Phosphatidic acid-functionalized monolithic stationary phase for reversed-phase/cation-exchange mixed mode chromatography

Article abstract of DOI:10.1039/c6ra21504a

A novel phosphatidic acid functionalized polymeric monolithic column was prepared through the thermally initiated co-polymerization of 12-methacryloyl dodecylphosphatidic acid (MDPA) and ethylene glycol dimethacrylate (EDMA) in the presence of 1,4-butanediol and isopropanol as porogens within 100 μm I.D. capillaries. The polymerization conditions of monolithic columns were systematically optimized in order to obtain good permeability, stability and column efficiency. The reproducibility of the optimized monolithic column was also satisfactory. The physicochemical properties of the monolithic column were evaluated by use of instrumental techniques including scanning electron microscopy, Fourier transform infrared spectra, ζ-potential analysis and micro-HPLC. A series of test compounds such as small peptides, alkylphenones, etc., were employed to investigate the retention mechanism on the poly(MDPA-co-EDMA) monolithic column. The results demonstrate that both hydrophobic and cation-exchange interactions could contribute to the overall retention of analytes. Furthermore, the novel reversed-phase/cation-exchange mixed mode monolithic column was applied to the separations of small peptides, phenols, water-soluble vitamins B, and pharmaceutical compounds. The successful applications indicate the potential of the poly(MDPA-co-EDMA) monolithic column in complex sample analysis.

Full text of DOI:10.1039/c6ra21504a

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Paper
Phosphatidic acid-functionalized monolithic stationary phase for
reversed-phase/cation-exchange mixed mode chromatography†
Received 00th January 20xx,
Accepted 00th January 20xx
Kun Peng,
Jiang*c and Fuhai Wu*b
‡ ‡
a,b,c Qiqin Wang, c Weijia Chen,c Donghai Xia,c Zhengyin Zhou,c Yuqiang Wang,c Zhengjin
DOI: 10.1039/x0xx00000x
A novel phosphatidic acid functionalized polymeric monolithic column was prepared through the thermally initiated co-
polymerization of 12-methacryloyl dodecylphosphatidic acid (MDPA) and ethylene glycol dimethacrylate (EDMA) in the
presence of 1,4-butanediol and isopropanol as porogens within 100 μm I.D. capillaries. The polymerization conditions of
monolithic column were systematically optimized in order to obtain good permeability, stability and column efficiency.
The reproducibility of the optimized monolithic column was also satisfactory. The physicochemical properties of the
monolithic column were evaluated by use of instrumental techniques including scanning electron microscopy, fourier
transform infrared spectra, ζ-potential analysis and micro-HPLC. A series of test compounds such as small peptides,
alkylphenones, etc, were employed to investigate the retention mechanism on the poly(MDPA-co-EDMA) monolithic
column. The results demonstrate that both hydrophobic and cation-exchange interactions could contribute to the overall
retention of analytes. Furthermore, the novel reversed-phase/cation-exchange mixed mode monolithic column was
applied to the separations of small peptides, phenols, water-soluble vitamins B, and pharmaceutical compounds. The
successful applications indicate the potential of the poly(MDPA-co-EDMA) monolithic column in complex sample analysis.
www.rsc.org/
isothiocyanatopropyltriethoxy modified ZOR-BAX Rx-SIL silica
particles for the separation of membrane peptides and
proteins with different size, charge and hydrophobicity. The
Introduction
Recently, phospholipid functionalized stationary phases have
received great attention and became an important tool for
pharmaceutical industry and academia with numerous
applications, such as predicting the drug-cell membrane
interaction, purification of membrane protein and separation
of small molecules.1-3 So far, only phosphatidylcholine (PC)
functionalized silica columns are commercially available. A few
attempts have been directed to develop other phospholipids
functionalized stationary phases.1,4,5 Phosphatidic acid (PA),
the simplest anionic diacylglycerophospholipid, possesses long
alkyl chains and phosphate groups and exhibits versatile
complexation abilities, which is essential for the proper
functioning of several transmembrane proteins and the
physiology of cell membrane.2,6 Davies-Tuck et al.2 developed a
retention of membrane peptides and proteins on this PA
functionalized column was governed by both hydrophobic and
cation-exchange interactions. More recently, Bocian et al.7
prepared a series of alkyl (C10 or C18)-phosphate functionalized
silica columns, which have an opposite spatial arrangement of
long alkyl chains and phosphate groups. Good peak shapes and
highly selectivity for β-blockers and nucleosides were obtained
on these columns. It was found that the hydrophobic
interaction is responsible for the retention and separation of
these analytes. Therefore, developing PA functionalized
stationary phases could not only enrich the type of
phospholipids functionalized column for better mimicking the
drug-cell membrane interactions, but also provide unique
reversed-phase/cation-exchange mixed mode columns for the
specific and selective separation of
a wide range of
a.School of Pharmacy, Guangdong Pharmaceutical University, Guangzhou 510006,
PR China.
compounds. The reversed-phase/cation-exchange mix mode
technique has been growing rapidly because it offers more
than one retention modes to improve the selectivity for
simultaneous separation of complicated samples.8,9 So far,
most reported PA functionalized stationary phases are silica
based columns. The residual silanol and/or amino groups on
these silica based stationary phases may influence their
stability and the chromatographic behavior of small
molecules.10,11 End-capping with glycidol/methylglycolate (MG)
or decanoic/propionic anhydride could be employed to
overcome this issue.1,2,7 However, the preparation process is
b.School of Public Health, Guangdong Pharmaceutical University, Guangzhou
510006, PR China.
c. Institute of Pharmaceutical Analysis, College of Pharmacy, Jinan University,
Guangzhou, Guangdong, 510632, China.
d.
