graft copolymer composed of cationic backbone and bottle brush-like side chains as a physically...

Research Article

Graft copolymer composed of cationicbackbone and bottle brush-like side chainsas a physically adsorbed coating for proteinseparation by capillary electrophoresis

To stabilize electroosmotic flow (EOF) and suppress protein adsorption onto the silica

capillary inner wall, a cationic hydroxyethylcellulose-graft-poly (poly(ethylene glycol)

methyl ether methacrylate) (cat-HEC-g-PPEGMA) graft copolymer composed of cationic

backbone and bottle brush-like side chains was synthesized for the first time and used as a

novel physically adsorbed coating for protein separation by capillary electrophoresis.

Reversed (anodal) and very stable EOF was obtained in cat-HEC-g-PPEGMA-coated

capillary at pH 2.2–7.8. The effects of degree of cationization, PEGMA grafting ratio,

PEGMA molecular mass, and buffer pH on the separation of basic proteins were inves-

tigated. A systematic comparative study of protein separation in bare and HEC-coated

capillaries and in cat-HEC-g-PPEGMA-coated capillary was also performed. The basic

proteins can be well separated in cat-HEC-g-PPEGMA-coated capillary over the pH range

of 2.8–6.8 with good repeatability and high separation efficiency, because the coating

combines good protein-resistant property of bottle brush-like PPEGMA side chains with

excellent coating ability of cat-HEC backbone. Besides its success in separation of basic

proteins, the cat-HEC-g-PPEGMA coating was also superior in the fast separation of other

protein samples, such as protein mixture, egg white, and saliva, which indicates that it is a

promising coating for further proteomics analysis.

Keywords: Capillary electrophoresis / Cationic hydroxyethylcellulose / Physicallyadsorbed coating / Poly(ethylene glycol) methyl ether methacrylate / ProteinseparationDOI 10.1002/jssc.201100597

1 Introduction

Capillary electrophoresis (CE), one of the most important

techniques for the analysis of charged biomolecules [1–3],

has been applied in Human Genome Project very success-

fully due to its high separation efficiency, high speed, and

easy automation [4–6]. It is expected that CE will continue

its contribution to the next research field, proteomics, which

attempts to explore the relationship between protein and

particular gene expression [7–10]. However, up to now, CE

has not totally reached the high-expected impact because

some problems still remain to be solved. In practice, the

separation efficiencies typically achieved in protein separa-

tions are considerably lower than theoretical values and the

repeatability is also poor, because there are adsorptions

caused by different types of interactions between positively

charged residues of the proteins and negatively charged

silanol groups of the inner surface of the bare fused-silica

capillaries [11–14].

Over the years, a lot of approaches have been developed

to suppress protein adsorption and control or stabilize elec-

troosmotic flow (EOF) and thus obtaining efficient and

reproducible separation: the use of extreme pH [15, 16], high

ionic strength [17], zwitterionic additives [18], and so on. But

these approaches may limit separation sensitivity and lead to

protein denaturation. The most common method is to use

coatings, which can be broadly categorized as [11, 14]:

covalently linked polymer coatings, physically adsorbed

polymer coatings, or small molecule additives. Covalently

attached coatings can be very effective; however, the

procedures adopted in preparing covalent coatings are

laborious and time consuming and may not be reproducible.

Dan ZhouLina XiangRongju ZengFuhu CaoXiaoxi ZhuYanmei Wang

CAS Key Laboratory of SoftMatter Chemistry, Department ofPolymer Science andEngineering, University ofScience and Technology ofChina, Hefei, P. R. China

Received July 7, 2011Revised August 20, 2011Accepted September 7, 2011

Abbreviations: CAN, ceric ammonium nitrate; cat-HEC,cationic hydroxyethylcellulose; cat-HEC-g-PPEGMA, cationichydroxyethylcellulose-graft-poly(poly(ethylene glycol) methyl-ether methacrylate); GTA, glycidyltrimethylammoniumchloride; HEC, hydroxyethylcellulose; PEGMA, poly(ethyleneglycol) methyl ether methacrylate

Correspondence: Professor Yanmei Wang, Department of Poly-mer Science and Engineering, University of Science andTechnology of China, Hefei 230026, P. R. ChinaE-mail: [email protected]: 186-551-3601592

& 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.jss-journal.com

J. Sep. Sci. 2011, 34, 3441–3450 3441

Physically adsorbed polymer coatings, which are adsorbed

onto the capillary via electrostatic, hydrogen bonding, and

hydrophobic interactions, have attracted more and more

interests in the last few years due to their simplicity to use,

possibility for automation, regeneration of the coating, and

good performances in terms of separation efficiency and

repeatability. Numerous neutral polymers [5, 19–24] have

been used as physical coatings to shield silanol groups of

capillary wall, decrease z-potential, and increase viscosity in

the layer of coating near the wall [11]. Among them,

hydroxyethylcellulose (HEC), a polysaccharide, has been

widely used for protein and DNA separation by CE with the

advantages of commercial availability, high separation

performance, and low viscosity [20, 21]. However, HEC still

faces some problems, e.g. narrow pH application range, and

poor repeatability and stability [25]. This is because HEC

cannot be adsorbed strongly to the capillary surface due to its

high hydrophilicity and thus can be easily rinsed away. Some

improved HEC coatings were developed to overcome these

problems in our lab [26–28]. Another common coatings,

poly(ethylene oxide) (PEO), poly(ethylene glycol) (PEG, the

low-molecular mass form of PEO), and their derivatives

(such as PEG methyl ether methacrylate (PEGMA)), are well

known to be the most desirable and effective molecules for

reducing the surface adsorption of proteins [29–31].

