Influence of polyelectrolyte capillary coating conditions on protein analysis in CE

Research Article

Influence of polyelectrolyte capillary coatingconditions on protein analysis in CE

CE of biomolecules is limited by analyte adsorption on the capillary wall. To prevent this,

monolayer or successive multiple ionic-polymer layers (SMILs) of highly charged poly-

electrolytes can be physically adsorbed on the inner capillary surface. Although these

coatings have become commonly used in CE, no systematic investigation of their

performance under different coating conditions has been carried out so far. In a previous

study (Nehme, R., Perrin, C., Cottet, H., Blanchin, M. D., Fabre, H., Electrophoresis 2008,

29, 3013–3023), we investigated the influence of different experimental parameters on

coating stability, repeatability and peptide peak efficiency. Optimal coating conditions

for monolayer and multilayer (SMILs) poly(diallyldimethylammonium) chloride/

poly(sodium 4-styrenesulfonate) coated capillaries were determined. In this study, the

influence of polyelectrolyte concentration and ionic strength of the coating solutions, and

the number of coating layers on coating stability and performance in limiting protein

adsorption was carried out. EOF magnitude and repeatability were used to monitor

coating stability. Coating ability to limit protein adsorption was investigated by moni-

toring variations of migration times, time-corrected peak areas and separation efficiency

of test proteins. The separation performance of polyelectrolyte coatings were compared

with those obtained with bare silica capillaries.

Keywords:

Adsorption prevention / Coating stability / Multilayer coating / Polyelectrolyte /Protein DOI 10.1002/elps.200800688

1 Introduction

Proteins are complex biopolymers susceptible to a wide

variety of degradation processes [2–4] and are characterized

by high micro-heterogeneity (especially glycoproteins [5]).

CE has found widespread use for protein analysis (structure

characterization, disease biomarkers identification, confor-

mation stability investigations, etc.) (see [6–15] and refer-

ences therein) because of its high resolving power and the

possibility to work under non-denaturing [8] conditions.

However, silanol groups on the inner surface of the

fused-silica capillaries are negatively charged at pH values

above 2 and have thus a high affinity for large organic

molecules such as proteins, which is detrimental to their

analysis [16–20]. Proteins interact with the capillary surface

by cooperative multi-point attachments due to the presence

on the protein of ionized, hydrophobic and bio-specific sites

[21–24]. Protein adsorption onto the capillary surface influ-

ences their physical properties [25] and leads – when

reversible – to peak broadening, low separation efficiency,

resolution and mass recovery, as well as poorly reproducible

migration times and peak areas [9, 21, 23, 26, 27].

Irreversible protein adsorption may also take place [21, 23,

28].

In order to obtain high efficiencies and repeatable

separations, analyte–capillary interactions must be elimi-

nated and the EOF appropriately controlled [29, 30]. Several

strategies [6, 13, 15, 21, 25, 29–38] have been employed,

aiming to create columbic repulsion between the proteins

and the capillary wall [21, 39] and/or to shield the silanol

groups [28, 40]: (i) use of BGE solution at extreme pH value

[41, 42]; (ii) use of high ionic strength (I) BGE [39, 43]; (iii)

coating of the capillary surface. The use of extreme pHs and

high ionic strength may limit selectivity and/or lead to

protein denaturation [31, 44]. Coating the capillary appears

to be the most flexible because a wide variety of chemical

substances can be employed.

The capillary coating agent (e.g. polyacrylamide) can be

covalently bound to the silanol groups [16, 18, 45–49] but

Reine NehmeCatherine PerrinHerve CottetMarie-Dominique BlanchinHuguette Fabre

Institut des Biomolecules MaxMousseron, UMR, UniversiteMontpellier 1, UniversiteMontpellier 2, CNRS,Montpellier, France

Received October 22, 2008Revised December 11, 2008Accepted January 7, 2009

Abbreviations: a-Lac, a-lactalbumin; CPA, time-correctedpeak areas; Cyt c, cytochrome c; Lys, lysozyme; Myo,

myoglobin; PDADMAC (C8H16ClN)n, poly(diallyldimethyl-ammonium) chloride; PSS (C8H7NaO3S)n, poly(sodium 4-styrenesulfonate); Rib A, ribonuclease A; SMIL, successivemultiple ionic-polymer layer

Correspondence: Dr. Catherine Perrin, Institut des BiomoleculesMax Mousseron, Faculte de pharmacie, Laboratoire de ChimieAnalytique, 15, avenue Charles Flahault, 34093 MontpellierCedex 5, FranceE-mail: [email protected]: 133-4-67-66-81-19

& 2009 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.electrophoresis-journal.com

Electrophoresis 2009, 30, 1888–18981888

this requires multiple time-consuming steps and an

inhomogeneous layer may be obtained [29, 50, 51]. Coating

agents can also be physically adsorbed to the capillary (see

[25, 32–34, 40, 52, 53] and references therein) via electro-

static, hydrogen and hydrophobic interactions [21, 25].

