,

Journal of Chromatography A, 1204 (2008) 104–109

Contents lists available at ScienceDirect

Journal of Chromatography A

journa l homepage: www.e lsev ier .com/ locate /chroma

Fast and easy coating for capillary electrophoresis based ona physically adsorbed cationic copolymer

José Bernala, Irene Rodríguez-Meizosoa, Carlos Elvirab, Elena Ibáneza, Alejandro Cifuentesa,∗

a Institute of Industrial Fermentations (CSIC), Juan de la Cierva 3, 28006 Madrid, Spainb Institute of Science and Technology of Polymers (CSIC), Juan de la Cierva 3, 28006 Madrid, Spain

a r t i c l e i n f o

Article history:Received 28 May 2008Received in revised form 7 July 2008Accepted 22 July 2008Available online 26 July 2008

Keywords:Coated capillariesPhysically adsorbed polymersProteinsBenzoic acidsAmino acidsNucleosides

a b s t r a c t

In this work, a new copolymer synthesized in our laboratory is used as physically adsorbed coating forcapillary electrophoresis (CE). The copolymer is composed of ethylpyrrolidine methacrylate (EPyM) andmethylmethacrylate (MMA). The capillary coating is easily obtained by simply flushing into the tubingan EPyM/MMA solution. It is demonstrated that the composition of the EPyM/MMA copolymer togetherwith the selection of the background electrolyte (BGE) and pH allow tailoring the direction and magnitudeof the electroosmotic flow (EOF) in CE. It is also shown that the EOF obtained for the EPyM/MMA-coatedcapillaries was reproducible in all cases independently on pH or polymer composition. Thus, RSD valueslower than 1.9% (n = 5) for the same capillary and day were obtained for the migration time, while therepeatability interdays (n = 5) was observed to provide RSD values lower than 0.5%. The stability of thecoating procedure was also tested between capillaries (n = 3) obtaining RSD values lower than 0.6%. It isdemonstrated with several examples that the use of EPyM/MMA coatings in CE can drastically reducethe analysis time and/or to improve the resolution of the separations. It is shown that EPyM/MMA-coatedcapillaries allow the separation of basic proteins by reducing their adsorption onto the capillary wall. AlsoEPyM/MMA-coated capillaries provide a faster separation of samples containing simultaneously positive

and negative analytes. Moreover, it is demonstrated that the use of EPyM/MMA-coated capillaries canincorporate an additional chromatographic-like interaction with nucleosides that highly improves the

f solu

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separation of this group o

. Introduction

Capillary electrophoresis (CE) has not totally reached the highxpected impact among the scientific community because stillome problems remain to be solved. These limitations include, rela-ively low concentration sensitivity, low quantitative repeatability,

igration time drifts and the existence of analyte–wall interac-ions.

Several of these problems rely on the capillary wall charge thatontrols among other parameters the electroosmotic flow (EOF)nd electrostatic interactions. Thus, the apparent mobility of ana-ytes is often less reproducible because of run-to-run variations inOF [1], while electrostatic interactions between solutes and cap-

llary wall ruin the CE separation mostly of compounds bearing aigh positive charge [2–12].

Many approaches have been proposed for reducing these dele-erious effects including variations of pH [13], ionic strength [14],

∗ Corresponding author. Tel.: +34 91 5622900; fax: +34 91 5618806.E-mail address: acifuentes@ifi.csic.es (A. Cifuentes).

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021-9673/$ – see front matter © 2008 Elsevier B.V. All rights reserved.oi:10.1016/j.chroma.2008.07.061

tes.© 2008 Elsevier B.V. All rights reserved.

r viscosity of the background electrolyte (BGE) [15], while otherpproaches have proposed the use of BGE additives [16] and alsohere are works related to the physical control of EOF by exter-al radial electric field [17,18]. The procedure most commonlysed to improve repeatability between injections while reduc-

ng solute–capillary wall interactions is to coat the inner capillaryall. One of the first works which dealt with the use of coated

apillaries to solve these problems was published in 1985 [19].hese coatings can basically be classified in two different groups20,21], namely covalently linked coatings [3,22–24] and physi-ally adsorbed coatings (static or dynamic) [2,25–36], and theyan be based on polymers [22–36] or small molecules [37–42]. Theevelopment of new coatings for CE still remains as an active areaf research, as it is demonstrated with the publication of severalapers in 2008 [43,44].