However, they are believed to coat the capillary surface only

by the hydrogen bonding between the ether oxygen of PEG

and the surface protonated silanol groups and are unstable at

higher pH due to the weaker interactions between the

polymer and the ionized negatively charged surface [14],

which is similar to HEC. In recent years, although the use of

neutral polymers as coatings has resulted in excellent protein

separations, attention has been shifted toward cationic

coatings [32–38] because they can strongly be adsorbed onto

capillary inner wall through electrostatic interaction to

improve stability of the hydrophilic coatings. The cationic

coatings can be used to adjust both the magnitude and the

direction of EOF or to improve the separation efficiency.

However, many of them cannot effectively suppress the

adsorption of proteins.

In this work, with the aim of obtaining a simple, fast,

and reproducible physically adsorbed coating with not only

good protein-resistant property but also excellent coating

ability, we designed and synthesized a series of graft copo-

lymers composed of cationic backbone and bottle brush-like

side chains for the first time, cationic hydroxyethylcellulose-

graft-poly (PEG methyl ether methacrylate) (cat-HEC-g-

PPEGMA). The suppression of EOF by the coating at

different pHs was studied. The effects of degree of cationi-

zation, PEGMA grafting ratio, PEGMA molecular mass, and

pH on the separation of basic proteins were investigated,

and a comparative study of basic protein separation in bare

and HEC-coated capillaries and in cat-HEC-g-PPEGMA-

coated capillary was performed. Repeatability experiments

were also carried out. Finally, the coated capillary was

applied to analyzing other protein samples, such as protein

mixture, egg white, and saliva.

2 Materials and methods

2.1 Materials

In this experiment deionized water was distilled three times

prior to use. HEC [Mv (viscosity–average molecular mass)]

90 000; DS (degree of substitution) 1.50; MS (average

number of moles of the substituent (hydroxylethyl) 2.50)

and PEGMA [Mn (number–average molecular mass) �300,

unless otherwise stated] were purchased from Aldrich

Chemical Company (USA). PEGMA was passed through

an activated basic alumina column to remove the inhibitor

before use. Trimethylamine, chloropropylene oxide, ceric

ammonium nitrate (CAN), citric acid monohydrate, sodium

phosphate dibasic, benzyl alcohol, sodium hydroxide,

hydrochloric acid, nitric acid, Tris (hydroxymethyl) amino-

methane hydrochloride (Tris-HCl), and other reagents were

obtained from Sinopharm Chemical Reagents (China).

Glycidyltrimethylammonium chloride (GTA) was prepared

by reacting chloropropylene oxide with trimethylamine at

451C for 6 h. Lysozyme from chicken egg white [pI(isoelectric point) 11.1, Mr (relative molecular mass)

14 300], cytochrome c from horse heart (pI 10.2, Mr

12 400), ribonuclease A from bovine pancreas (pI 9.3, Mr

13 700), a-chymotrypsinogen A from bovine pancreas (pI9.2, Mr 25 700), myoglobin from equine skeletal muscle (pI7.0, Mr 17 800), and trypsin inhibitor from soybean (pI 4.6,

Mr 22 000) were all obtained from Sigma Chemicals (USA)

and were used as received. Fresh eggs were purchased from

a local supermarket. All the solutions were filtered by

Millipore membrane filter (0.45-mm pore size) before use.

Eight phosphate-citrate buffers (Na2HPO4-citric acid)

with different pHs were prepared [39] for EOF measure-

ment and protein separation, and the ionic strength values

were calculated by using Peakmaster software. The

concentration and ionic strength values of the buffers are

listed in Table 1.