†
Electronic Supplementary Information (ESI) available: [details of any
supplementary information available should be included here]. See
DOI: 10.1039/x0xx00000x
*
Corresponding
jzjjackson@hotmail.com (Z. Jiang).
K.Peng and Q. Q. Wang contributed equally to this work.
authors
E-mail:
fuhaiwu@163.com
(F.
Wu),
‡
strategy
to
immobilize
PA
onto
3-
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rather complicated, which could limit its further applications Japan), while Ala-Val and Leu-Val were purchased from
involved self-lab preparation or chemical modification of silica- Bachem AG (Bubendorf, Switzerland). Vitamin B1 (VB1), vitamin
based phospholipid stationary phases.1 It is indeed meaningful B6 (VB6) and vitamin B4 (VB4) were obtained from Energy
to develop new strategies for preparing PA functionalized chemical (Shanghai, China). The chemical structures of some
stationary phases.
test compounds were presented in Fig. S1. Phosphorus
In the last decade, monolithic columns have proved to be an oxychloride was purchased from Aike Reagent Corp. (Chengdu,
effective alternative to packed columns and have attracted China). Cytochrome C (Cyt-C) was obtained from Sigma-Aldrich
considerable interest, owing to their facile preparation (St. Louis, MO, USA) and the tryptic digest procedure of Cyt-C
methodology, good column characteristics with respect to was according to previously reported method.20 HPLC-grade
permeability, efficiency, stability and selectivity.12-18 Few methanol (MeOH) and acetonitrile (ACN) were supplied by
attempts have been made to develop phospholipids Merck (Shanghai, China). The distilled H2O used throughout all
functionalized monolithic columns. Jiang et al.3,19 prepared experiments was deionized and purified using a Milli-Q system
single chain PC and mixed phospholipids (single PC and (Bedford, MA, USA) and filtered through a 0.22 μm membrane
phosphatidylserine (PS)) functionalized monolithic stationary before use. The fused silica capillaries with a dimension of 100
phases for predicting drug-cell membrane interactions and μm I.D. (375 μm O.D.) were purchased from Yongnian Optic
screening the phospholipidosis-inducing potential of Fiber Plant (Hebei, China).
pharmaceuticals, respectively. However, few studies have
Instrumentation
been focused on PA functionalized monolithic columns. Zou et
1H NMR spectra was carried out using tetramethylsilane (TMS)
as the internal standard in CDCl3 with a Bruker-AVANCE III
Digital NMR Spectrometer (Karlsruhe, Germany) at 300 MHz.
Mass spectra (MS) was performed on a Agilent 1260 Infinity
Hybrid SFC/UHPLC analytical system (Santa Clara, CA, USA)
al.20 prepared an ethylene glycol methacrylate phosphate
(EGMP) functionalized monolithic column and then coupled it
with a reversed phase column for online multidimensional
separation of the tryptic digest of yeast protein. Moreover,
EGMP functionalized monolithic column was also extensively
applied to develop immobilized metal ion affinity
chromatography for phosphopeptides enrichment.21 These
results demonstrated the possibility and prospect to prepare
PA functionalized monolithic columns with long alkyl chains.
In this study, a phosphatidic acid monomer 12-methacryloyl
dodecylphosphatidic acid (MDPA) was synthesized and
coupled with
a Agilent 6130 single quadrupole mass
spectrometry detector (Santa Clara, CA, USA). A Jinghong DK-
S22 water bath (Shanghai, China) was used for thermally
initiated copolymerization. A Leo 1530 VP Field Emission
Scanning Electron Microscope, equipped with Oxford INCA 400
energy dispersive X-ray microanalysis (Wiesbaden, Germany),
was employed to evaluate the morphology of the monolithic
stationary phases. The ζ-potential values of monolithic
materials were measured by Nano-ZS ζ-potential meter
(Malvern, UK). Fourier transform infrared (FT-IR) spectra were
obtained on a JASCO FT/IR-4600 spectrophotometer (Tokyo,
Japan) within the wavenumber range of 4000-400 cm-1 using
KBr pellets. All micro-HPLC experiments were performed on a
Dionex Ultimate 3000 RSLC nano system (Sunnyvale, CA, USA)
consisted of an Ultimate 3000 RS variable wavelength detector
with a 3 nL flow cell, an Ultimate 3000 Binary Rapid Separation
nano flow pump, an Ultimate 3000 RS autosampler and a four-
port injection valve with 20 nL internal loop (Houston, TX,
USA). Data acquisition and date handling were carried out
using Chromeleon 6.8. All chromatograms were converted into
.txt files and then redrawn using Microcal Origin 8.5. The pH
values were monitored using a Sartorius PB-10 pH meter
(Gottingen, Germany).
employed to prepare
dimethacrylate) (poly(MDPA-co-EDMA)) monolithic column
Fig. 1). The composition of the polymerization mixture was
a poly(MDPA-co-ethylene glycol
(
carefully optimized in order to obtain satisfactory column
permeability, efficiency and selectivity. The retention
mechanism of the poly(MDPA-co-EDMA) monolithic column
was also systemically investigated using a series of small
peptides as probe. Finally, the applicability of this monolithic
column was evaluated using a variety of analytes.
