Journal of Chromatography A, 1409 (2015) 132137

Contents lists available at ScienceDirect

Journal of Chromatography A
journal homepage: www.elsevier.com/locate/chroma

Biocompatible chiral monolithic stationary phase synthesized via

atom transfer radical polymerization for high performance liquid
chromatographic analysis
Huai-Song Wang a,b, , Xia-Yi Feng a , Ji-Ping Wei c
a

Department of Pharmaceutical Analysis, China Pharmaceutical University, Nanjing 210009, China

Key Laboratory of Drug Quality Control and Pharmacovigilance (Ministry of Education), China Pharmaceutical University, Nanjing 210009, China
c
Tianjin Modern Vocational Technology College, Tianjin 300350, China
b

a r t i c l e

i n f o

Article history:
Received 19 May 2015
Received in revised form 9 July 2015
Accepted 10 July 2015
Available online 14 July 2015
Keywords:
Restricted access material
Reverse atom transfer radical
polymerization
Chiral monolithic stationary phase
Enantio-separation
-CD
Biological samples

a b s t r a c t
Novel biocompatible chiral monolithic stationary phase was prepared by reverse and direct atom transfer radical polymerization (ATRP) methods. By taking advantages of the controlled/living property of
ATRP method, the chiral monolith was prepared by reverse ATRP (RATRP) rstly. An attractive feature of
RATRP is the prepared polymer containing a terminal radically transferable atom that can initiate another
post-polymerization reaction by direct ATRP. Then, the biocompatible poly(hydroxyethyl methacrylate)
(PHEMA) was grafted on the surface of the chiral monolith by direct ATRP as a diffusion barrier for proteins. This biocompatible chiral monolith was successfully used as restricted access stationary phase for
determination of enantiomers in biological samples with direct injection by high-performance liquid
chromatography (HPLC).
2015 Elsevier B.V. All rights reserved.

1. Introduction
Monolithic stationary phases have been widely used for HPLC
analysis because of their inherent advantages such as high column efciency and low consumption of samples [14]. In the past
decade, various monolithic chromatographic materials have been
developed including the organic polymer- and silica-based monoliths [57]. The monoliths based on organic polymers are typically
prepared from styrene, acrylamide or methacrylate monomers,
or are polymerized from ring-opening reaction [4]. Among them,
polymethacrylate polymers have been well used for preparing the
monolithic separation media, because their porous properties can
be easily tailored [8,9]. Furthermore, some polymethacrylate-based
chiral monolithic columns have been reported and received good
enantio-separation performance [912]. However, the analysis of
chiral drugs in biological uids is still a challenging task for the
organic polymer-based monolithic columns. Because, the sample
pretreatment procedures were needed to remove proteins by precipitation, liquidliquid extraction or solid phase extraction [13].

Corresponding author at: Department of Pharmaceutical Analysis, China Pharmaceutical University, Nanjing 210009, China. Tel.: +86 15094302563.
E-mail address: wanghuai1234@gmail.com (H.-S. Wang).
http://dx.doi.org/10.1016/j.chroma.2015.07.042
0021-9673/ 2015 Elsevier B.V. All rights reserved.

Recent years, the biocompatible chiral restricted access materials (RAMs) have been successfully utilized as chromatographic
stationary phase for direct injection of biological samples [14,15].
These chiral RAMs were mainly prepared based on porous silica
gel support, which were surface-modied with hydrophilic polymer networks (such as polyvinyl alcohol and albumin from bovine
serum) and inner-bonded with chiral selectors (such as -CD and
glycopeptide antibiotics) [1619]. As stationary phases for HPLC,
the chiral RAMs allow the analytes penetrating into the pores of
silica gel and interact with the chiral selectors bonded on the inner
surface, while proteins are eluted in the void volume. In that way,
the chiral drugs in biological samples can be analyzed with direct
HPLC injection, and the analytical efciency and accuracy of HPLC
separations can be well improved.
Herein, for the rst time, biocompatible chiral monoliths were
prepared and explored as novel stationary phases for analysis
of chiral drugs in biological samples with direct HPLC injection.
Reverse atom transfer radical polymerization (RATRP) with excellent controllability of the molecular weight and polydispersity
was employed to prepare -CD containing polymeric monolithic columns (CD-MC) [2023]. An attractive feature of RATRP
is the prepared polymer containing a terminal radically transferable atom that can initiate another post-polymerization reaction
by direct ATRP (Fig. S1). So, in present study, the direct ATRP,