2.2 Synthesis and characterization of cat-HEC-g-

PPEGMA

First, cat-HEC was synthesized by reacting HEC with GTA

using NaOH aqueous solution as a catalyst [26, 40], as

displayed in Fig. 1. In a typical procedure, 10 g of HEC

powder was fed into a three-necked flask containing 96 mL

of an 89% by mass aqueous solution of isopropyl alcohol

under stirring. Four grams of a 20% NaOH aqueous

Table 1. Concentration and ionic strength of phosphate-citrate

buffers

pH 2.2 2.8 3.6 3.8 4.6 5.8 6.8 7.8

Na2HPO4 (mM) 0.8 6.3 12.9 14.2 18.7 24.2 30.9 38.3

Citric acid (mM) 19.6 16.8 13.6 12.9 10.6 7.9 4.6 0.9

Ionic strength (mM) 4.8 13.9 27.7 31.2 46.1 69.6 92.4 114.9

J. Sep. Sci. 2011, 34, 3441–34503442 D. Zhou et al.


solution was then added into the resultant slurry and the

mixture was stirred at 251C for 2 h. Subsequently, GTA

aqueous solution was added as a cationizing agent to the

slurry and the resultant mixture was stirring for 1 h. Then,

the mixture was heated to 451C and stirred for additional 3 h

at this temperature. Finally, 4 g of a 20% HCl aqueous

solution was added to neutralize the mixture and stop the

reaction. Three cat-HEC polymers with different degrees of

cationization (different nitrogen contents) were obtained by

controlling the feed ratio (g/g) of HEC to GTA (10:2.5, 10:5.0,

10:7.5), signed as cat-HEC-1, cat-HEC-2, and cat-HEC-3,

respectively. The conditions and results are listed in Table 2.

Then cat-HEC-g-PPEGMA was prepared by grafting

PEGMA onto cat-HEC backbone using CAN initiator (in

nitric acid aqueous solution) [27, 28, 41], as shown in Fig. 1.

In a typical procedure, 0.5 g of cat-HEC was added into a

three-necked round bottom flask with a magnetic bar and

dissolved in 50 mL water. The reaction mixture was ther-

mostated to 351C and stirred vigorously and purged with

nitrogen gas for 1 h to remove dissolved oxygen. Then 0.05 g

CAN in 2.5 mL 1 M nitric acid aqueous solution was added

into the flask. After 10 min, PEGMA was added into the

reaction flask and the graft copolymerization was allowed to

proceed for 5 h under the nitrogen atmosphere. Finally, the

reaction was stopped by adding hydroquinone and the

synthesized copolymer was then purified by dialysis against

deionized water using a dialysis membrane with a mole-

cular mass cut-off 140 000 for about 96 h to get rid of ceric

ion, unreacted monomers, and also other low molecular

substance. The purified graft copolymer was then lyophi-

lized and recovered. A series of cat-HEC-g-PPEGMA copo-

lymers with different degree of cationization, PEGMA

grafting ratio, and PEGMA molecular mass were obtained,

respectively, by using cat-HEC with different degrees of

cationization, altering feed ratio of cat-HEC to PEGMA, and

using PEGMA with different molecular masses, as

summarized in Table 2.

The samples were characterized by using AVANCE 3001H-NMR Spectrometer (Bruker BIOSPIN AG, Switzerland),

Bruker EQUINOX55 Fourier Transform Infrared Spectro-

meter (Bruker, Germany), and Elementar Vario EL III

Universal CHNOS Elemental Analyzer (Elementar Analy-

sensysteme, Germany).

2.3 CE apparatus

Protein separation and EOF measurement were carried out

on a Beckman P/ACE MDQ Capillary Electrophoresis

System (Beckman Coulter Instruments, USA) with a

UV–vis detector working at 214 nm. The bare fused-silica

capillaries were obtained from Yongnian Optic Fiber Plant

(China), with effective/total length of 30/40 cm and id/od of

75/365 mm. Electrophoresis temperature was set to 251C.

Figure 1. Schematic diagramof the synthesis process forcat-HEC-g-PPEGMA.

Table 2. Preparation and properties of cat-HEC and cat-HEC-g-PPEGMA copolymers

Sample HECa)/GTA (g/g)b) N content in cat-HECc) (%) Degree of cationizationd) Cat-HEC/PEGMAe) (g/g)f) Grafting ratiog) (%)

Cat-HEC-1-g-PPEGMA-2 10:2.5 0.47 0.1 0.5:1.0 126

Cat-HEC-2-g-PPEGMA-1 10:5.0 1.55 0.4 0.5:0.5 74

Cat-HEC-2-g-PPEGMA-2 10:5.0 1.55 0.4 0.5:1.0 130

Cat-HEC-2-g-PPEGMA-3 10:5.0 1.55 0.4 0.5:2.0 254

Cat-HEC-2-g-PPEGMA1100-2h) 10:5.0 1.55 0.4 0.5:1.0 120

Cat-HEC-3-g-PPEGMA-2 10:7.5 2.55 0.7 0.5:1.0 140

a) Molecular mass of HEC is about 90 000, DS 5 1.50, MS 5 2.50.

b) Feed ratio (g/g) in the synthesis of cat-HEC.

c) Nitrogen content (%) in cat-HEC determined by Elemental Analyzer.

d) Number of moles of quaternary ammonium groups per anhydrous glucose unit. Degree of cationization 5 [N� (162144y)]/

[1400–151.5N], where N is the nitrogen content and y is the average number of moles of hydroxylethyl (MS 5 2.50) [40].

e) Molecular mass of PEGMA is about 300, unless otherwise stated.

f) Feed ratio (g/g) in the synthesis of cat-HEC-g-PPEGMA.

g) Calculated according to (w1�w0)/w0� 100%, in which w0 is the mass of cat-HEC and w1 is the mass of cat-HEC-g-PPEGMA.

h) Molecular mass of PEGMA is about 1100.