Experimental
Materials and chemicals
EDMA,
2,2'-azobis(2-methylpropionitrile)
(AIBN),
3-
(trimethoxysilyl)propyl methacrylate (γ-MAPS), 1,4-butanediol,
isopropanol, ammonium formate, formic acid, methacryloyl
chloride, 1,12-dodecanediol, trimethylamine, and some test
analysts (including toluene, thiourea, dimethylphthalate,
Synthesis of the monomer MDPA
anisole, naphthalene,
butyrophenone,
acetophenone, propiophenone,
hexanophenone,
MDPA was synthesized according to a previously reported
(Fig. 1
).22 Firstly, the
valerophenone,
method with proper modification
heptanophenone, octanophenone, vitamin B12 (VB12),
intermediate 1 (3.0 g, 10 mmol) was synthesized as described
previously,22 and then dissolved in 20 mL of phosphorus
oxychloride. The reaction mixture was stirred at room
temperature for 12 h, and it was then poured into 200 mL ice
water until a large amount of white precipitates formed. The
solution was directly extracted with ethyl acetate thrice
without filtration, and the organic layer was collected and
paracetamol,
acetylsalicylic
phenol,
acid,
p-nitrophenol,
p-bromophenol,
caffeine,
sulfanilamide,
benzenesulfonamide, phenacetin, coumarin, ranitidine, and
sulpiride) were purchased from Aladdin Chemicals (Shanghai,
China). Ala-Gly-Gly, Leu-Gly-Gly, Gly-Leu and Gly-Phe were
purchased from Tokyo Chemical Industry Co., Ltd. (Tokyo,
2 | J. Name., 2012, 00, 1-3
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concentrated under reduced pressure. The residue was into MeOH to give a concentration around 1 mg/mL. Both
purified by flash column chromatography on silica gel mobile phases and sample solutions were subjected to a
(CH2Cl2/MeOH = 30 : 1) to give the compound 2 as a straw filtration through 0.22 μm membrane filter prior to the HPLC
yellow oil (2.1 g, 60% yield). ESI-MS m/z: 350.9 [M+H]-. 1H NMR experiments. Chromatograms were recorded at wavelength of
(300 MHz, CDCl3) δ 6.11 (s, 1H), 5.56 (s, 1H), 4.14 (t, 2H), 3.78 214 or 254 nm.
(t, 2H), 1.96 (s, 3H), 1.75-1.60 (m, 4H), 1.43-1.18 (m, 16H).
Calculation
Permeability (K, m2) of the monolithic column was calculated
according to Darcy’s Law by using the following equation (1) 24
uLη
:
K =
(1)
∆P
where u (m/s), η (Pa/s), L (m) and ∆P (Pa) refer to the linear
velocity of mobile phase, the viscosity of mobile phase, the
length of the monolithic column and the pressure drop across
the monolithic column, respectively.
Chemical stability of the monolithic column can be
evaluated through the swelling propensity (SP) test. The SP
factor was calculated using equation (2)25:
Fig. 1. Synthesis of monomer MDPA and preparation of the
poly(MDPA-co-EDMA) monolithic columns. Reagents and
conditions: (i) Methacryloyl chloride, pyridine (rt); (ii) POCl3 (0 °C);
(iii) H2O, (from 0 °C to rt).
p(Solvent)-p(H2O)
SP =
(2)
p(H2O)
where the p value is the ratio of the mobile phase pressure
Preparation of the poly(MDPA-co-EDMA) monolithic column
drop and the corresponding viscosity. This SP factor
Prior to the copolymerization, the inner wall of the fused-silica characterizes the swelling degree of the monolithic material in
capillaries (100 μm I.D.) were pretreated with γ-MAPS various organic solvents compared to H2O.
according to the previously described method.23 The
polymerization mixtures used for the preparation of the
Results and discussion
poly(MDPA-co-EDMA) monolithic column was consisted of the
functional monomer (MDPA), the crosslinker (EDMA), the
initiator (AIBN, ~1 wt % with respect to the monomers) and
the porogens (1,4-butanediol and isopropanol). Before
introducing it into the capillary, the polymerization mixture
was degassed by sonicating for 15 min. The sonication also
facilitate the solubility of the monomers. The homogeneous
polymerization mixture was then manually introduced into the
pretreated capillary. Both ends of the capillary were sealed
with GC septa. The filled capillary and the sealed 2-mL vial
containing the rest of polymerization mixture were submerged
into a water bath at 60 °C for 12 h. The copolymerized
monolithic column was then rinsed with MeOH to flush out the
porogens and residual reagents.
Preparation of the poly(MDPA-co-EDMA) monolithic column
Because the composition of the polymerization mixture
significantly affects the permeability, morphology, efficiency
and selectivity of the monolithic column, several factors
including the composition and the weight content of porogens,
and the weight content of the crosslinker EDMA were
systemically optimized by evaluating the physicochemical
properties and morphology of the resulting monolithic
columns using micro-HPLC (Table S1) and SEM (Fig. 2).
The effect of the porogens composition was first
investigated by varying the weight ratio of isopropanol and
1,4-butanediol (w/w) from 86/14 (column C1) to 80/20
(column C3). It was noted that the minor change of the
porogens composition has a significant influence on the
column backpressure. As can be seen from Table S1, the
backpressure significantly increased from 2.3 to 8.0 MPa with
decreasing the weight content of isopropanol in the porogens
mixture from 86% to 80%, and column C2 exhibited the
highest column efficiency (20,000 plates/m). Therefore the
weight ratio of 83/17 between isopropanol and 1,4-butanediol
was selected for further experiments. It was also observed that
the crosslinker EDMA content in the monomer mixture has
some effects on the monolithic column properties. When the
weight content of EDMA increased from 40% (column C4) to
50% (column C5), the column backpressure was found to rise
from 2.4 MPa to 6.0 MPa and the column efficiency varied
from 17000 to 6700 plates/m. An EDMA weight content of
45% (column C2) in the monomer mixture provides the highest
column efficiency (20000 plates/m) and reasonable
A 1 cm length of monolithic column was cut for scanning
electron microscopy (SEM) analysis. The bulk polymer in the 2-
mL vial was cut into small pieces, Soxhlet extracted with MeOH
for 16 h, and then dried under vacuum at 60 °C. These
polymers were used for ζ-potential measurements and FI-IR
spectra.
