CHIRAL SEPARATION OF FUCOSE AND PIPECOLIC ACID USING CE
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and the mixture was heated for an additional 60 min at 90 ꢀC. Finally,
the samples were diluted with water to a concentration of 6 mM and
stored at –20 ꢀC.
In this study, simple, efficient, and rapid micellar electroki-
netic chromatography (MEKC), cyclodextrin electrokinetic
chromatography (CD-EKC), and CD-MEKC methods were
developed and are presented in this report for the baseline
separation of the enantiomers of pipecolic acid and fucose.
The methods involve simple derivatization of the analytes
with the derivatization agents FMOC-Cl and 5-amino-2-naph-
thalene-sulfonic acid (ANSA), respectively. For the separation
of the enantiomers of pipecolic acid and fucose, different poly-
meric surfactants and CDs were used as chiral selectors.
When the optimum chiral selector was determined for each
analyte, several parameters were varied, such as the concen-
tration of the chiral selector, the addition of modifiers, the
applied voltage, and of the chiral selector, the addition of
modifiers, the applied voltage, and the column temperature.
It is important here to note that in this study the ionic liquid
D-alanine tert-butyl ester lactate (D-alaC4NTf2), which was
synthesized according to a procedure reported by Bwambok
et al.,15 was used as an additive in the background electrolyte
(BGE) in order to improve the enantioseparation of pipecolic
acid. To the best of our knowledge, this is the first time that
an amino ester-based ionic liquid was used as an additive in
the BGE in order to improve enantioseparations.
Derivatization of Pipecolic Acid
For the derivatization of pipecolic acid, FMOC-Cl was used as the
proper reagent. Briefly, 10 mM of pipecolic acid and 10 mM of FMOC-
Cl were dissolved in borate buffer (40 mM borax, pH 9) and in acetonitrile
(ACN), respectively. Then, 200 mL of FMOC-Cl solution was mixed with
200 mL of pipecolic acid. After reacting at room temperature for 2 min,
the mixture was extracted with 0.5 mL of pentane to remove the excess
FMOC-Cl. The solution was diluted with water 10 times.
Synthesis of D-alanine tert-butyl ester Lactate
The ionic liquid D-alaC4NTf2 was synthesized according to the proce-
dure reported elsewhere.15 Briefly, the appropriate amount of D-alaC4Cl
was dissolved in methanol and an equimolar amount of silver lactate
was suspended separately in methanol. The two solutions were then
mixed and stirred at room temperature (RT). The obtained precipitated
was filtered and the remained clear solution was evaporated and purified
by crystallizing in methanol/diethyl ether.
RESULTS AND DISCUSSION
Derivatization of Fucose
It is very important here to point out the reason why a sat-
urated solution of fucose was used for the reaction. At equilib-
rium, the concentration of open-chain DL-fucose in a solution
is very small. Solutions of DL-fucose give no observable UV
absorption band for a carbonyl group, and solutions of
DL-fucose with Schiff’s reagent (ANSA) give a negative test.
When the fucose solution was not saturated (600 mM), the
reagent (ANSA 600 mM) did not react completely, so two
peaks were obtained instead of one, and they corresponded
to the derivatized fucose compound and the reagent ANSA.
On the other hand, when the fucose solution was saturated
(1.2 M), the reagent (ANSA 600 mM) reacted almost completely.
In this case, a high-intensity peak that corresponded to the ana-
lyte under study and a very low-intensity peak that corresponded
to the reagent were observed (data not shown). It is important
here to note that due to the above-mentioned problem, this
particular derivatizing agent cannot be used for the quantitation
of fucose in real samples.
MATERIALS AND METHODS
Instrumentation
All experiments were carried out on a PrinCE 760 Capillary Electropho-
resis System (Emmen, Netherlands), which consisted of a regulated
high-voltage power supply and DAD for UV detection. The wavelength
was set at 214 and 254 nm for the detection of the enantiomers of
pipecolic acid and fucose, respectively. The instrument was controlled
by DAx 3D software for the collection of all experimental data.