J. Sep. Sci. 2011, 34, 3441–3450 Other Techniques 3443


2.4 Preconditioning and coating of the capillary

The activation of silanol groups on the inner wall of the

capillary is important prior to its first use, in which the

capillary was washed with 1 M NaOH for 10 min, and

then rinsed with deionized water for another 10 min. Then

the preconditioned bare capillary could be used to

measure EOF or separate proteins after being washed

with buffer for 10 min. The next step was physical coating,

in which the preconditioned capillary was rinsed

with 0.2% m/v (2 mg/mL) copolymer aqueous solution for

10 min, and then statically stayed for 10 min to

ensure the copolymer to be adsorbed onto the capillary wall

adequately. Finally, the capillary was purged with running

buffer for 10 min to remove the excess uncoated copolymer.

All the rinse steps were taken under a high pressure of

138 kPa.

2.5 EOF measurement

EOF measurements were carried out with benzyl alcohol as

neutral marker at pH 2.2–7.8 according to the protocol [42].

Three replicates were taken for each EOF value.

2.6 Preparation and separation of protein samples

2.6.1 Basic proteins

A mixture of 0.5 mg/mL lysozyme, cytochrome c, ribonu-

clease A, and a-chymotrypsinogen A in water was injected at

a low pressure (3.5 kPa) for 3 s and separated in bare, HEC-

coated, and cat-HEC-g-PPEGMA-coated capillaries at 20 kV,

respectively. Phosphate-citrate buffers with different pHs

(2.8, 3.6, 4.6, 5.8, and 6.8) were used. Between runs, the

capillary was rinsed with buffer for 3 min to remove the

adsorbed proteins and obtain the pH balance.

2.6.2 Basic, neutral, and acidic protein mixture

A mixture of 0.5 mg/mL lysozyme, cytochrome c, ribonu-

clease A, a-chymotrypsinogen A, myoglobin, and trypsin

inhibitor in water was separated in bare and cat-HEC-2-g-

PPEGMA-2-coated capillaries at pH 2.8, respectively, and

other conditions were the same as in the basic protein

separation.

2.6.3 Egg white sample

The egg was purchased locally and egg white was diluted

with 20 mM Tris-HCl buffer (pH 7.4) in a 1:20 ratio [43].

The mixture was oscillated for 1 min and then centrifuged at

600� g for 15 min, and the resulting supernatant was

filtered through a Millipore membrane filter (0.45 mm). The

separation conditions were the same as in the protein

mixture separation.

2.6.4 Saliva sample

A saliva sample collected from a healthy adult was diluted

twofold with deionized water [44, 45], giving the sample A

(without lysozyme). Six microliters of 5 mg/mL lysozyme

was added into 1 mL sample A, giving the sample B (with

lysozyme). The processing method of the sample and the

separation conditions were the same as in the egg white

sample separation.

3 Results and discussion

3.1 Preparation and characterization of cat-HEC-g-

PPEGMA

During graft copolymerization, ceric (IV) ions are known to

create active sites on the cellulose backbone through single

electron transfer process; hence, PEGMA participated only

in graft copolymerization and its participation in the

formation of homopolymer was insignificant [41]. With

ceric-ion initiation, it is possible to use mild reaction

conditions, such as aqueous solutions, low temperature [46],

and short reaction time, to prevent PEGMA copolymer

crosslinking.1H-NMR spectra of HEC, cat-HEC, and cat-HEC-g-

PPEGMA using D2O as the solvent are shown in Fig. 2,

respectively. The peaks at 3.4–3.8 ppm (Fig. 2A, a) corre-

spond to methylene and methine protons of HEC. The

characteristic peaks at 3.3 ppm (Fig. 2B, b) are ascribed to

the methyl protons of quaternary ammonium groups,

indicating the formation of cat-HEC. Appearance of the

peaks at about 1.0, 2.0, and 4.1 ppm (Fig. 2C, e, d, and c) are

indicative of methyl and methylene protons on the PPEG-

MA main chains and methylene protons next to ester

groups on the PEG side chains, respectively, revealing

successful grafting of PEGMA onto cat-HEC. Furthermore,

Figure 2. 1H-NMR spectra of HEC (A), cat-HEC (B), and cat-HEC-g-PPEGMA (C) using D2O as the solvent.



the peaks of methylene protons of ethylene glycol units and

the peaks of HEC combine together and have a chemical

shift of 3.4–3.8 ppm (peak a), while the methyl protons of

PEG side chains and quaternary ammonium groups have

similar chemical shift (peak b).

