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Introduction
Abstract
An effort has been made to investigate the chromatographic behavior and to understand the
basic mechanisms in HILIC-based separation of water-soluble vitamins with highly varied
properties on a diol column. The water content of the mobile phase is of utmost importance
because it directly affects the type and extent of interactions of the solutes with the stationary
phase and with the buffered mobile phase. A mixed-mode partitioning-surface adsorption
mechanism enables most precise description of their chromatographic retention and separation. The point at which surface adsorption becomes apparent, however, depends on the
properties of the solutes on the given stationary phase, and on the presence of buffer salt
ions. Adjustment of mobile phase pH and use of different buffer salts can be used to modify
electrostatic interactions among the solutes, active silanols, and counter-ions. The role of
hydrogen bonding was clarified by substitution of ACN by solvents with moderate to strong
hydrogen bonding potential. Analytes which are neutral at the working pH start to interact
with the stationary phase when the ACN content is increased to 80%. Negatively charged
analytes are adsorbed on the stationary phase when the ACN content is approximately 86%,
because augmentation of the counter-ions weakens electrostatic repulsion by the active
silanol groups. On the other hand, the electrostatic attraction of thiamine contributes
significantly to its retention even when using mobile phases with high water content.
Keywords
Column liquid chromatography
Hydrophilic interaction liquid chromatography
Diol column
Chromatographic behavior
Retention models
Water-soluble vitamins
Original
DOI: 10.1365/s10337-010-1564-3
0009-5893/10/05
751
Experimental
Chemicals, Reagents,
and Solutions
752
Chromatography
Liquid chromatographic analysis of the
water-soluble vitamins was conducted
with a Shimadzu (Duisburg, Germany)
HPLC system comprising an LC20AD
pump, a CTO 10AS column oven, and
Original
Fig. 2. Eect of organic modier on separation of the WSV. Conditions: Inertsil diol column
with 10:90 (v/v) aqueous ammonium acetate buer (10 mM, pH 5.0) organic solvent as mobile
phase; ow rate 0.6 mL min-1; column temperature 25 C. 1 = nicotinamide, 2 = pyridoxine,
3 = riboavin, 4 = nicotinic acid, 5 = L-ascorbic acid, 6 = thiamine
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Fig. 3. Eect of pH on the retention factors (k) of the WSV. Mobile phase ACNwater 90:10
(v/v); ow rate 0.6 mL min-1; column temperature 25 C
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mobile phase pH from 3.0 to 6.0. Nicotinic acid has two pKa values
(pKa1 * 2.2 for the carboxyl group and
pKa2 * 4.8 for the pyridinyl group) and
hence, in the pH range 3.04.0 the
carboxylic acid functionality is deprotonated, the amine functionality is partially protonated, and the molecule exists
as a zwitterion. At pH >5.0, the amine
functionality is deprotonated and the
molecule is negatively charged. L-Ascorbic acid, also, has two pKa values in
water (pKa1 * 4.1 and pKa2 * 11.7). At
mobile phase pH 3.0, retention of nicotinic acid and L-ascorbic acid is similar
to that of pyridoxine and riboavin,
causing co-elution of these four analytes
(Fig. 3). On increasing the mobile phase
pH to 4.0, L-ascorbic and nicotinic acids
are charged to a dierent extent and
discriminated from pyridoxine and
riboavin, because they are more polar.
At mobile phase pH 5.0, nicotinic and
L-ascorbic acids are fully resolved with
inversion of the order of elution and
with L-ascorbic acid the later-eluting
compound. At this pH, ascorbic acid
becomes fully deprotonated and, hence,
more polar than at pH 4.0. This may
account for the inversion in the order of
elution of the peaks for this pair of
analytes when changing from pH 4.0 to
pH >5.0. Thiamine is positively
chargedit carries a permanent positive
charge on thiazolium ring (pKa1 of
the pyrimidinyl group *4.80)thus,
undergoing ion-exchange interactions
with residual silanols which leads to
greater retention.
Original
Fig. 4. Eect of buer concentration on separation of the WSV. Conditions: Inertsil diol
column with 90:10 (v/v) ACNwater containing 5, 10, or 20 mM ammonium acetate (pH 5.0) as
mobile phase; ow rate 0.6 mL min-1; column temperature 25 C. Peak assignment as for Fig. 2.
Peaks 5 and 6 at 20 and 5 mM, respectively, do not elute in the time scale of the chromatograms
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Fig. 5. Plots of log k against volume fraction of water in the mobile phase. Conditions: Inertsil
diol column with ACN10 mM aqueous ammonium acetate, pH 5.0, as mobile phase; ow rate
0.6 mL min-1; column temperature 25 C
AS
log u
nw
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Original
Original
Fig. 6. Plots of log k against the logarithm of the volume fraction of water in the mobile phase.
Conditions as for Fig. 5
ln /
RT
R
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aa
Nicotinamide
Pyridoxine
Riboavin
Nicotinic acid
L-Ascorbic acid
Thiamine
-1.29
-1.46
-2.03
-0.64
0.74
0.34
ba
0.23
0.12
0.26
0.28
0.28
0.23
-0.655
-0.954
-1.39
-1.37
-1.08
-1.91
ca
0.093
0.050
0.11
0.15
0.19
0.29
-0.69
-0.36
-1.52
-4.78
-8.07
-7.66
0.37
0.19
0.42
0.61
0.76
0.91
R2
RESmean
RESSD
0.9951
0.9991
0.9985
0.9986
0.9986
0.9952
0.024674
0.012975
0.025509
0.035012
0.043962
0.041138
0.017
0.009
0.023
0.019
0.029
0.034
Nicotinamide
Pyridoxine
Riboavin
Nicotinic acid
L-Ascorbic acid
Thiamine
a
b
ACNwater 80:20a
ACNwater 90:10a
ACNwater 90:10b
R2
DH (kJ mol-1)
R2
DH (kJ mol-1)
R2
DH (kJ mol-1)
0.9975
0.9939
0.9957
0.9926
0.9934
0.9924
-6.67
-9.04
-11.9
-13.6
-21.9
-8.00
0.9966
0.9900
0.9951
0.9847
0.9870
0.9524
-5.65
-8.53
-10.4
-9.53
-19.9
-6.14
0.9983
0.9937
0.9932
0.9929
0.9973
0.9848
-8.47
-13.0
-16.9
-16.4
-32.6
-8.44
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Conclusion
The mechanism of retention of WSV in
HILIC is multimodal. Apart from partitioning, specic interactions, that may
predominate, depend strongly on the
structure of the analytes, the nature of
the stationary phase, and the composition of the mobile phase. Deviation
from the partition mechanism when
highly organic mobile phases are used is
evidence of the occurrence of both
partitioning and surface adsorption.
Data gathered from this study indicate
that above 80% ACN there is a change
in the relative contributions of these
two mechanisms and adsorption becomes more signicant. The contribution of the adsorption mechanism for
the charged WSV depends on the extent
Original
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