Chromatographic conditions
The mobile phases were prepared by mixing appropriate
proportions (v/v) of buffer solution, H2O and ACN. The stock
buffer solutions of ammonium formate were prepared by
dissolving an appropriate amount of the salt in deionized H2O
and adjusted to desired pH with formic acid. Unless otherwise
stated, the mobile phase pH values mentioned in this research
refer to the aqueous portion only. The stock sample solutions
were prepared by dissolving appropriate amount of samples
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backpressure (3.1 MPa). The influence of the porogens content different linear velocities. Thiourea was selected as the dead-
in the polymerization mixture was also evaluated. As can be time marker to determine the linear velocities. Fig. 3 shows
seen in Table S1, the weight content of porogens was the linear relationships between linear velocity and column
increased from 55% (column C6) to 65% (column C7) (w/w), backpressure using four different mobile phases. The excellent
both the column backpressure and efficiency decreased. linearity (R2 ≈ 0.999) attests the good mechanical stability of
Finally the polymerization conditions used to produce column the poly(MDPA-co-EDMA) monolithic column over the
C2 were selected for all further studies.
pressure range from 1 to 12 MPa. As can be seen in Table S2
,
the calculated permeability values for column C2 were 2.25 ×
K
Characterization of the poly(MDPA-co-EDMA) monolithic
10−13, 1.54 × 10−13, 1.20 × 10−13 and 0.09 × 10−13 m2 using ACN,
MeOH, H2O/ACN (50/50, v/v) and H2O as mobile phase,
respectively. The comparable permeability, when different
solvents were passed through the column, also indicates that
the monolithic column does not swell or shrink obviously in
solvents of different polarity. The SP factor of column C2 was
also calculated, and the measured SP factors are 0.24, 0.03
and 0.13 for ACN, MeOH and H2O/ACN (50/50, v/v),
SEM, FI-IR, and surface charge characterization. The morphology
of the optimized poly(MDPA-co-EDMA) monolithic column C2
was characterized using SEM. As can be seen from Fig. 2, the
monolithic matrix attached well to the inner wall of capillary
and large clusters of polymer were formed with
interconnected voids, which provides a strong evidence for the
successful copolymerization reaction. The presence of MDPA
in the monolithic stationary phase was confirmed by the FT-IR
spectra. As shown in Fig. S2, the characteristic absorbance at ν
≈ 3,300-3,600 cm-1, 1,244 cm-1, and 1,153 cm-1 are assigned to
the O-H vibrations in hydroxyl groups, the P=O and P-O-alkyl
stretching of monomer MDPA, respectively. These results
confirmed that phosphatidic acid functionalities were
successfully introduced into the monolithic matrix.
respectively. According to equation (2),
a non-swelling
material will have an SP value of 0. The higher the SP factor is,
the more the material swells , and shrinkage is indicated by a
value below 0.27 Therefore, these values further confirm that
the prepared monolithic materials were rigid and no significant
swelling and shrinkage was observed.
It is well known that the surface charge of monolithic
stationary phases could play a decisive role in the retention
characteristics of charged analytes.
12
8
R2=0.9989
H O
2
H O/ACN (50/50,v/v)
2
R2=0.9998
ACN
MeOH
R2=0.9999
R2=0.9997
4
0
Fig. 2. Scanning electron microphotographs of the poly(MDPA-
co-EDMA) monolithic column.
0
4
8
12
Linear velocity (mm/s)
Fig. 3. Correlation of backpressure and linear velocity for
The materials with PA functionality have a negative surface
charge over a wide pH range (Fig. S3).26 In order to further poly(MDPA-co-EDMA) monolithic column with different
confirm the surface charge of the poly(MDPA-co-EDMA)
monolithic column, which contains the phosphoric acid groups,
ζ-potential measurements of the bulk polymer were
performed in pH-dependent manner over a pH range of 3 to 7.
As shown in (Fig. S3), the ζ-potential values significantly
decreased with increasing pH due to the amount of
deprotonated phosphate group. This may also indicate the
phosphatidic acid functionalities were immobilized successfully
on the polymeric surface but not embodied into matrix.
mobile phases. Conditions: column dimensions, 140 mm × 100
μm I.D.; mobile phase, ACN, MeOH, H2O and H2O/ACN (50/50,
v/v); UV detection wavelength, 214 nm; injection volume, 20
nL; Samples: toluene.
The reproducibility of the optimized poly(MDPA-co-EDMA)
monolithic column was finally assessed by measuring the
relative standard deviations (RSDs) for the retention factors (k)
of dimethyl phthalate, anisole and naphthalene (thiourea as
the t0 marker) (Table S3). A mixture of H2O/ACN (60/40, v/v)
was used as the mobile phase. The RSD values for the run-to-
run reproducibility (n = 10) of dimethyl phthalate, anisole and
naphthalene are 0.51%, 0.34% and 0.34%, respectively, while
the day-to-day reproducibility (n = 3) are 2.06%, 2.09% and
2.31%, respectively. Notably, the separation performance of
the poly(MDPA-co-EDMA) monolithic column does not appear
to deteriorate after 100 injections over 60 days. These results
confirm the satisfactory robustness of the monolithic column.