HEC, cat-HEC, and cat-HEC-g-PPEGMA were char-

acterized by FTIR spectrometer. The characteristic absorp-

tion bands also give evidence that the cat-HEC-g-PPEGMA

has been synthesized successfully.

3.2 Stabilization of EOF in coated capillary at

different buffer pHs

The magnitude, direction, and repeatability of EOF reflect

the property of the capillary inner surface and the effect of

coating. The pH of the running buffer affects the EOF

greatly. Figure 3 compares the EOF mobilities in bare and

HEC-coated capillaries with those in cat-HEC-2-g-PPEGMA-

2-coated capillary at different pH values ranging from 2.2 to

7.8. As can be seen from Fig. 3, the EOF in a bare capillary

shows a strong and typical dependence on pH, that is, EOF

is close to zero at pH 2.2 and then increases sharply as the

pH increases from 2.2 to 7.8 due to ionized silanol groups.

Coating the bare capillary with neutral HEC can suppress

the EOF to very low values, but HEC coating is easily

washed off from the capillary because of high hydrophilicity

and weak hydrogen bonding interaction, so the repeatability

of EOF is poor and the relative standard deviation (RSD) of

EOF is high (Fig. 3). It should be noted that when cat-HEC-

2-g-PPEGMA-2 was used as the coating material, very stable

(pH-independent) and reversed (anodal) EOF is observed

(Fig. 3). Speculation as to why the repeatability of EOF in

cat-HEC-2-g-PPEGMA-2-coated capillaries is more excellent

than those in bare and HEC-coated capillaries is proposed in

Fig. 4A: we believe that cat-HEC-2-g-PPEGMA-2 is firmly

adsorbed onto capillary inner wall through the strong

electrostatic interaction between positive quaternary ammo-

nium groups on cat-HEC-2-g-PPEGMA-2 and negative

silanol groups as well as the hydrogen bonding interaction

between HEC and silanol groups on capillary inner wall.

Furthermore, the cat-HEC-2-g-PPEGMA-2 with high

concentration of 0.2% m/v shows excess positive charges

to neutralize or shield the negative silanol groups on the

capillary inner wall and thus results in stable and reversed

EOF over the investigated pH range of 2.2–7.8.

Moreover, when the degree of cationization of cat-HEC-

g-PPEGMA-2 copolymers increases from 0.1 to 0.7, the

absolute values of EOF mobilities increase about 20% due to

more excess positive charges on capillary inner surface.

However, no significant difference appears in EOF

suppressing ability among the synthesized cat-HEC-2-g-

PPEGMA copolymers with different PEGMA grafting ratios

and molecular mass over the pH range of 2.2–7.8. This

might be explained by the fact that when the degree of

cationization is fixed (such as 0.4), PEGMA content and

molecular mass have little effect on EOF.

3.3 Separation of basic model proteins

Another way to characterize coatings is the separation of

proteins, where high separation efficiency (number of

theoretical plates) and excellent repeatability should be

achieved. The separation efficiency (N) can be calculated by

the following equation according to the peak width at half-

height method:

N ¼ 5:54ðt=wÞ2

where t and w are migration time and full peak width at half

height of selected peaks, respectively. Proteins with a pIabove 8 and/or with a molecular mass larger than 50 kDa

have been identified as difficult to analyze on bare fused-

silica capillaries [47]. The performances of cat-HEC-g-

PPEGMA coatings were assessed based on the separation of

four basic proteins (lysozyme, cytochrome c, ribonuclease A,

and a-chymotrypsinogen A) recommended as model

analytes [48] due to their strong adsorption on the capillary

wall, which results in low separation efficiency, low protein

recovery, and poor repeatability in migration time [8].

3.3.1 Effect of degree of cationization on protein

separation

The cationic agent (quaternary ammonium groups) can

greatly affect the separation performance, so three cat-HEC-

g-PPEGMA copolymers with different degrees of cationiza-

tion (0.1, 0.4, and 0.7) were prepared and used in

comparative separation. When PEGMA grafting ratio is

kept constant (about 130%), the migration time of proteins

in cat-HEC-g-PPEGMA-coated capillaries at pH 4.6 would

increase with degree of cationization of polymers due to the

Figure 3. Effect of pH on EOF mobility in bare, HEC-coated, andcat-HEC-2-g-PPEGMA-2-coated capillaries. Error bars corre-spond to standard deviation of three EOF measurements ineach EOF value. Conditions: capillary, id/od of 75/365 mm andeffective/total length of 30/40 cm; applied voltage, 10 kV for1 min; injection, 1.4 kPa for 3 s; buffer, phosphate citrate at pH2.2, 3.8, 5.8, and 7.8.