Fused-silica capillaries of 57.0 cm (50.0 cm effective length) x 50 mm i.d.
were purchased from Polymicro Technologies (Phoenix, AZ). New capil-
laries were rinsed with water for 10 min, followed by 1 M NaOH for 1 h,
again with water for 10 min, and finally with the BGE for 30 min. Between
runs the capillary was rinsed with 0.1 M NaOH for 2 min, water for 2 min,
and finally with BGE for 5 min. The temperature of the capillary cassette
ranged from 15 to 25 ꢀC and the applied voltage ranged from 15 to 25 kV.
Samples were injected hydrodynamically by applying a pressure of
50 mbar for 6 sec.
Chemical Reagents and Materials
Chiral Separation of Fucose
The racemic mixture of pipecolic acid, the enantiomers D- and L-
pipecolic acid, the enantiomers D- and L-fucose, sodium tetraborate
decahydrate (borax, B4Na2O7.10H2O), a-, b- and g-cyclodextrins (a-, b-
and g-CDs), ANSA (C10H9NO3S), silver lactate, D-alaline tert-butyl ester
hydrochloride, and sodium cyanoborohydride were purchased from
Sigma-Aldrich (Steinheim, Germany). FMOC-Cl and SDS were pur-
chased from Fluka (Steinheim, Germany). Isopropanol (IPA), methanol,
pentane, ACN, hydrochloride (HCl), and diethyl ether were purchased
from Merck (Darmstadt, Germany). The polymeric surfactants poly
(sodium N-undecanoyl-LL-leucyl-valinate) (poly-LL-SULV), poly (sodium
N-undecanoyl-LL-valine-valinate) (poly-LL-SUVV), and poly (sodium N-
undecanoyl-LL-alanine-valinate) (poly-LL-SUAV) were synthesized at
Louisiana State University by professor I.M. Warner’s research group.
An appropriate amount of sodium tetraborate decahydrate
was dissolved in water to give a concentration of 40 mM,
and the pH of the BGE was adjusted using 1 M NaOH at
pH 9.5. Different chiral selectors, such as poly(sodium N-
undecanoyl-LL-leucine-valinate) (poly-LL-SULV), poly-LL-SUVV,
poly-LL-SUAV, a-, b- and g-CDs, were examined for the baseline
separation of the enantiomers of fucose. Enantioselectivity was
observed only with b-CD (data not shown). The fucose chain
protruded outwards, interacting with the C-3 hydroxyl group
on the b-CD molecule via its C-2 hydroxyl group, which appears
to govern this chiral recognition. Varying the concentration of
b-CD in the BGE optimized the separation of the ANSA-DL-
fucose enantiomers. Three concentrations were examined (10,
15, and 20 mM) and the electropherograms obtained are demon-
strated in Figure 1. An increase in b-CD concentration from
10 mM to 15 mM resulted in a minor decrease in electrophoretic
migration, probably due to an increase in complexation. When
b-CD concentration becomes equal to or larger than 10 mM,
the analytes are probably fully complexed and a further in-
crease in concentration has minor effects on migration times.16
Chirality DOI 10.1002/chir
Derivatization of Fucose
Fucose was derivatized by a Schiff-Base (ANSA). The reaction was
performed between the aromatic amine of the reagent and the aldehyde
form of fucose, followed by reduction of the Schiff base. Fucose was
dissolved in distilled water to give a concentration of 1.2 M and stored
in a freezer prior to derivatization. To 200 mL of the reagent solution
(600 mM ANSA, pH 6, adjusted with 1 M HCl) 200 mL of fucose solution
was added. The mixture was heated at 90 ꢀC for 10 min. Then, 80 mL of
the reducing agent, sodium cyanoborohydride (0.3 mg/mL), was added