In this work, a new copolymer synthesized at our laboratory is

ested as physically adsorbed coating in CE. The main characteris-ics of any physically adsorbed coating should include [20,21]: (i)implicity of coating formation; (ii) possibility of coating regen-ration; and (iii) access to a priori knowledge on coating polymerroperties.

J. Bernal et al. / J. Chromatogr. A 1204 (2008) 104–109 105

bCofl

ne(camclcpawiica

2

2

h3mcn(dpaOd2rrc

iwv(n

Table 1Composition and pH of the background electrolytes (BGEs) used in this work(between parenthesis volumes of the dissolutions described in Section 2.3)

pH NaOH Acid

2.2 43 mM (2.15 ml) 86 mM H3PO4 (4.3 ml)3.8 50 mM (2.5 ml) 75 mM HCOOH (3.75 ml)

2

sma4a6acPmalc

2

dt1(Cis

2

E1fiatpa

f2t

wf3

Fig. 1. Structure of the EPyM/MMA copolymer used in this work.

Our research group has already developed a new polymer foreing used as coating in CE [45], demonstrating its suitability for theE analysis of different analytes [10–12] or even the compatibilityf the polymer with other analytical technique such as supercriticaluid chromatography [46].

The goal of this work is to demonstrate the advantages of aew copolymer synthesized in our laboratory and composed ofthylpyrrolidine methacrylate–methylmethacrylate (EPyM/MMA)see Fig. 1) as coating for CE. Thus, different EPyM/MMA copolymersontaining different percentages of the monomers EPyM and MMAre synthesized. One interesting characteristic of these copoly-ers is that the number of positive charges can be modified by

hanging the percentage of EPyM and with that the coated capil-ary wall properties (i.e., EOF, adsorption, etc.). Moreover, the BGEomposition and pH can also help to modify the capillary wallroperties. Several conditions were tested by varying the percent-ge of EPyM together with different BGEs. Results were comparedith those obtained using a bare fused silica capillary includ-

ng the repeatability that both types of capillaries provide. Somenteresting advantages are derived from the use of these coatedapillaries including faster separations, better resolved and reducednalyte–capillary wall adsorption.

. Materials and methods

.1. Chemicals

Sodium hydroxide, acetone, formic acid and ammoniumydroxide were obtained from Merck (Darmstadt, Germany).-(Cyclohexylamino)-1-propanesulfonic acid (CAPS) and 2-(N-orpholino)ethanesulfonic acid (MES), bovine cytochrome c, horse

ytochrome c, ribonuclease A, tryptophan, tyrosine, phenylala-ine, 2-hydroxybenzoic acid, 2′-deoxyadenosine monohydratedA), 2′-deoxyguanosine monohydrate (dG), 2′-deoxycytidine (dC),eoxythymidine (dT) and 5-methyldeoxycytidine (mdC) were sup-lied from Sigma–Aldrich Chemie (Steinheim, Germany). Boriccid was purchased from Riedel-de Haën (Hannover, Germany).rtophosporic acid was from Panreac (Barcelona, Spain). Water waseionized with a Milli-Q system from Millipore (Bedford, MA, USA).-Ethyl-(2-pyrrolidine) methacrylate (EPyM) was synthesized byeaction of 1-(2-hydroxyethyl) pyrrolidine with metacryloyl chlo-ide, both from Fluka (Buchs, Switzerland) and purified by columnhromatography, as previously described [47].

Methyl methacrylate (MMA) was provided by Across Organ-cs (Geel, Belgium). 2,2-Azobisisobutyronitrile (AIBN) from Flukaas purified by fractional crystallization from ethanol. The sol-

ents used were tetrahydrofuran (THF) from Sharlau ChemieBarcelona, Spain) and ethanol from RP Normapur, Prolabo (Fonte-ay, France).