H.-S. Wang et al. / J. Chromatogr. A 1409 (2015) 132137

133

Fig. 1. Illustration of the synthesis process of RA-CD-MC.

surface-initiated from the polymeric monoliths, was employed for

grafting biocompatible polymer brushes, which can signicantly
improve their surface hydrophilicity and acted as a protective layer
to prevent proteins in biological samples from being adsorbed
irreversibly on the monoliths. The chiral separation ability of
the biocompatible restricted access monolithic column (RA-CDMC, Fig. 1) was evaluated by reversed-phase HPLC method with
direct injection of biological samples. And the monolithic stationary phases gave well resolution for the separation of drugs in human
plasma. Meanwhile, good protein recovery was obtained.

acetate buffer solutions (TEAA solution) were prepared according

to the reference [19]. All reagents were analytical grade and used
as received unless otherwise stated.

2. Materials and methods

2.3. Synthesis of m-nitrophenyl methacrylate (NPMA)

2.1. Reagents and chemicals

NPMA was prepared by adding 5 g of acrylyl chloride to a solution of 2.5 g of potassium hydroxide and 4 g of m-nitrophenol in
200 mL of water at 0 C. The product was recrystallized in ether
(yield 75%). NMR: H (300 MHz; CDCl3 ; Me4 Si) 8.138.09 (1H, m,
HAr), 8.038.01 (1H, t, HAr), 7.607.54 (1H, t, HAr), 7.507.45 (1H,
m, HAr), 6.39 and 5.83 (2H, m, C CH2 ), 2.08 (3H, m, CH3 ). C
(100 MHz; CDCl3 ; Me4 Si) 164.8 (C O), 151.1, 148.7, 130.0, 128.2,
120.8 and 117.6 (HAr), 135.1 and 128.5 (C C), 19.4 (CH3 ) (Fig. S3
and Fig. S4).

-CD was purchased from Aoboxing Biotech. Co. Ltd. (Beijing,

China). N,N,N ,N ,N -pentamethyl diethylenetriamine (PMDETA),
hydroxyethyl methacrylate (HEMA), ethylene glycol dimethacrylate (EGDMA), o-, m-, p-nitrophenol, azobisisobutyronitrile (AIBN)
and methacryloyl chloride were obtained from SigmaAldrich (St.
Louis, USA). Bovine serum albumin (BSA) was from Solarbio Science
& Technology Co. Ltd. (Beijing, China). Chlorthalidone, propranolol, aminoglutethimide, benzoin, amlodipine, chlorpheniramine,
phenylalanine and ibuprofen were obtained from National Institute
for the Control of Pharmaceutical and Biological Products (Beijing, China). 2-Bromoisobutyryl bromide (BIBB) was from Hengye
Zhongyuan Chemical Co. Ltd. (Beijing, China). Triethylammonium

2.2. Instrumentation
The HPLC system consisted of a Shimadzu LC-20 AD pump and
a SPD-20A UV detector. NMR spectra were recorded on a Bruker
AVANCE III instrument. The SEM images were recorded on a Hitachi
S-3400N II scanning electron microscope. The IR spectra were
obtained from an AVATAR-360 FTIR instrument (Nicolet, USA).

2.4. Preparation of monolithic column (MC) via RATRP

The monolithic column (MC) was prepared via in situ RATRP
using a binary solvent (acetonitrile/lauryl alcohol = 9/1, v/v)

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H.-S. Wang et al. / J. Chromatogr. A 1409 (2015) 132137