Physical coatings present several advantages [29, 54, 55]: (i)

simplicity of the procedure, (ii) low cost (no use of organic

solvents), (iii) possibility for automation and regeneration of

the coating and even (iv) EOF regulation [56, 57].

Physical coatings can be obtained by either a dynamic or

a static approach. With the dynamic approach [25, 32], the

coating agent (surfactant [52], mono- and oligo-amines [53],

polymer [58], etc.) is added to the BGE to prevent the

bleeding of the coating. However, the presence of coating

agent in the BGE can alter/limit selectivity, and may inter-

fere with the sample causing protein denaturation [29, 33],

and with analyte detection (e.g. spectrometric detection)

[14, 59]. With the static approach, coating of the capillary is

performed before the analysis run. When strongly charged

high molecular weight polyelectrolytes are used as coating

agents, ordered, uniform, repeatable and quasi-stable coat-

ings [30, 60–65] can be obtained [1, 66]. To further improve

the coating stability, Katayama et al. [66, 67] introduced a

successive multiple ionic-polymer layer (SMIL) coating

procedure, in which ‘‘a cationic polymer is sandwiched

between an anionic polymer and the uncoated negative

fused-silica capillary’’. A variety of polyelectrolytes can be

used ([1] and references therein, [68–70]) to produce very

highly stable multilayer coatings of silica surfaces in general

[60, 71–73], and of fused-silica capillaries in particular

[1, 66, 67, 74].

Although static physical coatings with polyelectrolytes

are frequently used for the CE analysis of proteins, no

systematic investigation of their performance under differ-

ent coating conditions has been carried out so far. In a

previous study [1], we investigated the influence of coating

conditions on coating stability and efficiency in preventing

peptide adsorption and determined optimal coating condi-

tions for monolayer poly(diallyldimethylammonium) chlor-

ide (PDADMAC (C8H16ClN)n) and SMIL PDADMAC/

poly(sodium 4-styrenesulfonate) (PSS (C8H7NaO3S)n)

coated capillaries. Because proteins are more likely to adsorb

onto the capillary walls than peptides, a systematic investi-

gation of the performance of monolayer and SMIL (5 – and

11– layers) coatings to limit protein adsorption has also been

carried out. The influence of polyelectrolyte concentration

and ionic strength of the coating solutions, and the number

of coating layers on analysis repeatability and separation

efficiency of a protein test mixture was systematically

investigated. As the selected proteins are positively charged

at the pH of investigation (pHBGE 5 2.5opI), the last

deposited layer was always cationic to avoid untoward elec-

trostatic interactions between proteins and the coatings [24].

Coating stability was evaluated by measuring the magnitude

and repeatability of the EOF. The ability of the coating to

prevent protein adsorption was investigated by monitoring

peak efficiencies and the repeatability of migration times

(tm) and time-corrected peak areas (CPAs). The effect

of the preparation procedure of the protein solutions

on their stability and electrophoretic behavior was

investigated.

2 Materials and methods

2.1 Chemicals and materials

Doubly distilled water, produced in house from a glass

apparatus, was used throughout. PDADMAC (High Mole-

cular Weight: Mr�4.105–5.105) 20% w/w in water, PSS

(average Mr�10.105) 10% w/w in water, cytochrome c (Cyt c)

from bovine heart, a-lactalbumin (a-Lac) from bovine milk,

lysozyme (Lys) from chicken egg white, myoglobin (Myo)

from horse heart, ribonuclease A (Rib A) from bovine

pancreas and PBS solution (pH 7.2) were purchased from

Sigma-Aldrich (Lyon, France). Tris (purity Z99.7%) was

from Fluka (Buchs, Switzerland). DMF, HCl (37% w/w in

water) and NaOH (purity Z97%) were from Carlo Erba (Val

de Reuil, France). Orthophosphoric acid (85% w/w in water)

was from Prolabo (Paris, France). NaCl, (purity Z99.5%)

was purchased from different suppliers. All chemicals were

used as received. Nylon PuradiscTM Syringe Filters, pore

size 0.45 mm were purchased from Whatman (Versailles,

France).

2.2 Solutions

Coating solutions were prepared by dissolving the cationic

(PDADMAC) or anionic (PSS) polyelectrolyte at the required

concentration in a 20 mM Tris aqueous solution adjusted to

pH�8.3 with HCl 0.01 M. The ionic strength (I) of this

solution is 0.01 M, neglecting the polyelectrolyte concentra-

tion. It was set to the required ionic strength (ranging from

0.01 to 4.5 M) by NaCl addition. Coating solutions were used

within 1 wk and stored at 41C when not in use.