2

CmI

6.1 50 mM (2.5 ml) 100 mM MES (10 ml)6.9 36 mM (1.8 ml) 22 mM H3PO4 (1.1 ml)8.8 50 mM (2.5 ml) 100 mM H3BO3 (20 ml)

10.7 50 mM (2.5 ml) 75 mM CAPS (15 ml)

.2. Polymers synthesis and characterization

In this work, EPyM/MMA copolymers (see Fig. 1) were synthe-ized at our laboratory using three different percentages of theonomers (25/75, 50/50 and 75/25 of EPyM/MMA with number-

verage molecular masses (Mn) and polydispersity values of 43 200,2 500, 39 900 and 2.2, 2.2, 2.3, respectively). The monomers EPyMnd MMA were copolymerized by free radical copolymerization at0 ◦C for 48 h of reaction time, using AIBN ([I] = 1.5 × 10–2 mol/L) asradical initiator and THF as solvent to reach a final monomer con-entration equal to 1.0 mol/L. Polymerization was carried out in ayrex ampoule under an oxygen-free N2 atmosphere. The reactionixture was precipitated in a large excess of hexane, filtered off

nd vacuum-dried to obtain the EPyM/MMA copolymer as a yel-ow powder (average reaction yield = 53%). The copolymers wereharacterized by H NMR spectroscopy.

.3. Preparation of BGEs

To study the EOF mobilities from the different capillaries atifferent pHs, using normal or reverse polarity, six background elec-rolytes (BGEs) were employed. Each one was made up by mixingM NaOH with the adequate volume of the following solutions

1 M H3PO4, 1 M formic acid, 0.5 M MES, 0.25 M H3BO3 and 0.25 MAPS) in water till a 50 ml total volume, trying to obtain a similar

onic strength for all of them (50 mM). The buffers employed areummarized in Table 1.

.4. Capillary coating

Several copolymers composed of 25/75 EPyM/MMA, 50/50PyM/MMA and 75/25 EPyM/MMA were tested as coatings for CE.2 mg of each copolymer was first dissolved in 200 �L of 100 mMormic acid, an equivalent volume of 100 mM ammonium hydrox-de and leaded to a final concentration of polymer of 1 mg/mL incetone (for acetone solutions) or ethanol (for ethanol solutions),est were made with acetone and ethanol because solvents of theolymer solutions could affect the composition of the capillary wallt the solid–liquid interface.

The procedure followed to obtain a reproducible coating was asollows [12]: each new capillary was washed with 0.1 M NaOH for0 min, then flushed with the polymer solution for 10 min, and lefto stand overnight inside the capillary.

At the beginning of the day, the capillary was washed for 10 minith the polymer solution to prepare the coating. Between the dif-

erent runs, the capillary was washed with the polymer solution formin, water for 2 min and then the buffer for 3 min.

.5. Separation conditions and samples

A P/ACE 2050 instrument with UV detection from Beckmanoulter (Fullerton, CA, USA) was used to perform the CE experi-ents. The capillaries used were made of fused silica with 50 �m

.D. and 360 �m O.D. (Composite Metal Services, Worcester, UK),

1 atogr. A 1204 (2008) 104–109

we

mdpsp

c(wtH

aawiwaa

ficnT1apamw

3

3c

(cocaicMtcw

caEirftsupllc

TimtFvswdsEw

3

asfTvmtwfwm(acetone and ethanol).

The intraday repeatability and the interday repeatability of thecoating procedure were evaluated next. Thus, three capillaries werecoated with a 50/50 EPyM/MMA copolymer in acetone. The EOF

Table 2RSD values of the EOF values obtained for bare fused silica and different EPyM/MMA-coated capillaries at the different pH values used in this work

pH RSD (%, n = 5) of EOF values

Bare silica EPyM/MMA inacetone (%EPyM)

EPyM/MMA inethanol (%EPyM)

25% 50% 75% 25% 50% 75%

2.2 2.5 1.0 0.5 0.3 0.7 0.3 0.1

06 J. Bernal et al. / J. Chrom

ith 370 mm of total length (300 mm detection length.) For all thexperiments the capillary was thermostated at 30 ◦C.