porogen system. In the synthesis, the NPMA (monomer), EGDMA

(cross-linker), AIBN, CuBr2 and PMDETA were dissolved in porogenic solvent under ultrasonication. After deaerated with nitrogen,
the pre-polymerization solution was injtected into a stainless steel
column (150 mm 4.6 mm, i.d.). And then the both ends of the
stainless-steel column was sealed with plastic tubes. The polymerization was performed in water bath at 80 C for 24 h. After the
polymerization, the plastic tubes were removed from the column.
The prepared MC was provided with ttings, then connected to
HPLC pump and ushed with MeOH.
2.5. Modifying -CD on the monolithic column (CD-MC)
Before the reaction, the MC was connected to HPLC pump and
washed with MeOH/H2 O (1/9, v/v) for 30 min. Then the carbonate buffer (pH 11) containing -CD (17 mM) was pumped into
MC, and reacted in water bath at 50 C for 4 h. This reaction
procedure was repeated for three times. After the reaction, the prepared CD-MC was connected to the HPLC system and washed with
MeOH/H2 O (1/1, v/v) to remove unreacted -CD and the generated
m-Nitrophenol.
2.6. Grafting hydrophilic brushs on CD-MC via ATRP
A solution containing HEMA (0.5 mM), 2-bromoisobutyryl bromide (free initiator, BIBB), CuBr and PMDETA in MeOH was
deaerated with nitrogen and pumped into CD-MC by a HPLC pump.
The polymerization was performed at 60 C for 4 h in a water bath.
Then, the CD-MC grafted with hydrophilic poly(HEMA) (RA-CDMC) connected to the HPLC system and washed with MeOH to
remove unreacted monomer.
2.7. Chromatographic evaluation
A Shimadzu HPLC instrument was employed for evaluating the
chromatographic properties of CD-MC and RA-CD-MC as described
in our previous work [18]. The column hold-up time (t0 ) was
determined with methanol. The separation factor was calculated by = (t2 t0 )/(t1 t0 ). The resolution (Rs ) was calculated by
Rs = 2(t2 t1 )/(w1 + w2 ), where, t1 and t2 are the retention times,
and w1 and w2 are the peak width of the enantiomers.
3. Results and discussion
For grafting the chiral selector (-CD) on the monolithic column
(MC, Fig. 1), the functional monomer (m-nitrophenyl methacrylate,
NPMA) was synthesized (Fig. 2) [24,25]. After the polymerization,
the m-nitrophenyl groups on the prepared MC can react with -CD
via an inclusion-acylation procedure occurred selectively at one of
the hydroxyl groups of -CD under mild conditions.
During the synthesis of MC by RATRP (Fig. S2), the ratio
of monomer/initiator/catalyst plays an important role for the

Fig. 2. Synthesis of m-nitrophenyl methacrylate (NPMA).

Table 1
Preparation of CD-MCs and evaluation of the CD-MC column performance by using
the positional isomer (o-, m-, p- NP).a
CD-MC

NPMA/AIBN/CuBr2
(molar ratio)

-CD
density/mol g1

k O-NP

Rs b

I
II
III
IV
V

200:1:0
200:1:1
200:1:2
300:1:2
400:1:2

37
36
41
39
43

2.73
2.85
3.44
3.82
3.84

1.09
1.34
1.47
1.56
1.26

a
The molar ratios of NPMA/EGDMA = 1:1 and CuBr2 /PMDETA = 1:2 was xed for
preparing MCs by RATRP. The ratio of monomer (NPMA)/porogen was 2:3 v/v.
b
The Rs for o- and m-NP.

morphology of MC and the pore size of polymeric monolith. In

this work, different monomer (NPMA)/initiator (AIBN)/CuBr2 ratios
were investigated to obtain MCs with suitable structures, including
pore and globule size, for chromatography after the immobilization
of -CD (Table 1). The evaluation of the CD-MC column performance was carried out by using the positional isomer (o-, m-,
p-nitrophenol, NP). The results have shown that the CD-MCs with
AIBN/CuBr2 ratio of 1:2 have well separation ability for o-, m- and
p-NP. But during the separation of positional isomer, the column
backpressure on CD-MC-III was about 3.5 MPa, which is lower than
that on column CD-MC-IV (4.4 MPa) and on CD-MC-IV (4.7 MPa).
This indicates CD-MC-IV and CD-MC-V columns containing well
uniformly distributed pores. It was found that when the ratio of
NPMA/AIBN was 300:1, the CD-MC-IV exhibits well column efciency (1.2 104 plates/m) which was the results of narrow pore
and globule size distribution (Fig. 3 and Fig. S5). The chiral selectivity of CD-MC-IV for several structurally diverse compounds was
evaluated by HPLC method in reversed-phase mode (Table 2). We
found the concentration of buffer solution in the mobile phase
has a signicant effect for the retention and chiral separation. For
instance, during the separation of chlorthalidone, 0.3% triethylammonium acetate (TEAA) was used as the aqueous composition in
the mobile phases. Although the peak tailing can be improved
by using relatively high concentration (1% TEAA), the retention
time and separation efciency were both decreased. So compromise between the separation efciency and peak tailing has to be
considered. And we found better chiral separation efciency (for
chlorthalidone) and protein excluding ability can be obtained by
using 0.3% TEAA in the buffer solution in mobile phase. Generally, the peak tail during chiral separation can be decreased by
changing the value of PH of the mobile phase, type or concentration of buffer solution. According to Table 2, the chlorthalidone and
chlorpheniramine were baseline separated on CD-MC-IV. So, the