Column stability, permeability, and reproducibility. The
mechanical stability of monolithic column is of great
importance for their application in liquid chromatography. The
mechanical stability, the permeability of the poly(MDPA-co
-
EDMA) monolithic column were measured by pumping ACN,
MeOH, H2O/ACN (50/50, v/v) and H2O through the column at
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antilogarithm of the slope of a plot of the natural logarithm of
retention factor (ln ) vs. the carbon number ( ) of a set of
alkylphenones.28 A mixture of H2O/ACN (50/50, v/v) was
employed as the mobile phase. Under this condition, a
baseline separation of all seven alkylphenones can be obtained
within 14 min(Fig. 5). As depicted inFig. S4 , a good linear
Furthermore, the column-to-column and batch-to-batch
reproducibilities were also investigated with the same batch or
different batches of polymerization mixture. The column-to-
column reproducibility (n = 3) were 1.38%, 1.33% and 1.38%
for the retention factors of dimethyl phthalate, anisole and
naphthalene. The batch-to-batch reproducibility (n = 3) of
three analytes were 2.82%, 2.29% and 2.11%, respectively.
Moreover, RSD values for their reproducibility based on
quantitative performance (peak height and peak area) were
also satisfactory (Table S3). These results further confirm the
good reproducibility of the optimized poly(MDPA-co-EDMA)
monolithic columns.
k
n
correlation between ln
k and n was observed and the
calculated methylene selectivity is 1.53 for the poly(MDPA-co
-
EDMA) monolith. This further confirm the typical reversed
phase retention characteristics on the poly(MDPA-co-EDMA)
monolithic column.
Separation mechanisms
2.0
Hydrophobic interaction. It was expected that hydrophobic
interaction could contribute to the overall retention of non-
polar analytes on the poly(MDPA-co-EDMA) monolithic column
because of the presence of long alkyl chains of MDPA. In order
to confirm the deduction, the retention behavior of toluene
and thiourea was systemically studied by varying the ACN
content in the mobile phase. As can be seen in Fig. 4, the
retention time of non-polar toluene decreased dramatically
with increasing ACN content from 20% to 90% and then
levelled off when the ACN content further increased to 95%.
However, the retention time of polar thiourea almost
remained constant when ACN content varied from 20% to 90%,
further increase of ACN content (> 90%), the retention time of
thiourea increased slightly. These results demonstrated that a
typical RP-HPLC mechanism was exhibited on the poly(MDPA-
co-EDMA) monolithic column when the ACN content is below
90%.
1
1.6
1.2
2
3
0.8
4
5
0.4
6
7
0.0
0
3
6
9
12
15
Time (min)
Fig. 5. Chromatogram of seven alkylphenones separation.
Conditions: column dimensions, 140 mm × 100 µm I.D.; mobile
phase, H2O/ACN (50/50, v/v); detection wavelength, 214 nm; flow
rate, 600 nL/min; samples, (1) acetophenone; (2) propiophenone;
(3) butyrophenone; (4) valerophenone; (5) hexanophenone; (6)
heptanophenone; (7) octanophenone.
30
Cation-exchange interaction. As shown by the ζ-potential
experiments, a negatively charged surface was observed for
the poly(MDPA-co-EDMA) monolithic column over a wide pH
range, and therefore ionic interaction is expected to play an
important role in the retention of charged analytes. In order to
toluene
thiourea
25
20
15
10
5
verify the ion exchange interactions on the poly(MDPA-co
-
EDMA) monolithic column, six small peptides (Ala-Gly-Gly, Leu-
Gly-Gly, Ala-Val, Gly-Leu, Gly-Phe, and Leu-Val) were selected
as test analytes. Some parameters (p
Ka, logD) of small
peptides were calculated and listed inTable S4.
The effect of buffer pH on the retention of small peptides
was first studied by varying the pH of ammonium formate
buffer from 3.0 to 6.4, while remaining the salt concentration
at 10 mM in H2O/ACN (90/10, v/v). As shown inFig. 6a, the
increase in buffer pH clearly led to a reduction in retention of
six small peptides on the poly(MDPA-co-EDMA) monolithic
column. Such behaviors could be explained by cation-exchange
interactions. When the mobile phase pH increased from 3.0 to
6.4, the peptides became less positively charged and even
negatively charged, while their hydrophobicity did not change
significantly as listed inTable S4. At the same time, the
negative charge of the monolithic column increased. As a
0
20
40
60
80
100
ACN(%)
Fig. 4. Relationship between retention time and ACN concentration
on the poly(MDPA-co-EDMA) monolithic column. Conditions:
column dimensions, 140 mm
× 100 μm I.D.; mobile phase,
H2O/ACN; flow rate, 600 nL/min; UV detection wavelength, 214 nm;
injection volume, 20 nL; Samples: toluene, thiourea.
The hydrophobicity of the poly(MDPA-co-EDMA) monolithic
column was further evaluated through determining its
methylene selectivity, which is calculated from the
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results, the electrostatic attraction interaction between
peptides and the poly(MDPA-co-EDMA) monolithic stationary
phase became weaker or even change to electrostatic
repulsion interaction, and thus led to a shorter retention time.
These results indicate that cation-exchange interaction plays a
key role in the retention of small peptides on the poly(MDPA-
co-EDMA) monolithic column.