increase in the absolute values of EOF (opposite direction of

protein electrophoresis), such as the migration time of

a-chymotrypsinogen A of about 14.1, 15.3, and 17.2 min for

cat-HEC-1-g-PPEGMA-2, cat-HEC-2-g-PPEGMA-2, and cat-

HEC-3-g-PPEGMA-2 coating, respectively. All three copoly-

mers possess both good coating ability of cat-HEC (via

electrostatic and hydrogen bonding interactions) and

excellent anti-protein property of bottle brush-like PPEGMA

chains, which results in high separation efficiency. As

depicted in Table 3, the separation efficiencies increase with

an increase in degree of cationization from 0.1 to 0.4 due to

more positive charges helpful for stronger adsorption of

coating onto capillary wall, and cat-HEC-2-g-PPEGMA-2

results in separation efficiency up to 372 000 theoretical

plates/m for a-chymotrypsinogen A at pH 4.6. However,

when the degree of cationization reaches 0.7, the separation

efficiencies decrease obviously (Table 3), which indicates

that too high content of cationic agent in copolymer is

possibly bad for separation.

3.3.2 Effect of PEGMA grafting ratio on protein

separation

To investigate the effect of PEGMA grafting ratio on

protein-resistant property of copolymer coatings, three

copolymers (cat-HEC-2-g-PPEGMA-1, -2, and -3) with

different grafting ratios (74, 130, and 254%) were synthe-

sized. When the degree of cationization is fixed (0.4), three

capillaries coated with cat-HEC-2-g-PPEGMA-1, -2, and -3

display similar migration times of proteins at pH 4.6 as a

result of approximate EOF as discussed in Section 3.2.

However, the separation efficiencies in three coated

capillaries are very different, and the highest separation

efficiencies are obtained in cat-HEC-2-g-PPEGMA-2-coated

capillary in which the copolymer with a moderate PEGMA

grafting ratio (130%) was used as the coating material, as

displayed in Table 3. Once the cat-HEC is adsorbed onto the

capillary inner surface, bottle brush-like PPEGMA side

chains stretch to the solution and generate a hydrophilic

domain on the PPEGMA/aqueous interface to resist

proteins, as speculated in Fig. 4A. The ability of PEG

coatings to reduce protein adsorption has been attributed to

the molecule’s high mobility, so the surface-immobilized

PEG chains can sway very fast, which results in steric

repulsion for proteins from the surface [49, 50]. However,

if the surface density of PPEGMA is too low (very thin

coating) or too high (densely packed and near crystalline),

the surface will not be able to effectively prevent protein

adsorption, and the optimum density should provide the

best protein resistance [51]. Thus, cat-HEC-2-g-PPEGMA-2-

coated capillary possesses the best protein separation

performance.

3.3.3 Effect of PEGMA molecular mass on protein

separation

As listed in Table 3, cat-HEC-2-g-PPEGMA-2 (PEGMA

molecular mass of 300) results in higher separation

efficiencies than cat-HEC-2-g-PPEGMA1100-2 (PEGMA

molecular mass of 1100), which indicates that PEGMA

molecular mass plays an important role in protein separa-

tion. Under similar PEGMA grafting ratio (130 and 140%),

the former can form a loose and thick coating which is

beneficial to keep high mobility of PEG and thus prevent

Figure 4. Proposed schematic diagrams ofadsorption of cat-HEC-g-PPEGMA (A) andcat-HEC-g-PPEGMA1100 (B) onto capillarysurface and resistance of protein adsorptiononto capillary surface.

Table 3. Separation efficiency (theoretical plates/m) of basic proteins separated in cat-HEC-g-PPEGMA-coated capillaries at pH 4.6a)

Sample Lysozyme Cytochrome c Ribonuclease A a-Chymotrypsinogen A

Cat-HEC-1-g-PPEGMA-2b) 139 000 180 000 149 000 257 000

Cat-HEC-2-g-PPEGMA-1b) 95 000 126 000 89 000 164 000

Cat-HEC-2-g-PPEGMA-2b) 101 000 236 000 187 000 372 000

Cat-HEC-2-g-PPEGMA-3b) 72 000 92 000 50 000 94 000

Cat-HEC-2-g-PPEGMA1100-2c) 106 000 160 000 56 000 98 000

Cat-HEC-3-g-PPEGMA-2b) 42 000 103 000 97 000 140 000

a) Conditions: capillary, id/od of 75/365 mm and effective/total length of 30/40 cm; separation voltage, 20 kV; temperature, 251C; detection

wavelength, 214 nm; injection, 3.5 kPa for 3 s. Sample: 0.5 mg/mL.

b) Molecular mass of PEGMA is about 300, unless otherwise stated.

c) Molecular mass of PEGMA is about 1100.



protein adsorption (Fig. 4A), whereas the latter can produce

a dense and thin polymer coating which does not efficiently

suppress protein adsorption (Fig. 4B).

3.3.4 Effect of buffer pH on protein separation

The buffer pH strongly affects the protein separation

performance because it can remarkably change the net

charges and the properties of both proteins and capillary

wall. And wide pH application range is seen as a criterion

for a good coating. The pI values of the studied proteins

range from 9 to 11. Therefore, the separation was

investigated at acidic pH values (2.8–6.8) at which these

proteins were cationic. Considering the effect of degree of

cationization, PEGMA grafting ratio and molecular mass on

the protein separation, cat-HEC-2-g-PPEGMA-2 was chosen

and used for further investigations.