Fig. 3. SEM images of CD-MC-I (A), CD-MC-II (B), and CD-MC-IV (C). The scale bar is 1.0 m.

H.-S. Wang et al. / J. Chromatogr. A 1409 (2015) 132137

135

Table 2
Enantio-separation of chiral compounds on CD-MCs.
Analyte

CD-MC-I
k 1

CD-MC-III

Rs

7.5

1.07

0.68

7.8

1.04

0.51

17.7

k 1

CD-MC-IV

Rs

9.3

1.46

1.43

8.8

1.04

19.4

1.18

0.44

8.6

1.06

16.2

k 1

Mobile phase

Rs

9.6

1.66

1.58

0.53

8.6

1.05

0.56

1.09

1.28

20.6

1.11

1.37

18.1

1.20

0.64

20.1

1.21

0.66

1.19

8.7

1.10

1.21

8.6

1.10

1.27

1.09

1.17

18.1

1.16

1.42

18.5

1.17

1.49

2.1

1.06

0.16

2.3

1.07

0.24

2.3

1.07

0.25

20.2

1.15

0.43

22.1

1.17

0.48

Chlorthalidone

Propranolol

Aminoglutethimide

Benzoin

Amlodipine

Chlorpheniramine

D, L-Phenylalanine

Ibuprofen
k 1 is the capacity factor of the former compound, is the relative retention and Rs is the resolution of the separation. Flow rate: 1.0 mL min1 , detection wavelength was
254 nm. The mobile phases were: A: (0.3% TEAA, pH 4.9)/MeOH (85/15, v/v); B: MeOH/(0.3% TEAA, pH 5.4) (gradient elution: 050 min, 2/81/1, v/v); C: MeCN/(0.3% TEAA,
pH 6.8) (gradient elution: 020 min, 5/953/7, v/v); D: MeCN/(0.3% TEAA, pH 6.8) (gradient elution: 040 min, 1/93/7, v/v); E: 0.3% TEAA, pH 4.9; F: MeOH/(0.3% TEAA, pH
4.9) (9/1, v/v).

CD-MC-IV was used in the following preparation of biocompatible

chiral monolithic stationary phase.
A unique property of the resulting polymers prepared by RATRP
is the preservation of the active end group that can further

initiate a second round of polymerization by direct ATRP. In this

study, we aim to prepare biocompatible HPLC stationary phase that
can be used for determination of chiral drugs in biological samples with direct injection. However, CD-MCs as HPLC columns are

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H.-S. Wang et al. / J. Chromatogr. A 1409 (2015) 132137

Fig. 4. Structure of RA-CD-MC (A) and FTIR spectra (B) of RA-CD-MC-3 (a), RA-CD-MC-2 (b) and RA-CD-MC-1 (c).

Fig. 5. The protein exclusion mechanism of RA-CD-MC (A), and liquid chromatograms of human plasma samples spiked with chlorthalidone (B) and chlorpheniramine (C).
Mobile phases: MeOH/(0.3% TEAA, pH 4.9) (1/9, v/v) for chlorthalidone; MeCN/(0.3% TEAA, pH 6.8) (gradient elution: 040 min, 10/9025/75, v/v) for chlorpheniramine. The
ow rate was 1.0 mL min1 , the column backpressure was 4.34.7 MPa. Detection was at 254 nm.