A buffer/water/acetone (20/75/5) was employed as sample toeasure the EOF mobilities; it was injected for 3 s at 3450 Pa and

etected at 254 nm. The voltage applied was 20 kV. Each run waserformed by triplicate. For the study of the different coatings theame sequence of BGEs was used (i.e., from the lowest to the highestH) reproducing in that way any possible hysteresis effect.

Proteins sample consisted of bovine cytochrome c, horseytochrome c and ribonuclease A all dissolved in bufferHCOOH/NaOH at pH 3.8)/water (1/4, v:v) at 50 �g/mL. The proteinsere analyzed at 30 kV. The sample was injected for 10 s at 3450 Pa;

he detection wavelength was set at 200 nm. The BGE employed wasCOOH/NaOH at pH 3.8.

The test mixture contained tryptophan, tyrosine, phenylalaninend 2-hydroxybenzoic acid, all dissolved in buffer (H3PO4/NaOHt pH 2.2)/water (1/4 v/v) at 0.5 mM. The analysis of the test mixas performed at 20 kV (normal polarity) with the bare silica cap-

llary and at −20 kV (reverse polarity) with the coated capillary. Theavelength used was 214 nm, and the mixture was injected for 3 s

t 3450 Pa. The BGE used to analyze this test mix was H3PO4/NaOHt pH 2.2.

Two nucleoside mixtures were also studied in this work. Therst one included dA, dC, dT and mdC and the second one wasomposed of the four previously cited nucleosides plus dG, bothucleosides mixtures were dissolved in deionized water at 1 mM.hey were analyzed using two different BGEs: CAPS/NaOH at pH0.7 and H3PO4/NaOH at pH 2.2. Thus, at pH 10.7 the same volt-ge and polarity (20 kV) was used for both types of capillaries. AtH 2.2, a 20 kV (normal polarity) was used with the silica capillarynd −20 kV (reverse polarity) with the coated capillary. Nucleosideixtures were injected for 5 s at 3450 Pa. The detection wavelengthas 254 nm.

. Results and discussion

.1. Electroosmotic flow provided by EPyM/MMA-coatedapillaries

Unlike other positive polymers already used as CE coatingspolyethyleneimine [2], polybrene [48,49], etc.) the EPyM/MMAopolymer (see structure in Fig. 1) allows adjusting the numberf positive charges by modifying in the synthesis the initial EPyMontent. Thus, this quality of EPyM/MMA copolymers together withn adequate selection of the BGE can allow tailoring the CE behav-or of the EPyM/MMA-coated capillaries. For this reason, differentopolymer compositions were studied as coatings in this work.oreover, since it was thought that the solvent used to dissolve

he polymer could influence the behaviour of the coating (e.g., byontrolling the interaction between the copolymer and the silicaall) this point was also studied.

In order to investigate these points, three different EPyM/MMAopolymers were synthesized (namely composed of 75/25, 50/50nd 25/75 EPyM/MMA) and dissolved using acetone or ethanol.ach capillary coating was prepared as described in Section 2.4 andts CE behaviour studied using the six BGEs described in Table 1anging from pH 2.2 to 10.7. The electroosmotic mobility of a bareused silica was determined as reference employing the same men-ioned BGEs. The results obtained for the different capillaries arehown in Fig. 2. As can be seen, the EOF is highly modified by

sing EPyM/MMA capillaries showing a different behavior com-ared with the EOF from a bare silica capillary. Thus, at pH values

ower than 6.1 the observed EOF for the EPyM/MMA-coated capil-aries is clearly reversed independently on the composition of theopolymer and the solvent used to dissolve it (acetone or ethanol).