The BGE solution was a 100 mM Tris-phosphate buffer

pH 2.5 prepared by mixing 100 mM H3PO4 with 73.64 mM

Tris, without pH adjustment. The ionic strength of this

solution is 0.07 M.

The EOF marker solution was a 0.05% v/v DMF solution

in water.

The test solution used to evaluate the coating perfor-

mance in terms of peak efficiency and adsorption preven-

tion was a mixture of five acidic and basic proteins. A stock

mixed protein solution, 0.2 g/L a-Lac (pI 4.3), 0.1 g/L Myo

(pI 7.3), 0.5 g/L Rib A (pI 8.7), 0.1 g/L Cyt c (pI 10.5) and

0.1 g/L Lys (pI 11) was prepared in PBS, diluted to have a

similar ionic strength as the BGE (0.07 M). Proteins were

dissolved in PBS by gentle agitation because vigorously

shaking protein solutions (by vortex, ultrasonication, etc.)

may cause thermal denaturation as well as mechanical

degradation in particular when the protein solutions

incorporate air bubbles on which proteins can adsorb and

Electrophoresis 2009, 30, 1888–1898 CE and CEC 1889


undergo conformational changes [2, 3]. Protein solutions

were filtrated through 0.45 mm pore size filters, divided into

aliquots, and then stored at �201C. Each aliquot was

defrosted at a constant temperature of 371C (bain-marie) for

25 min, heated at 501C (bain-marie) for 25 min and then

allowed to stand at 251C for 30 min before analysis. Protein

solutions were used within 3 days and stored at 41C when

not in use. Injection of individual proteins enabled peak

identification through mobility matching.

2.3 Instrumentation and operating conditions

All experiments were performed on a P/ACE MDQ

instrument (Beckman, Fullerton, CA, USA) equipped with

a photodiode array detection system.

Capillaries used for all experiments, 31.2 cm total length

(21 cm to the detector window) and 50 mm internal diameter

were prepared from bare fused-silica tubing (TSP, Composite

Metal Services, Hallow, UK) and housed in a cartridge with a

(100� 800 mm) detection window. A new capillary was

prepared for each set of experiments. New capillaries were

initially conditioned by performing the following rinse cycles

to produce a surface capable to bind polyelectrolytes homo-

geneously: 1 M NaOH for 15 min; 0.1 M NaOH for 15 min;

water for 2 min. All rinse cycles were carried out at 20 psi.

For coated capillaries, the EOF was reversed. Cathodic

injections were done and the analysis sequence was: (i)

injection of the EOF marker solution to monitor coating

stability; (ii) injection of the test protein solution with co-

injection of EOF marker solution to monitor both coating

efficiency and EOF stability. The respective injected volumes

of test and DMF marker solutions were 9.88 nL (0.5 psi, 5 s)

and 2.37 nL (0.3 psi, 2 s). Separations in the BGE were

carried out at �10 kV and 251C using 214 nm as detection

wavelength. For protein analysis, the BGE solution of

separation vials was changed every five runs. Between runs,

the capillary was flushed for 2 min with the BGE.

2.4 Capillary coating, storage and regeneration

conditions

PDADMAC and PSS were respectively chosen as the

polycation and the polyanion coating agents. The general

monolayer and multilayer coating, storage and regeneration

procedures used all through this study are given in Table 1

(for details see reference [1]).

2.5 Measurements and calculations

2.5.1 Electroosmotic mobility determination

For coated capillaries, a strong reversed anodic EOF is

obtained. Its mobility (meo) was calculated from the

migration time of DMF used as neutral marker.

For non-coated silica capillaries, another approach

was used to calculate EOF mobility as at pH 2.5, it takes

about 100 min for the neutral marker to pass the detection

window due to the weak EOF generated. The method

described by Williams and Vigh [75] was used (for

details see [1]). The EOF mobility in bare fused-silica capil-

laries (normal polarity, 110 kV) was determined to be

12.85.10�6 cm2 V�1 s�1 with an RSD of 22% (n 5 9).

2.5.2 Repeatability studies

Coating stability was controlled directly after coating

deposition by repeated injections of the EOF marker,

and during the course of protein analysis by co-injection

of the EOF marker solution. Possible analyte adsorption

was monitored by repeated measurements of protein

tm and CPAs, and EOF mobility. Repeatability was

expressed as RSDs where ‘‘n’’ is the number of repeated

injections.

2.5.3 Coating performance

The coating performance was expressed in terms of

separation efficiency by injecting the test mixed protein

solution, ‘‘n’’ times successively. Considering that peak

broadening is solely due to longitudinal diffusion, efficiency

expressed as the number of theoretical plates, N, can be

calculated from the Eq. (1):

N ¼ l2

2 :D: tmð1Þ

with ‘‘l’’ the migration length (cm), ‘‘D’’ the diffusion

coefficient (cm2.s�1) and tm the analyte migration time (s).

