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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: caperrin@univ-montp1.frFax: 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
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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
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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
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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.
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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.
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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.
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& 2009 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.electrophoresis-journal.com
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.
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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
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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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