Fig. 2. Electroosmotic mobility as function of pH and type of capillary.

his behaviour indicates that the global charge of the capillary walls positive due to the ionization of the amine groups of the EPyM

onomer at these pH values. On the other hand, at pH values higherhan 6.9 the EOF is towards the cathode as can be deduced fromig. 2. This indicates that the positive charges are lower at basic pHalues and, that the remaining positive charges would be compen-ated by the number of ionized silanol groups on the fused silicaall bearing negative charge. Moreover, the EOF obtained for theifferent copolymers gave a similar behaviour also at this pH range,howing that at pH about 6.5 it is possible to achieve a nearly zeroOF. Next, a study on the stability and repeatability of these coatingsas carried out.

.2. Repeatability of the coating

The repeatability provided by the seven capillaries (six coatednd one bare silica capillary) used in the experiment of Fig. 2 wastudied by measuring the RSD of the EOF mobility obtained at 6 dif-erent pH (n = 5 injections). The obtained RSD values are shown inable 2. It can be observed that at the low pH range (2.2–6.1) the RSDalues were improved by using the EPyM/MMA-coated capillaries;eanwhile from pH 6.9 to 10.7 the RSD values were better using

he bare silica capillary. In all the cases the RSD values obtainedere lower than 2% for the coated capillaries and lower than 2.5%

or the bare silica capillary. Moreover, not significant differencesere observed in the RSD values obtained for the different copoly-ers (75/25, 50/50 and 25/75) or between the two tested solvents

3.8 1.8 1.1 0.7 1.0 1.3 1.1 0.76.1 1.2 0.9 0.8 0.4 1.8 1.3 1.46.9 0.8 1.4 1.5 1.5 1.1 1.2 1.68.8 0.4 1.9 1.0 1.7 0.1 0.9 1.9

10.7 0.4 1.5 0.1 1.8 1.4 1.0 1.7

J. Bernal et al. / J. Chromatogr. A 1204 (2008) 104–109 107

Table 3Repeatability of the coating procedure (3 capillaries, same day) and interday repeatability (5 days, same capillary)

pH Coating repeatability (3 capillaries, same day) Interday repeatability (same capillary, 5 days)

�eo (m2 V−1 s−1) RSD (%, n = 5) �eo (m2 V−1 s−1) RSD (%, n = 5)

C d RSD

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3

wpa

3

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3

tabc

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aa

required nearly 25 min in a bare silica capillary at low pH (2.2).At the same pH, the use of a 50/50 EPyM/MMA-coated capillaryreduced drastically the analysis time at 10 min while improvingthe efficiency for dT and dA (see Fig. 5b). Moreover, it can also beobserved that the migration order has clearly changed for some

2.2 −3.85 × 10−8 0.610.7 4.04 × 10−8 0.1

apillaries were coated using a 50/50 EPyM/MMA copolymer. EOF mobility (�eo) an

obility was determined for each capillary at two different pH val-es (2.2 and 10.7; n = 5 injections at each pH) and the results arehown in Table 3. As it can be seen, the results showed that practi-ally there are not differences among the three capillaries for bothH values, with RSD values lower than 0.6%. So it can be concludedhat the coating procedure is reproducible.

The interday repeatability was checked using a 50/50PyM/MMA-coated capillary for 5 days. The EOF mobility and RSDalues obtained at pH 2.2 and 10.7 are shown in Table 3. It can beeduced from the analysis of the results, that there are slight vari-tions in the RSD values but they are not significant (RSD < 0.5%).herefore, these results seem to show that the use of the selectedoating procedure provided stable coated capillaries.

In summary, from the result shown above it can be concludedhat the behavior was practically the same for all the copolymer-oated capillaries tested, and although there were slight differencesmong them, the trend of the EOF was very similar in all the cases.oreover, the coating procedure and its interday repeatability have

lso been demonstrated to be high. Since their CE behavior wasndependent on the percentage of EPyM or the solvent used to dis-olve the copolymer, a coated capillary using the 50/50 EPyM/MMAopolymer was selected for the following studies. On the otherand, acetone was chosen as solvent instead of ethanol becausehe copolymer was dissolved more easily using that solvent.