Figure 5 shows the effect of pH on the separation of

four basic model proteins in a cat-HEC-2-g-PPEGMA-2-

coated capillary. The migration time increases with pH

increase (from 2.8 to 6.8) because the decrease in the net

positive charges on the proteins can result in the decrease in

the electrophoretic mobility of proteins. This reduction in

mobility is stronger for ribonuclease A and a-chymo-

trypsinogen A because of their lower pI values. On the other

hand, we can see from Fig. 5 that the peaks become wider

and weaker at higher pHs, especially a-chymotrypsinogen A

at 6.8, which indicates that the adsorption of proteins on the

capillary wall takes place to an enhanced extent with the pH

increasing. The number of negative charges of ionized

silanol groups on the capillary surface increase at higher

pHs, which leads to the decrease in the net positive charge

of cat-HEC-2-HEC-g-PPEGMA-2-coated capillary, as can be

seen from Fig. 3 that the absolute values of reversed EOF

become slightly lower with the increase of pH. Hence, the

decreases in the net positive charges of both the coated

capillary and the basic proteins at higher pH values result in

a decrease of the repulsion forces between the coated

surface of the capillary and the basic proteins, which causes

adsorption of the proteins [26, 27]. Although there are

protein adsorptions at higher pHs, the separation effi-

ciencies in cat-HEC-2-g-PPEGMA-2-coated capillary at pH

6.8 are still high (291 000, 360 000, and 473 000 theoretical

plates/m for lysozyme, cytochrome c, and ribonuclease A,

respectively) because of the presence of the bottle brush-like

PPEGMA layer suppressing protein adsorption. Further-

more, as shown in Fig. 5, the migration order of cytochrome

c and lysozyme changes when pH ranges from 3.6 to 4.6,

whereas at higher pH values, the proteins appear to migrate

in the order of their pI (lysozyme first and a-chymo-

trypsinogen A last).

3.3.5 Comparison of basic protein separation in bare,

HEC-, and cat-HEC-2-g-PPEGMA-2-coated capillaries

To demonstrate the effectivity of the synthesized copolymer

coating, the separation performances in cat-HEC-2-g-PPEG-

MA-2-coated capillary were compared with those in bare and

HEC-coated capillaries at pH 5.8. As shown in Fig. 6, no

ideal peak can be observed in bare capillary, which reveals

serious adsorption of proteins onto the capillary. When the

capillary was coated with HEC, three peaks occur (Fig. 6).

However, although improved separation efficiency

(32 000–165 000 theoretical plates/m) can be achieved by

using fresh HEC-coated capillary, HEC is ineffective to

reduce the protein adsorption after several runs because the

HEC coating can be washed off easily due to its high

hydrophilicity and poor coating ability only through weak

hydrogen bonding. Thus, the HEC coating yields poor

migration time repeatability (about 3%, n 5 3). The similar

results were observed [26, 27]. Furthermore, hydrogen

bonding is affected by pH to a great degree, when the

operational pH value becomes greater than 3, the risk of

protein adsorption becomes quite real in HEC-coated

capillaries [20], so the applicable pH range of HEC is very

narrow (o5). However, the use of cat-HEC-2-g-PPEGMA-2

can solve these problems mentioned above. From Fig. 6,

although the migration time is slightly long because

Figure 5. Effect of pH (2.8, 3.6, 4.6, 5.8, and 6.8) on separation offour basic proteins in cat-HEC-2-g-PPEGMA-2-coated capillary.Conditions: capillary, id/od of 75/365 mm and effective/totallength of 30/40 cm; separation voltage, 20 kV; temperature,251C; detection wavelength, 214 nm; injection, 3.5 kPa for 3 s.Sample: 0.5 mg/mL; peak identification: 1, lysozyme; 2, cyto-chrome c; 3, ribonuclease A; and 4, a-chymotrypsinogen A.



reversed EOF and protein electrophoresis are directed

oppositely (counterelectroosmotic migration), good baseline

separations of four proteins are obtained, the adsorption of

proteins is greatly decreased, and the separation efficiency

and repeatability are also remarkably improved. The

excellent performance in terms of high separation efficien-

cies (up to 544 000 theoretical plates/m) and low RSDs

(0.11–1.71%, n 5 3) in the copolymer-coated capillary can be

attributed to the properties and the specific structure of cat-

HEC-2-g-PPEGMA-2 that forms on the capillary wall as

proposed in Fig. 4A, which may be helpful for basic protein

separation. On the one hand, cat-HEC backbone is strongly

anchored on the negatively charged capillary inner wall

through electrostatic and hydrogen bonding interactions,

which offers improved repeatability of EOF and migration

time and long-term stability of the coating; on the other

hand, PEG-derived bottle brush-like grafting chains (PPEG-

MA) protrude to the solution and generate a hydrophilic

interface, which forms the protein-resistant layer to reduce

protein adsorption extremely due to the molecule’s high

mobility and thus steric repulsion. Furthermore, the

repulsive interactions between the positively charged cat-

HEC and basic proteins reduce the adsorption of proteins

on the wall, improving the separation efficiency and RSD as

a result.