not suitable for direct injection analysis, because biological matrices are incompatible with their hydrophobic surface, and proteins
can precipitate and adsorb irreversibly on the CD-MCs. In order to
preparing biocompatible chiral monolithic column, the alkyl halide
groups on CD-MCs were employed for grafting the hydrophilic
polymer chains, as a diffusion barrier for proteins, by direct
ATRP.
In the synthesis, the CD-MC-IV was chosen as starting material and hydroxyethyl methacrylate (HEMA) was selected as
the monomer of hydrophilic chain. Some free initiator (2bromoisobutyryl bromide) was added in the reaction system to
generate enough Cu(II) for better control of the polymerization. The
polymer chain length was controlled by the concentration of HEMA.
The resulting biocompatible restricted access monolithic columns
(RA-CD-MCs, Fig. 4A) were evaluated by FTIR. According to Fig. 4B,
the 1725 cm1 peak from ester carbonyl in the RA-CD-MCs was
increased with the increase of HEMA concentration. This indicates
the length of hydrophilic PHEMA chains was increased. Furthermore, the relationship between the hydrophilic polymer length and
the protein exclusion ability was evaluated by the recovery of BSA
(Table 3). We found the BSA recoveries were achieved above 95% on
RA-CD-MC-2 and RA-CD-MC-3. Considering the longer hydrophilic
polymer chains might have an inuence on the chiral selectivity.
We used the RA-CD-MC-2 as HPLC column for chiral separation via
direct injection of biological samples.
According to the SEM image (Fig. S6), the morphology of
RA-CD-MC-2 has no obvious change compared with CD-MC-IV.

This indicates there was no bulk polymerization occurred during the second round of polymerization by direct ATRP, and the
PHEMA chains were grafted on the surface of CD-MC-IV. The RACD-MC-2 column was tested by the direct injection of human
plasma sample spiked with racemic chlorthalidone and chlorpheniramine. As shown in Fig. 5, proteins can be totally excluded from
the column (retention time 2.56.0 min), and the enantiomers
were baseline separated from each other [18,26]. Compared
with the commercial cyclodextrin-bonded silica stationary phases
[27], the chiral separation ability of the RA-CD-MC column
is inferior. But the bi-functional RA-CD-MC column combines
both of the enantio-separation and restricted-access properties.
The hydrophilic PHEMA layers on RA-CD-MC can decrease the
Table 3
Protein exclusion ability of the RA-CD-MC synthesized by different conditions.a
RA-CD-MC

RA-CD-MC-1
RA-CD-MC-2
RA-CD-MC-3

Initiator b /CuBr/PMDETA/
BIBB/HEMA (molar ratio)

1:4:8:5:200
1:4:8:5:400
1:4:8:5:800

Recovery of BSA (%)c

5 mg mL1

10 mg mL1

79.7
96.9
98.5

84.3
98.2
99.7

The reaction was performed at 60 C for 4 h in a water bath.

The amount of initiator is the total amount (moles) of halide groups in the RACD-MC-IV.
c
HPLC column size was 150 4.6 mm. The mobile phase was MeOH/(0.3% TEAA,
pH 5.43) (1/9, v/v). The BSA solution (20 L) was injected and detected at 280 nm.
a

H.-S. Wang et al. / J. Chromatogr. A 1409 (2015) 132137

hydrophobicity of the chiral stationary phase. So, the retention

factors (k) and resolution factors (Rs ) of all enantiomers were
accordingly decreased. The separation conditions can be optimized
by varying the polarity, buffer concentration and pH value of the
mobile phase. Enhancing mobile phase polarity or changing the pH
value in a certain range can increase k and Rs of chiral compounds.
These results demonstrated that the restricted access monolithic
stationary phase can be used for HPLC analysis of biological samples
with direct injection.
4. Conclusion
In conclusion, chiral monolithic stationary phase was synthesized via reverse ATRP. The -CD was immobilized in the
monolithic column as chiral selector. In order to improve the biocompatible of the monolithic column, hydrophilic polymer brushes
were grafted on the surface of monolithic column via a second
round polymerization via direct ATRP. This biocompatible monolithic column was used as chiral restricted access stationary phase
for direct injection analysis of enantiomers in biological samples.
The results indicated that this multifunctional material can be
applied in the determination of chiral drugs in plasma with direct
HPLC injection of biological samples. It will facilitate the well analytical efciency (more than 1 104 plates/m) in the biological
sample chiral analysis.
Acknowledgement
This work was supported by the Fundamental Research Funds
for the Central Universities (Grant No. 2015PY010).
Appendix A. Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.chroma.2015.07.
042
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