Because in our different experiments, EOF mobility

(and hence protein tm) could depend on the experimental

conditions used, the product N.tm was employed (instead

of N) to compare efficiencies obtained in different coating

Table 1. Capillary coating, storage and regeneration conditions

(from [1])

Coating conditions Storage and regeneration

conditions

Coating procedure: Storage:

1. Rinse water (2 min, 20 psi) Rinse water (10 min, 20 psi)

and store in water2. Rinse coating solution

(10 min, 20 psi)

3. Rinse BGE (2 min, 20 psi)

Repeat steps 2 and 3 for

SMIL coatings

Stabilization after coating: Regeneration after storage:

1. 110 kV/10 min in BGE 1. Rinse water (5 min, 20 psi)

2. 10 min wait time in BGE for

SMIL coatings performed

with 0.2% w/v poly

electrolyte concentration

2. Rinse BGE (5 min, 20 psi)

3. Apply 110 kV/10 min in BGE

Electrophoresis 2009, 30, 1888–18981890 R. Nehme et al.


conditions. The SD on N.tm was calculated from the

Equation 2:

SD ¼ffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi

s2tm

t2m

þ s2N

N2

� �:ðN:tmÞ2

sð2Þ

with stm, SD on analyte migration time; sN, SD on the

number of theoretical plates.

3 Results and discussion

First of all, the effect of the preparation procedure of the

protein solutions on their stability and electrophoretic

behavior was investigated. Then, the influence of the

polyelectrolyte (PDADMAC and PSS) concentration and

the ionic strength of coating solutions, and the number of

the coating layers (1, 5 and 11 layer(s)) on the protein

analysis was studied.

Although the number of proteins is huge, the aim of

this study was to make some broad deductions concerning

the influence of different experimental parameters on the

efficiency of the polyelectrolyte capillary coating to quench

protein adsorption. In this work, no attempt was done to

optimize protein separation by modifying, for example, the

BGE properties, sample stacking, etc. On the contrary, in

this study, sample stacking was minimized by adjusting the

analyte and the BGE solutions to the same ionic strength.

Thus, any improvement in the protein peak efficiencies

when analyzed with coated capillaries could be largely

ascribed to the coating conditions.

3.1 Protein preparation procedure

A protein in unfolded (denaturized) state has more

hydrophobic amino acid residues exposed to water than in

folded (native) state and thus must reduce its contact with

water [2, 76]. Consequently, unfolded proteins tend to

adsorb to surfaces, e.g. the inner capillary wall when

analyzed in CE. In other terms, a denaturized protein has

higher affinity to the inner capillary wall than in its folded

(native) conformation [23]. Changes in the residues exposed

on the surface also cause self-association of protein

molecules into an aggregate that may remain in solution

or precipitate [2]. Inter-molecular interactions with other

constituents in the sample matrix (i.e. with the other

proteins in the case of a protein mixed solution) and with

the separation medium [77–80] are also possible. These

possible interactions change the effective net charge of the

protein and its hydrodynamic radius that are, with the

protein molar mass, the major parameters that influence

protein migration time and separation performance in CE

(see [81] and references therein). Therefore, when proteins

are studied in CE it is crucial to avoid untoward physical

denaturation of the protein. It is established that the folded

structure of a protein strongly depends on its environment,

viz solvent, pH, temperature (heating, freezing), additives,

pressure, etc. [2, 3], and that the unfolding in aqueous

solutions is easily induced [76]. Hence, the effect of the

protein solvent and of the procedure used for defrosting

aliquots was investigated to establish optimal conditions

preserving proteins from denaturation during sample

preparation.

3.1.1 Protein solvent

In this work, lyophilized protein samples were used. Freshly

distilled water, BGE (Tris-phosphate; pH 2.5) and PBS

solution (pH 7.2) were foreseen as potential protein solvent.

Water was not selected for several reasons: (i) poor solubility

of Myo in water; (ii) general need of a saline or a buffer

solution to reconstitute lyophilized proteins in their native

(folded) state [2]; (iii) sample stacking with this solvent of

low conductivity may mask the influence of coating

conditions on analyte-peak efficiency; and (iv) possible

inter-molecular interactions between the different proteins

in the mixed solution which are more important in the

absence of salt in the solvent [2, 4, 82].

To eliminate the stacking effect, proteins were dissolved

in the BGE (pH 2.5). Analyzed in CE (in uncoated and

cationic coated capillaries), numerous spikes were obtained

on the electropherograms. This may be due to protein preci-

pitation in the solvent. Actually, proteins tend to denaturize –

and precipitate – at extreme pH values (2.5 in this case) [3].