3.3.6 Repeatability

It is well known that excellent repeatability for run-to-run,

day-to-day, and capillary-to-capillary is necessary to obtain

reliable analytical results. In many cases, physically

adsorbed coatings show poor stability, which limits their

routine applications. As mentioned above, at higher pH 5.8,

the RSD values (0.11–1.71%) of migration time of four basic

proteins in cat-HEC-2-g-PPEGMA-2-coated capillary were

much lower than those in HEC-coated capillary (about 3%).

To further study the application value, the repeatability

experiments at lower pH 3.6 in cat-HEC-2-g-PPEGMA-2-

coated capillary were also performed. The RSDs were o1.52,

2.01, and 2.77% for run-to-run (n 5 30), day-to-day (n 5 3),

and capillary-to-capillary (n 5 3), respectively. The coating

shows good repeatability of the migration times for these four

proteins, which confirms that the coating is stable enough.

3.4 Applications of copolymer coating in separation

of other protein samples

Besides the separation of basic model proteins, it is very

important to evaluate the applicability of cat-HEC-g-PPEG-

MA copolymer coating in the analysis of neutral and acidic

proteins as well as real complex samples.

3.4.1 Separation of basic, neutral and acidic protein

mixture

The protein mixture, which included four basic, one neutral

and one acidic proteins, was separated in bare and cat-HEC-

2-g-PPEGMA-2-coated capillaries at pH 2.8, as displayed in

Fig. 7A. It is evident that the adsorption between the

proteins and the capillary wall is so strong that the analysis

of protein mixtures in bare capillary is impossible, whereas

good baseline separation can be obtained in coated capillary

due to anti-protein property of the copolymer coating.

3.4.2 Separation of egg white sample

Egg white was used as a real complex sample, in which the

major protein components are positively charged at pH 2.8,

and thus it is impossible to be well separated in a bare

capillary. Using the proposed copolymer coating, both acidic

(conalbumin and ovalbumin) and basic (lysozyme) proteins

in egg white can be analyzed in a single run, as shown in

Fig. 7B. Glycoisoforms were also found in this complex

sample [52].

3.4.3 Separation of saliva samples

Human body fluids are great sources of potential biomar-

kers for disease. Thus, the analysis of these body fluids is of

high interest from a clinical point of view. A saliva sample

from a healthy adult was analyzed and the results are shown

in Fig. 7C. A great number of peaks are observed for the

tested saliva sample, which most likely corresponds to

proteins, peptides, and so on. After 0.03 mg/mL lysozyme is

spiked into the saliva sample, it is observed with good peak

shape. This indicates that the coating allows basic protein

analysis in a complex sample. Accordingly, the coating

Figure 6. Electropherograms of four basic proteins in bare (A),HEC-coated (B), and cat-HEC-2-g-PPEGMA-2-coated (C) capillaryat pH 5.8. Conditions and peak identification are the same as inFig. 5.



shows its potential to be applied in biological sample and

further proteomics studies.

4 Concluding remarks

A series of cat-HEC-g-PPEGMA graft copolymers composed

of cationic backbone and bottle brush-like side chains were

synthesized for the first time, and all of these cationic

coatings could produce reversed and very stable EOF over a

pH range of 2.2–7.8. The investigation results demonstrate

that basic proteins could be well separated in copolymer-

coated capillaries at pH 2.8–6.8 with good repeatability and

high separation efficiency, because the coating combines

good protein-resistant property of bottle brush-like PPEG-

MA with excellent coating ability of cat-HEC. Cat-HEC-2-g-

PPEGMA-2-coated capillary resulted in the best perfor-

mance due to moderate degree of cationization, PEGMA

grafting ratio, and molecular mass. Finally, the successful

fast separations of protein mixture, egg white, and saliva

samples in cat-HEC-2-g-PPEGMA-2-coated capillary reveal

the practicability of this new copolymer coating for protein

analysis in complex samples and indicate that it is a

promising coating for further proteomics analysis due to the

separation provided with high separation efficiency, speedi-

ness, excellent repeatability, simplicity of coating procedure,

and a wide pH range of operation.

Other parameters will be optimized in the future to

further improve the protein separation performances, and

one of our interests is to use the new coating to separate

other real complex samples by CE.

We greatly acknowledge the support by the National NaturalScience Foundation of China (Grant No. 21074124) andMinistry of Science and Technology of China (Grant No.2007CB936401). We also thank Professor Hua Cui andMs. Yuan Ge for providing Beckman P/ACE MDQ systeminstrument.

The authors have declared no conflict of interest.

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