.3. Applications of EPyM/MMA-coated capillaries

The benefits of employing EPyM/MMA-coated capillaries in CEere investigated through the study of three different types of sam-les, namely: (i) basic proteins, (ii) a sample containing cationic ornionic solutes, and (iii) nucleosides.

.3.1. Separation of basic proteinsAdsorption of basic proteins onto fused silica capillary wall has

een one of the main problems in protein analysis by CE. A possi-le solution is to use coated capillaries to obtain either a neutralr a positively charged surface reducing in this way the adsorptionroblem and allowing basic proteins separation. To demonstratehe possibilities of the EPyM/MMA-coated capillaries for the sepa-ation of basic proteins, a mixture composed of bovine cytochrome, horse cytochrome c and ribonuclease A with isoelectric pointsf 10.5, 10.2 and 9.3, respectively, was analyzed using both a bareilica capillary and a 50/50 EPyM/MMA-coated capillary. As cane observed in Fig. 3, the use of the EPyM/MMA-coated capil-

ary allows a complete and fast separation of these basic proteinshat could no be achieved by using bare silica capillaries due todsorption problems. So, it can be concluded that the adsorptionf analytes bearing a high positive charge (like basic proteins) isvoided by using the EPyM/MMA-coated capillary.

.3.2. Separation of amino acids including benzoic acids

The application of EPyM/MMA-coated capillaries was also inves-

igated for the analysis of both negatively and positively chargednalytes in the same run. Namely based on the study of the EOFehaviour vs. pH of Fig. 2, it could be deduced that the analysis timesould be shortened by using coated capillaries for these charged

F2a3

−3.80 × 10−8 0.54.09 × 10−8 0.2

values were obtained at pH 2.2 and 10.7.

olutes at pH values lower than 7, since in this case the EOF woulde strongly anodal and the electrophoretic mobility of the mostegative compound (benzoic acid) would be also anodal, while theobility for the group of amino acids would be cathodal but lower

han the EOF. To probe this point, a mixture of four compoundstryptophan, tyrosine, phenylalanine and 2-hydroxybenzoic acid)as injected in the CE system and analyzed using a bare silica

apillary and a 50/50 EPyM/MMA-coated capillary.As can be seen in Fig. 4, baseline separation of this group

f compounds was achieved in less than 5 min by using thePyM/MMA-coated capillary, meanwhile migration time of 2-ydroxybenzoic acid (peak 4) was higher than 15 min using theare silica capillary due to the high cathodal migration of thisompound in the fused silica capillary (data not shown). Also, asxpected, the migration order of the compounds changed using theoated capillary because the polarity was reversed. So, this exam-le demonstrates that EPyM/MMA-coated capillaries can providedequate and fast separations of positively and negatively chargednalytes in a single run.

.3.3. Separation of nucleosidesThe last application of the coated capillary was focused on the

nalysis of nucleosides at low (2.2) and high pH (10.7) using asbove bare silica capillary and a 50/50 EPyM/MMA-coated capillary.

As it can be observed in Fig. 5a, the nucleosides separation

ig. 3. Comparison of the separation of a group of basic proteins (1, ribonuclease A;, bovine cytochrome c; and 3, horse cytochrome c) using a bare silica capillary and50/50 EPyM/MMA-coated capillary using in both cases a HCOOH/NaOH BGE at pH.8.

108 J. Bernal et al. / J. Chromatogr. A 1204 (2008) 104–109

F(p5

niitclc

os

F+H

FlC

F

ig. 4. Comparison of the separation of tryptophan (1), tyrosine (2), phenylalanine3) and 2-hydroxybenzoic acid (4) employing in both cases a H3PO4/NaOH BGE atH 2.2 together with a bare silica capillary and +20 kV as running voltage; and a0/50 EPyM/MMA-coated capillary and −20 kV as running voltage.

ucleosides in the coated capillary. Thus, the expected reversionn the migration order is not observed comparing the bare sil-ca and the coated capillary. For instance, the nucleoside dA inhis case seems to have been more “retained” using the coatedapillary, what could indicate some additional chromatographic-ike interaction between these solutes and the EPyM/MMA

oating.