PBS solution was found to be the most appropriate

solvent for the selected proteins. It contains NaCl and

phosphate ions, and is buffered to the same pH value as the

physiological medium (pH 7.2). Protein physical stability, in

particular thermal stability can be enhanced by adding salts

(NaCl, ammonium phosphate, etc.), sugars, glycerol, etc.[2, 3]. Thus, in PBS solution the folded structure of the

proteins is stabilized and their conformational stability is

enhanced versus frosting–defrosting cycles. In addition,

protein solubility is enhanced by the salting-in effect of NaCl

ions. Another advantage is that protein charges are shielded

by NaCl, which reduces protein intra- and inter-interactions

with each others and with the BGE components [4].

3.1.2 Protein defrosting procedure

Frozen proteins were defrosted at a constant temperature of

371C (physiological temperature). After defrosting, samples

were heated at 501C for 25 min and then left for 30 min to

refold at 251C. In fact, freezing may compromise protein

physical stability and causes denaturation [76]. Physical

denaturation is generally a reversible phenomenon; when

the denaturizing source is removed, protein refolds.

However, non-native kinetic traps may exist and causes

protein refolding in a non-native state (for more details see

[83] and references therein). Heating is used to avoid

possible kinetic traps which is crucial for fast protein folding

into their native conformation [84]. Proteins dissolved in

PBS solution are expected to resist to possible denaturation



at 501C. This was evidenced by the constant electrophoretic

mobility of the proteins analyzed before freezing and after

defrosting at 501C.

Having stable protein solutions is crucial to assure

consistent starting conditions for each analytical run.

Heating applied while defrosting yielded reproducible

results in terms of protein migration times (RSDtmo2%)

and CPAs (RSDCPAso5%) between the different protein

aliquots analyzed with monolayer or multilayer coated

capillaries (optimal coating conditions determined in refer-

ence [1] were used in this investigation).

Furthermore, when using this sample preparation

protocol, the five proteins of the test mixture solution (Rib

A, Lys, Cyt c, a-Lac and Myo) could be detected in bare

fused-silica capillaries whereas it was reported [67] that,

because of adsorption phenomena, the peaks of Rib A, Lys

and Cyt c, dissolved in water, could not be detected

when analyzed with uncoated fused-silica capillaries in

similar separation conditions (phosphate buffer, pH�3.0,

I 5 0.05 M).

3.2 Influence of polyelectrolyte concentration and

ionic strength of coating solutions on coating

stability and separation efficiency

When coating silicone wafers, polyelectrolyte concentration

was reported to increase the polyelectrolyte amount

deposited on the surface until the saturation of the silanol

groups [85, 86]. There is also a ‘‘strong dependence of layer

thickness on polymer concentration’’ [71].

Moreover, it has been shown that the ionic strength

of the coating solution has a major impact on the layer

thickness and thus on their efficiency to mask silanol

groups of silicone wafers [73, 87] and of fused-silica

capillaries [1].

The influence of PDADMAC and PSS concentrations

and ionic strength of coating solutions on coating stability

and protein peak efficiency was systematically investigated.

The PDADMAC and PSS concentrations studied were 0.04

and 0.2% w/v. At high polyelectrolyte concentrations

(40.7% w/v), no stable SMIL coatings could be obtained [1].

The ionic strengths studied were 0.01 and 1.5 M. Other

coating conditions were used at their optimum determined

in our previous investigation, for a peptide test mixture [1].

Capillaries were modified by a monolayer of polycation

(PDADMAC) or a SMIL (5- and 11-layers) of PDADMAC

and PSS.

3.2.1 PDADMAC monolayer coating

For monolayer coated capillaries, a stable anodic EOF

(RSDmeoo1%, n 5 9) was obtained immediately after coat-

ing whatever the coating conditions (Table 2a and b).

However, its stability was higher at high ionic

strength (RSDmeoo0.1%). EOF magnitude is slightly

higher for low ionic strength of the coating solution.

Consequently, better resolution of proteins was obtained at

I 5 1.5 M.

In the course of protein analysis, EOF decreased

severely for both ionic strengths when capillaries were

coated at a low PDADMAC concentration 0.04% w/v

(Table 2a). Protein analysis was not possible unless the

coating is regenerated between successive runs, by rinsing

the capillary with the coating solution for 0.5 min followed

by a 0.5 min wait time in the PDADMAC solution.

Table 2. Influence of the ionic strength and the electrolyte concentration of the coating solution on the EOF magnitude and repeatability

Ionic strength (M) Number of layers EOF measurements

Immediately after capillary coating In the course of protein analysis

meo (�10�4 cm2 V�1 s�1) % RSD (n 5 9) meo (�10�4 cm2 V�1 s�1) % RSD (n 5 13)

(a) Polyelectrolyte concentration 0.04% w/v

1 3.52 0.34 3.58a) 0.66

0.01 5 3.21 0.80 3.53a) 0.53

11 3.12 0.46 3.16a) 0.37

1 2.94 0.05 2.83a) 0.99

1.5 5 2.97 0.07 3.03 0.59

11 2.82 0.20 2.86 1.24

(b) Polyelectrolyte concentration 0.2% w/v

1 3.40 0.86 3.54 0.35

0.01 5 3.04 0.25 3.16 0.59

11 3.04 0.42 3.15 0.70

1 2.96 0.10 3.04 0.38

1.5 5 2.90 0.18 2.99 0.35

11 2.83 0.22 b) b)

a) Results obtained with regeneration of the coating between successive protein runs.

b) No protein could be detected under these conditions.