45
36
27
18
9
(a)
(b) 35
Leu-Val
Gly-Phe
Gly-Leu
Ala-Val
Leu-Gly-Gly
Ala-Gly-Gly
Leu-Val
Gly-Phe
Gly-Leu
Ala-Val
Leu-Gly-Gly
Ala-Gly-Gly
28
21
14
7
0
0
3
4
5
6
7
9
12
15
18
21
Concentration (mmol/L)
pH
The effect of ionic strength on the retention of small
peptides was also investigated by varying the concentration of
ammonium formate (pH 4.3) in the mobile phase of H2O/ACN
(90/10, v/v). At pH 4.3, all small peptides are positively
charged. As shown inFig. 6b, the retention of small peptides
decreased as the ammonium formate concentration increased
from 10 to 20 mM. These behaviors could be attributed to the
inhibition effect of high salt concentration in the mobile phase
on the cation-exchange interaction between small peptides
and the poly(MDPA-co-EDMA) monolithic stationary phase.
(c)
(d)
20
16
12
8
0.6
Leu-Val
Gly-Phe
Gly-Leu
Ala-Val
Leu-Gly-Gly
Ala-Gly-Gly
0.4
0.2
5
3
1
2
4
0.0
6
4
-0.2
0
8
16
24
32
0
6
12
18
24
30
ACN (%)
Time (min)
Fig. 6. Influence of mobile phase pH, salt concentration and ACN
content on retention factors (plot a, b and c) and chromatogram of
small peptides separation (plot d). Conditions: column dimensions,
To further investigate its cation-exchange characteristics, a
simple empirical stoichiometric displacement model was
180 mm
× 100 µm I.D.; flow rate, 400 nL/min; detection
wavelength, 214 nm; injection volume, 20 nL; mobile phase, plot a,
H2O/ACN (90/10, v/v) containing 10 mM ammonium formate with
various pH, plot b, H2O/ACN (90/10, v/v) containing ammonium
formate pH 4.3 at various concentration, plot c, H2O/ACN
containing 10 mM ammonium formate pH 4.3 at various ACN
content, plot d, 10 mM ammonium formate, pH 4.3, in H2O/ACN
(80/20, v/v); Samples, (1) Ala-Gly-Gly; (2) Leu-Gly-Gly; (3) Ala-Val;
(4) Gly-Leu; (5) Gly-Phe; (6) Leu-Val.
employed. The model predicts a linear dependence between
and
k
1/[C]
given
by
equation
(3)29:
s
K =
+α
(3)
[C]
where s and α are constants, [C] is the concentration of ammonium
formate. The slope values of the plots depend on the type of
interactions between the analytes and the monolithic stationary
phases.30 When hydrophobic interaction plays a major role, the
slope would be close to 0, while the slope would be steeper where
the ion-exchange interaction dominates. The intercept value
highlights the contribution of RP-HPLC mechanism at infinite buffer
concentration.30 As can be seen inFig. S5, these tested analytes
exhibited different slopes ranging from 7.736 to 218.416 and
positive intercepts from 0.240 to 5.268, indicated a mixed-mode
mechanism of hydrophobic interaction and cation-exchange
interaction.
Application
Phenols are important contaminants with moderate toxicity
and persistence in the environment.31 The applicability of the
poly(MDPA-co-EDMA) monolithic column for the separation of
phenols was investigated. As shown inFig. 7, a fast separation
of four phenols was achieved within 3 min using a mixture of
In addition, the ACN content in the mobile phase has a significant
influence on the retention of small peptides. As can be seen inFig.
6c, their retention decreased with increasing the ACN content as
other conditions were kept constant, implying a typical reversed-
phase retention mechanism in this case. The contribution of
hydrophobic interaction to the retention could also be evidenced by
their elution order, which is consistent with the order of calculated
logD4.3 values.
H2O/ACN (50/50, v/v) as the mobile phase. Paracetamol (p
9.38, log 1.08) was eluted first and followed with phenol (p
9.86, log
bromophenol (p
K
K
a
a
P
P
1.48),
p
-nitrophenol (p
K
a 7.13, log
2.49) in turns. This elution order
values. Since the
-nitrophenol) are uncharged
P 1.91), and p-
K
a 9.34, logP
is consistent with the order of their log
majority of phenols (apart from
P
p
at pH 7.0, the contributions from cation-exchange interactions
are negligible under this separation conditions. Therefore, the
hydrophobic interaction may dominate the retention
behaviour of these phenols.
All these results indicate that both cation-exchange and
hydrophobic interactions could contribute significantly to the
overall retention of charged analytes on the poly(MDPA-co-EDMA)
monolithic column. This could increase the separation power and
degree of freedom in adjusting the separation selectivity compared
to single cation-exchange or reversed-phase mode. By optimizing
the pH and ionic strength, as well as organic solvent concentration,
The group of water-soluble vitamins B are important
micronutrients. 32 In order to evaluate the separation power of
the poly(MDPA-co-EDMA) monolithic column in the micro-LC
mode for water-soluble vitamins B, four vitamins (VB1, VB4,
a good separation was obtained for the six small peptides within 20 VB6 and VB12) were employed as test analytes. As depicted
min(Fig. 6d).
inFig. 8, a baseline separation of all vitamins B was obtained
using a 10 mM ammonium formate (pH 3.0) in H2O/ACN
(10/90, v/v) as mobile phase. Vitamins B were eluted in the
order of VB12 (p
1.20) VB6 (p a 5.58, log
according to their p a values but not log
K
a 1.59, log
P
-0.91)
＜
VB1 (p
a 9.80, log
P values. Due to the
K
a 4.80, log
P
＜
K
P
-1.00)
＜
VB4 (p
K
P
-0.03)
K
high ACN content in the mobile phase, the hydrophobic
6 | J. Name., 2012, 00, 1-3
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interaction is unlikely to contribute to the retention of
analytes. The cation-exchange interaction may play
a
8
6
4
2
0
dominate role. At pH 3.0, VB12, VB1 and VB6 will be partially
deprotonated and present in negative form with different
degree of charge, while VB4 will be completely protonated and
1
fully positively charged. As
a result, four water-soluble
2
vitamins B can be separated due to the different degree of the
electrostatic interaction.