In order to further study this effect the same group of nucle-sides plus mdC was analyzed at high pH value (10.7) using bareilica and a 50/50 EPyM/MMA-coated capillary. As it can be seen in

ig. 5. Separation of nucleosides (dA, dC, dT, dG) using (a) a bare silica capillary at20 kV and (b) a 50/50 EPyM/MMA-coated capillary at −20 kV using in both cases a3PO4/NaOH BGE at pH 2.2.

nltcam

wotiatithtilitttlomtaoTtb

ig. 6. Separation of nucleosides (dA, dC, dT, dG, mdC) using (a) a bare silica capil-ary and (b) a 50/50 EPyM/MMA-coated capillary using in both cases +20 kV and aAPS/NaOH BGE at pH 10.7.

ig. 6a, when the bare silica capillary was used two of the studieducleosides comigrated (mdC, dC) while dG and dA were not base-

ine resolved. The use of the 50/50 EPyM/MMA-coated capillary athe same pH and run voltage clearly improved the resolution asan be seen in Fig. 6b. A The nucleosides dA and dC seem to inter-ct with the EPyM/MMA coating what would explain their higherigration time under these conditions.Nucleosides contain several ionizable groups in their structure

ith pK values ranging from 1.6 to 12.9 [50] while the pK valuesf their amino groups are in the range 1.6–4.3 [51,52]. Therefore,heir global electrical charge at the separation pH (2.2) will be pos-tive what seems to preclude any ionic interaction between solutesnd the EPyM/MMA coating. Thus, some other kind of interac-ion (e.g., hydrophobic, Van der Waals) should explain the changen the separation selectivity observed in Fig. 5. In order to fur-her study this point the same group of nucleosides plus the moreydrophobic mdC were analyzed at pH 10.7. Under these condi-ions the nucleosides should bear a low negative charge. As shownn Fig. 5, comparing the results achieved with the bare silica capil-ary and the EPyM/MMA-coated capillary, similar “retention” effects observed (mainly for dA and dC) what seems to corroboratehe results obtained at pH 2.2. Therefore, one possible explana-ion is that nucleosides are separated in the coated capillary dueo a combination of capillary zone electrophoresis and open tubu-ar capillary electrochromatography what would explain the resultsbserved at pH 2.2 and 10.7. However, the result observed for theethylated nucleoside mdC (i.e., a more hydrophobic compound

han dC) does not seem to corroborate a possible hydrophobic inter-

ction between the solute and the coating since the migration orderf this compound was not affected by using the coated capillary.herefore, at the moment we do not have a clear explanation forhis chromatographic-like interaction and more experiments wille run at our laboratory to study it.

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J. Bernal et al. / J. Chrom

. Conclusions

In this paper, a simple and reproducible process to coat capillar-es has been presented. It has been proved that the EOF is clearly

odified using the EPyM/MMA capillaries compared to fused silicanes. These coated capillaries show an anodal EOF at low pH val-es, a nearly zero EOF at pH between 6 and 7, and a cathodal EOF atHs higher than 7. It has been demonstrated that the adsorption ofasic proteins is avoided by using EPyM/MMA-coated capillary; thiseparation could not be achieved by using bare silica capillaries. Itas also been demonstrated that EPyM/MMA-coated capillaries canrovide much faster separations of a group of charged (positive andegative) analytes than bare silica capillaries. EPyM/MMA-coatedapillaries, improve the analysis time, efficiency and resolution dur-ng the separation of nucleosides compared to bare silica tubing.

oreover, the use of EPyM/MMA-coated capillaries can incorporaten additional chromatographic-like interaction with the nucleo-ides that improves the separation of this group of solutes.

cknowledgements

J.B. would like to thank the Ministerio de Educación y CienciaSpain) for a Juan de la Cierva contract. This work was supportedy projects AGL2005-05320-C02-01 and Consolider Ingenio 2010SD2007-00063 FUN-C-FOOD (both from Ministerio de EducaciónCiencia, Spain) and S-505/AGR-0153 (ALI-BIRD, Comunidad deadrid, Spain).

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