A stable EOF was then obtained (RSDmeoo1%; n 5 9)

(Table 2a) whatever the ionic strength of the coating solu-

tion. Coatings obtained at 0.2% w/v concentration were

highly stable during protein analysis without any need of

regeneration with a RSDmeoo0.40% (n 5 9) (Table 2b).

Similar observations were made by Cordova et al. [61]

who successfully replaced the recoating steps between runs

by an increase of the polyelectrolyte concentration in the

coating solution.

Protein analysis repeatability (Table 3) is clearly

improved when the polyelectrolyte concentration is

increased particularly for migration times (RSDtmo1.2%,

n 5 13). From Table 3, it can be seen that for protein

analysis with monolayers coated at I 5 0.01 M, slightly better

results for migration time and CPA repeatability were

obtained compared with monolayer coatings performed at

1.5 M ionic strength. However, monolayers at I 5 1.5 M

were found to give remarkably higher protein-peak effi-

Table 3. Influence of the ionic strength and the polyelectrolyte concentration of the coating solution on protein analysis repeatability

(n 5 13)

Ionic strength

(M)

Number of layers

1 5 11

RSD % range

for tm

RSD % range

for CPAs

RSD % range

for tm

RSD % range

for CPAs

RSD % range

for tm

RSD % range

for CPAs

(a) Polyelectrolyte concentration 0.04% w/v

0.01a) 0.99–2.14 6.29–8.76 0.81–1.49 4.53–7.18 0.93–1.45 1.47–9.97

1.5 2.35–4.02a) 5.42–12.02a) 1.48–1.77 6.76–10.90 0.88–1.85 5.38–10.20

(b) Polyelectrolyte concentration 0.2% w/v

0.01 0.52–0.89 3.61–7.35 1.34–1.95 3.50–7.90 1.12–1.72 7.07–14.22

1.5 0.66–1.12 3.58–10.61 0.37–0.48 4.15–8.02 b) b)

a) Results obtained with regeneration of the coating between successive protein runs.

b) Not possible (unstable EOF during protein analysis).

Figure 1. Effect of the ionicstrength of the coating solutionon protein peak efficiency.Monolayer coating: polyelec-trolyte concentration 0.04%(A) and 0.2% w/v (B). Five-layercoating: polyelectrolyte con-centration 0.04% (C) and0.2% w/v (D). Error bars indicatethe SD on N.tm for ‘‘n’’ differentexperiments. For the values of‘‘n’’, see Table 2, and for coat-ing conditions see Table 1.



ciency. Fig. 1A and B shows that ‘‘N.tm’’ obtained with

coatings in the presence of salt are between two and five

times higher than those obtained when coating at

I 5 0.01 M. Although slightly improved at 0.2% w/v, the

effect of the polyelectrolyte concentration on peak efficiency

(Fig. 2) appeared to be rather limited compared with the

effect of the ionic strength.

3.2.2 PDADMAC/PSS multilayer coating

Coatings with 5- and 11-layers at 0.01 M and 1.5 M ionic

strengths were found to give a stable anodic EOF

(RSDmeoo1%) after coating (Table 2).

At low polyelectrolyte concentration (0.04% w/v), no

stable SMIL coatings were obtained during protein

analysis when the ionic strength of the coating solution is

0.01 M. Regeneration (procedure similar to that used

for monolayers) was needed to obtain repeatable

analysis. However, salt addition (I 5 1.5 M) in the coating

solutions assured a stable coating at this low polyelectrolyte

concentration all through the protein analysis unlike what

was observed for monolayer coatings in the same

conditions.

Coatings obtained at 0.2% w/v polyelectrolyte concen-

tration were stable but protein analysis repeatability with

5-layer coatings was clearly improved by the presence of salt

in the coating solution (Table 2). However, whereas a stable

EOF was obtained immediately after coating with 0.04% w/v

concentration, a wait time of 10 min is essential to obtain a

stable EOF rapidly when coating with 0.2% w/v polyelec-

trolyte solutions. 11-layer coatings at 1.5 M and 0.2% w/v

polyelectrolyte concentration could not be stabilized (see

Section 3.4).