3
In order to further evaluate the separation ability of the
poly(MDPA-co-EDMA) monolithic column, a mixture of eight
pharmaceutical compounds with MW ranging between 146
and 342 g/mol (including acidic drugs: aspirin; neutral drugs:
coumarin, phenacetin and ranitidine; basic drugs:
sulfanilamide, benzene sulfonamide and sulpiride), was
4
0
2
4
6
8
10
Time (min)
selected as test analytes. Some parameters (pKa, logD7.4) of
Fig. 8. Chromatogram of water-soluble vitamins B separation.
Conditions: column dimensions, 140 mm × 100 µm I.D.; mobile
phase: H2O/ACN (10/90, v/v) containing 10 mM ammonium
formate, pH 3.0; flow rate, 600 nL/min; detection wavelength, 254
nm; injection volume, 20 nL; samples, (1) VB12; (2) VB1; (3) VB6; (4)
VB4.
pharmaceutical compounds were calculated and listed inTable
S5. Ammonium formate (50 mM, pH 7.4) buffer and 100% ACN
were used as mobile phase component A and B, respectively.
As shown inFig. 9, all pharmaceutical compounds were
baseline separated on the poly(MDPA-co-EDMA) monolithic
column using a 18 min gradient of ACN from 10% to 100% in
the mobile phase. The peak shapes of all pharmaceutical
compounds are acceptable for further application. However,
this elution order is neither consistent with the order of their
90
6
pKa values nor that of logD7.4 values. The observation could be
2
60
attributed to that the comprehensive influence of reversed-
phase/cation-exchange mixed interactions.
4
5
Finally, the separation of a tryptic digestion sample of Cyt-C
was performed on the monolith to evaluate its practical utility.
Fig. S6 shows more than ten resolved peaks. These results
further confirm the potential applicability of the poly(MDPA-
co-EDMA) monolithic column for biological samples.
30
0
1
7
3
8
0
5
10
15
20
25
8
Time (min)
2
Fig. 9. Chromatogram of pharmaceutical compounds separation.
Conditions: column dimensions, 256 mm × 100 μm I.D.; mobile
phase, Eluent A, 50 mM ammonium formate, pH 7.4 in H2O, eluent
B, ACN; linear gradient, 0 min/10% B, 15.0 min/100% B, 18.0
min/100% B, 18.5 min/10% B, 26.0 min/10% B; detection
wavelength, 214 nm; flow rate, 600 nL/min; injection volume, 20
nL; Samples, (1) Acetylsalicylic acid (Aspirin), (2) Caffeine, (3)
Sulfanilamide, (4) Benzene sulfonamide, (5) Phenacetin, (6)
Coumarin, (7) Ranitidine, (8) Sulpiride.
6
4
4
3
2
1
0
0
1
2
3
4
5
Conclusions
Time (min)
In this study, a novel porous poly(MDPA-co-EDMA) monolithic
column was prepared via a one-step copolymerization of
MDPA and EDMA using isopropanol and 1,4-butanediol as
porogens. The optimized monolithic column showed
satisfactory properties with respect to morphology,
permeability, mechanical and chemical stability, and
chromatographic performance. Reversed-phase and cation-
exchange mixed-mode retention mechanism was observed on
the poly(MDPA-co-EDMA) monolithic column. Furthermore,
the resulting monolithic column was successfully applied to
Fig. 7. Chromatogram of phenols separation. Conditions: column
dimensions, 140 mm × 100 µm I.D.; mobile phase, H2O/ACN (50/50,
v/v); flow rate, 600 nL/min; detection wavelength, 214nm; injection
volume, 20 nL; samples, (1) Paracetamol, (2) Phenol, (3) p-
Nitrophenol, (4) p-Bromophenol.
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17. R. D. Arrua and E. F. Hilder, RSC Adv., 2015, 5, 71131-71138.
18. R. Poupart, D. N. E. Houda, D. Chellapermal, M.
the separation of a wide range of compounds, such as small
peptides, phenols, water-soluble vitamins B, pharmaceutical
Guerrouache, B. Carbonnier and B. L. Droumaguet, RSC Adv.,
2016, 6, 13614-13617.
compounds. This research not only developed
a novel
phospholipid functionalized monolithic column for the
separation of small molecular, but also provided an
opportunity for mimicking the cell membrane environment
containing PA.
19. X. Zhao, W. Chen, Z. Liu, J. Guo, Z. Zhou, J. Crommen, R.
Moaddel and Z. Jiang, J. Chromatogr. A, 2014, 1367, 99-108.
20. F. Wang, J. Dong, X. Jiang, M. Ye and H. Zou, Anal. Chem.,
2007, 79, 6599-6606.
21. H. Zhou, M. Ye, J. Dong, E. Corradini, A. Cristobal, A. J. Heck,
H. Zou and S. Mohammed, Nat. Protoc., 2013, 8, 461-480.
22. J. Seuring, P. Reiss, U. Koert and S. Agarwal, Chem. Phys.
Lipids, 2010, 163, 367-372.
