to remove any remaining solvent, then placed in a freezer
overnight. As the oil warmed to room temperature, a hard off-
white precipitate formed. This solid was dissolved with heating
and stirring in a mixture of acetonitrile (∼30 mL) and ethanol
(∼10 mL) that had been dried over MgSO4. The solution was
layered with anhydrous diethyl ether (∼10 mL) and placed into
the freezer overnight. The next day, a white precipitate had
formed. It was isolated by vacuum filtration and placed under
vacuum for 24 h. (mp ) 65-69° C).
1 B-3 MI-TFB and 1 B-3 MI-HFP . The ionic liquids were
prepared by a slight modification of a literature procedure.36 The
chloride salt (1B-3MI-Cl) was added to ∼50 mL of acetone,
forming two layers. One equiv of the appropriate salt (NaBF4 or
KPF6) was added, and the mixture was stirred at room temper-
ature for 24 h. The resulting NaCl or KCl precipitate was then
filtered off, and the solvent was removed by rotary evaporation to
leave a yellowish, clear liquid that was filtered through activated
charcoal to lighten the color. (fp ) -76 °C for BF4 and -8 °C for
PF6, both within the reported ranges).2,14
(17) Tsuchiya, H.; Sato, M.; Kato, H.; Okubo, T.; Juneja, L. R.; Kim, M. J.
Chromatogr., B 1 9 9 7 , 703, 253-258.
(18) Ho, Y.; Lee, Y. L.; Hsu, K. Y. J. Chromatogr., B 1 9 9 5 , 665, 383-389.
(19) Hollon, T. Scientist 2 0 0 0 , 14, 21.
(20) Strick, R.; Strissel, P. L.; Borgers, S.; Smith, S. L.; Rowley, J. D. Proc. Natl.
Acad. Sci. U.S.A. 2 0 0 0 , 97, 4790-4795.
(21) Haslam, E. Practical Polyphenolics, From Structure to Molecular Recognition
and Physiological Action; Cambridge University Press: Cambridge, 1998; p
10.
(22) Revilla, I.; Perez-Magarino, S.; Gonzalez-San Jose, M. L.; Beltran, S. J.
Chromatogr., A 1 9 9 9 , 847, 83-90.
(23) Bartolome, B.; Hernandez, T.; Bengoechea, M. L.; Quesada, C.; Gomez-
Cordoves, C.; Estrella, I. J. Chromatogr., A 1 9 9 6 , 723, 19-26.
(24) Dalluge, J. J.; Nelson, B. C.; Thomas, J. B.; Sander, L. C. J. Chromatogr., A
1 9 9 8 , 793, 265-274.
(25) Collier, P. D.; Mallows, R. J. Chromatogr. 1 9 7 1 , 57, 29-45.
(26) Andrade, P.; Seabra, R.; Ferreira, M.; Ferreres, F.; Garcia-Viguera, C. Z.
Lebensm Unters-Forsh., Teil A 1 9 9 8 , 206, 161-164.
(27) Prasongsidh, B.; Skurray, G. R. Food Chem. 1 9 9 8 , 62, 355-358.
(28) Horie, H.; Mukai, T.; Kohata, K. J. Chromatogr., A 1 9 9 7 , 758, 332-335.
(29) Nelson, B. C.; Thomas, J. B.; Wise, S. A.; Dalluge, J. J. J. Microcolumn Sep.
1 9 9 8 , 8, 671-679.
(30) Tomas-Barberan, F. A.; Garcia-Viguera, C. Analusis 1 9 9 7 , 25, M23.
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766-770.
Equipment. UV spectra were obtained using a Hewlett-
Packard model 8453 UV-vis spectrophotometer and a 1-cm quartz
cell. Solutions for absorbance measurements were prepared in
30-mL plastic bottles. All of the CE experiments were carried out
using either a Bio-Rad BioFocus 2000 or a Bio-Rad BioFocus 3000
(35) Dyson, P. J.; Grossel, M. C.; Srinivasan, N.; Vine, T.; Welton, T.; Williams,
D. J.; White, A. J. P.; Zigras, T. J. Chem. Soc., Dalton Trans. 1 9 9 7 , 3465-
3469.
(32) Harrold, M. P.; Wojtusik, M. J.; Riviello, J.; Henson, P. J. Chromatogr. 1 9 9 3 ,
640, 463-471.
(33) Quang, C.; Khaledi, M. G. Anal. Chem. 1 9 9 3 , 65, 3354-3358.
(34) Yanes, E. G.; Gratz, S. R.; Stalcup, A. M. Analyst 2 0 0 0 , 125, 1919-1923.
(36) Suarez, P. A. Z.; Dullius, J. E. L.; Einloft, S.; DeSouza, R. F.; Dupont, J.
Polyhedron 1 9 9 6 , 15, 1217-1219.
Analytical Chemistry, Vol. 73, No. 16, August 15, 2001 3839