For protein analysis (Table 3), best migration

time repeatabilities were obtained with 5-layer coated

capillaries at 1.5 M ionic strength and 0.2% w/v polyelec-

trolyte concentration with RSDtmo0.5% (n 5 13) and

RSDCPAso8% (n 5 13). CPA repeatability obtained in these

conditions is similar to that obtained with 5-layer films

coated at 0.01 M ionic strength. In terms of protein

separation efficiency, coatings at I 5 1.5 M with 5-layers

(Fig. 1C and D) were found to give at least two times higher

protein-peak efficiencies than coatings at 0.01 M. Polyelec-

trolyte concentration had a limited influence on peak

efficiency (Fig. 2C and D).

These results demonstrate that both the polyelectrolyte

concentration and the ionic strength of the coating solution

play an important role in coating stability of and repeat-

ability of protein analysis. The coating instability observed

during protein analysis in the case of coatings performed at

low polyelectrolyte concentration and/or low ionic strength

coating solutions, is very likely to be attributed to protein

adsorption. At low ionic strength, a strong electrostatic

repulsion exists between polyelectrolyte chains [71, 88],

which leads to the depletion of polyelectrolytes at the

capillary coating surface. Also, a low ionic strength

engenders thin surface coatings [73, 87] unable to mask

efficiently silanol groups especially when low insufficient

Figure 2. Effect of thepolyelectrolyte concen-tration of the coatingsolution on protein peakefficiency. Monolayercoating: polyelectrolyteconcentration 0.04% (A)and 0.2% w/v B). Five-layer coating: polyelec-trolyte concentration0.04% (C) and 0.2% w/v(D). Error bars indicatethe SD on N.tm for ‘‘n’’different experiments.For the values of ‘‘n’’ seeTable 2, and for coatingconditions see Table 1.



polyelectrolyte concentrations are used (in this case,

0.04% w/v). In these conditions, it seems that the coverage

of the capillary surface with polyelectrolytes is incomplete

resulting in a capillary surface that still expose many free

silanol groups on which proteins can adsorb resulting in

poor EOF and analysis repeatabilities [23, 26, 89]. Coating

regeneration between runs by rinsing the capillary with the

PDADMAC coating solution followed by a wait time in the

coating solution, probably removes any adsorbed protein. In

fact, it has been reported [90] that rinsing the coating with

high molecular weight polyelectrolytes (PDADMAC in this

case) is capable to stripe off low molecular weight poly-

electrolytes (proteins in this case) from the coating surface.

Quasi-soluble polyelectrolyte complexes are thus formed

and then washed away. The highest coating stability

(evidenced by a stable EOF) during protein analysis can be

obtained by adding salt to coating solutions (I 5 1.5 M). The

presence of salt induces a dramatic change in the polyelec-

trolyte chain configuration producing thick films [71, 87]

able to mask more efficiently silanol groups, thus prevent-

ing more efficiently analyte adsorption on the capillary wall

[1]. This is evidenced by the remarkable improvement of

peak efficiency obtained at I 5 1.5 M compared with

I 5 0.01 M (at least two times higher – Fig. 1) because

protein adsorption on surfaces causes their denaturation

and thus loss of peak efficiency [9, 31, 61, 82]. Similar

observations were made when we studied, in the same

conditions, the effect of the ionic strength on peptide peak

efficiency [1].

Although less significant, silanol coverage is also im-

proved by the use of 0.2% w/v polyelectrolyte concentration.

3.3 Influence of the coating layer number on coating

stability and separation efficiency

Monolayers as well as multilayers coated from low polyelec-

trolyte concentration (0.04% w/v) solutions were found to be

unstable in the absence of salt. However, multilayers coated

at 0.04% w/v and I 5 1.5 M showed very good stability and

efficiency in limiting protein adsorption (Tables 2 and 3).

This high stability is to be explained by the fact that

polyelectrolyte chains adopted a coiled configuration in the

presence of salt [87, 91] leading to strong interactions

between polyelectrolytes of opposite signs and extensive

layer interpenetrations [1, 60, 71–73]. In the absence of salt,

polyelectrolyte chains are fully extended [87, 91] which limits

the layer interpenetrations [71] and therefore the coating

stability. The film thickness increases with the number of

layers deposited [87, 91, 92] covering more efficiently the

capillary inner surface. These observations are confirmed by

the results obtained at 0.2% w/v. Repeatability is improved

for 5-layer coated capillaries by comparison with monolayers

only when salt is added to the coating solution.

When coating solutions are used at low ionic strength,

protein peak efficiencies increase with the number of layers

deposited; 11-layer coatings gave the best peak efficiencies

(Fig. 3A and C).

Figure 3. Influence of thenumber of the coatinglayers on protein peakefficiency. Polymer concen-tration 0.04% w/v: Ionicstrength 0.01 M (A) and1.5 M (B). Polymer concen-tration 0.2% w/v: Ionicstrength 0.01 M (C) and1.5 M (D). Error bars indi-cate the SD on N.tm for ‘‘n’’different experiments. Forthe values of ‘‘n’’ seeTable 2, and for coatingconditions see Table 1.