23. N. Li, Y. Shen, L. Qi, Z. Li, J. Qiao and Y. Chen, RSC Adv., 2015,
Author Contributions
K.P. and Q.Q.W. contributed equally to this work. Q.Q.W.,
F.H.W. and Z.J.J. supervised the project and designed the
experiments. K.P., Q.Q.W., W.J.C., D.H.X. and Z.Y.Z. performed
experiments and analysed data. Z.J.J., Y.Q.W. and F.H.W.
contributed to modification of the manuscript. K.P. and Q.Q.W.
wrote the manuscript. All authors participated in discussion of
scientific ideas.
5
, 61436-61439.
24. R. Yu, W. Hu, G. Lin, Q. Xiao, J. Zheng and Z. Lin, RSC Adv. ,
2015, , 9828-9836.
5
25. F. Nevejans and M. Verzele, J. Chromatogr. A, 1985, 350
145-150.
,
26. D. Marsh, CRC handbook of lipid bilayers, CRC Press: Boca
Raton, Florida, 1990.
27. X. Lin, W. Jia, S. Feng, J. Lin and Z. Xie, RSC Adv. , 2013, 3,
21888-21895.
28. Q. Duan, C. Liu, Z. Liu, Z. Zhou, W. Chen, Q. Wang, J.
Crommen and Z. Jiang, J. Chromatogr. A, 2014, 1345, 174-
181.
29. J. Ståhlberg, J. Chromatogr. A, 1999, 855, 3-55.
30. Z. Zhang, J. Xu, D. Hussain and Y.q. Feng, J. Chromatogr. A,
2016, 1453, 71-77.
31. M. Cledera-Castro, A. Santos-Montes and R. Izquierdo-
Hornillos, J. Chromatogr. A, 2005, 1087, 57-63.
32. H. Panahi, H. Kalal, A. Rahimi and E. Moniri, Pharm. Chem. J.,
2011, 45, 125-129.
Acknowledgements
We gratefully appreciate the financial support from the
National Natural Science Foundation of China (Grants:
81503030), the Natural Science Foundation of Guangdong
Province, China (Grant No. 2015A030310175).
Notes and references
1. F. Tsopelas, T. Vallianatou and A. Tsantili-Kakoulidou, Expert
Opin. Drug Discovery, 2016, 11, 473-488.
2. M. Davies-Tuck, T. Lee, A. Apffel and M.I. Aguilar, J.
Chromatogr. A, 2007, 1156, 167-173.
3. X. Zhao, W. Chen, Z. Zhou, Q. Wang, Z. Liu, R. Moaddel and Z.
Jiang, J. Chromatogr. A, 2015, 1407, 176-183.
4. C. Pidgeon, S. Ong, H. Choi and H. Liu, Anal. Chem., 1994, 66
2701-2709.
,
5. S. Ong, S. J. Cai, C. Bernal, D. Rhee, X. Qiu and C. Pidgeon,
Anal. Chem., 1994, 66, 782-792.
6. E. E. Kooijman and K. N. Burger, Biochim. Biophys. Acta, Mol.
Cell Biol. Lipids, 2009, 1791, 881-888.
7. S. Bocian, A. Nowaczyk and B. Buszewski, Anal. Bioanal.
Chem., 2012, 404, 731-740.
8. K. Hu, W. Zhang, H. Yang, Y. Cui, J. Zhang, W. Z, A. Yu and S.
Zhang, Talanta, 2016, 152, 392-400.
9. Z. Jiang, N.W. Smith, P.D. Ferguson and M. R. Taylor, J. Sep.
Sci., 2008, 31, 2774-2783.
10. A. Taillardat-Bertschinger, A. Galland, P.A. Carrupt and B.
Testa, J. Chromatogr. A, 2002, 953, 39-53.
11. M. Amato, F. Barbato, P. Morrica, F. Quaglia and M. I. La
Rotonda, Helv. Chim. Acta, 2000, 83, 2836-2847.
12. Q. Wang, J. Feng, H. Han, P. Zhu, H. Wu, M. L. Marina, J.
Crommen and Z. Jiang, J. Chromatogr. A, 2014, 1363, 207-
215.
13. Q. Wang, P. Zhu, M. Ruan, H. Wu, K. Peng, H. Han, G. W.
Somsen, J. Crommen and Z. Jiang, J. Chromatogr. A, 2016,
1444, 64-73.
14. N. Li, Y. Shen, L. Qi, Z. Li, J. Qiao and Y. Chen, RSC Adv. , 2015,
5, 61436-61439.
15. H. Wu, Q. Wang, M. Ruan, K. Peng, P. Zhu, J. Crommen, H.
Han and Z. Jiang, J. Pharm. Biomed. Anal., 2016, 121, 244-
252.
16. Q. Wang, E. Sanchez-Lopez, H. Han, H. Wu, P. Zhu, J.
Crommen, M. L. Marina and Z. Jiang, J. Chromatogr. A, 2016,
1428, 176-184.
8 | J. Name., 2012, 00, 1-3
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Phosphatidic acid-functionalized monolithic stationary phase for reversed
phase/cation-exchange mixed mode chromatography
Kun Peng,
‡
a,b,c Qiqin Wang,
‡
c Weijia Chen,c Donghai Xia,c Zhengyin Zhou,c Yuqiang Wang,c Zhengjin Jiang*c and
Fuhai Wu*b
A reversed-phase and cation-exchange mixed-mode poly(MDPA-co-EDMA) monolithic column was
prepared, and the resulting monolithic column was successfully applied to the separation of a wide
range of compounds, such as small peptides, phenols, water-soluble vitamins B, pharmaceutical
compounds.
This journal is © The Royal Society of Chemistry 20xx
J. Name., 2013, 00, 1-3 | 9