However, when the coating solution is set to a 1.5 M

ionic strength, peak efficiencies are only slightly improved

for 5-layer coatings (Fig. 3B and D). For 11-layer coatings,

efficiencies decreased at low polyelectrolyte concentration

and unstable coatings are obtained at 0.2% w/v. It seems

that these coatings are very thick and not suitable for protein

analysis. Indeed, Graul et al. [74] successfully analyzed

proteins in CE with 13-layers coated capillaries but only the

last seven layers were build-up from coating solutions

containing only 0.5 M salt. In these conditions, this film is

largely thinner than the 11-layer coatings at 1.5 M we used

in this work.

We believe that the instability of the 11-layer coatings

(0.2% w/v and I 5 1.5 M) is due to the presence of salt in the

multilayer originating from the BGE solution [90, 93] used

in the rinse step performed after each layer deposition.

Multilayers containing salt ions are reported to be less

interpenetrating than those containing no salt. Thus, indi-

vidual chains would have more mobility in the presence of

BGE ions and yields less stable coatings [71, 90, 94].

However, it has been shown when coating silicone wafers,

that the effect of the presence of salts in the multilayer is

observed for coatings of at least six polyelectrolyte layers and

in particular for salt concentrations above 0.5 M [95, 96].

Because in this study, the BGE concentration is only about

0.1 M, its effect on coating stability is limited. This

instability of the 11-layer coatings was observed only in the

case of protein analysis and not when peptides were

analyzed [1] which shows that proteins do interact more

strongly with polyelectrolyte coatings than peptides.

3.4 Comparison with uncoated fused-silica

capillaries

Protein analysis was performed in uncoated silica capillaries

for comparison. Figure 4 shows the dramatic improvement

of repeatability obtained for protein analysis using 5-layer

coatings prepared from 0.2% w/v polyelectrolyte concentra-

tions and 1.5 M ionic strength solutions, compared with

fused-silica capillaries.

Because incomplete peak resolution was obtained for

Myo, a-Lac and Rib A, only results for Cyt c and Lys were

reported in Fig. 3.

3.5 Intermediate (inter-day) precision

Inter-day precision was estimated for 5-layer coatings at

1.5 M ionic strength and 0.2% w/v polyelectrolyte concen-

tration by analyzing the protein test mixture solution on

four different days. Within each day, the protein test

mixture solution was analyzed three times under repeat-

ability conditions. The inter-day RSDs were less than 1% for

migration times and less than 10% for CPAs (Table 4).

4 Concluding remarks

The ionic strength of the coating solution, the polyelec-

trolyte concentration and the number of the coated layers

have shown to have a great influence on the coating stability

and performance in limiting protein adsorption.

It has been shown that to obtain efficient and highly

stable coatings, it is essential to use coating solutions

containing salt, preferentially at a 1.5 M ionic strength. But

when working at low polyelectrolyte concentrations

(0.04% w/v), salt addition to the coating solutions is not

sufficient to produce stable coatings versus protein adsorp-

Figure 4. Repeatability of protein analysis. Electropherogramsobtained for the first, seventh and fifteenth injection using (A) a5-layer coated capillary (optimal coating conditions) and (B) anuncoated fused-silica capillary. Peak identification: 1, Rib A; 2, a-Lac; 3, Myo; 4, Lys and 5, Cyt c. For capillary electrophoreticconditions see Section 2.

Table 4. Estimated inter-day precision for protein analysis with

5-layer coatings at 1.5 M ionic strength and 0.2% w/v

polymer concentration

Protein

Ribo A Lac Myo Lys Cyt c

RSD % for tm 0,43 0,41 0,93 0,65 0,93

RSD % for CPAs 2,70 5,44 8,90 4,93 9,84



tion, unless SMIL coatings are performed. For multilayer

coatings at 1.5 M, strong interactions and extensive inter-

penetrations between the polyelectrolytes of the different

layers result in the formation of thick and highly stable

coatings that mask efficiently the silanol groups responsible

for protein adsorption. Separation efficiency appeared to

depend mainly on the presence of salt in the coating

solution. The best results for protein analysis were

obtained when using 5-layer coatings at 1.5 M ionic

strength and ‘‘sufficient’’ polyelectrolyte concentrations

(0.2% w/v).

However, the presence of BGE ions in the multilayer

coatings seems to be detrimental to the coating stability

when an important number of layers is performed (11 layers

in this study). Therefore, it can be foreseen that even higher

stability of the coating could be obtained by rinsing with

water after each deposited layer instead of BGE solution

during the coating procedure.

The authors have declared no conflict of interest.

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Influence of polyelectrolyte capillary coating conditions on protein analysis in CE

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