· solution. The capillary was ﬂushed with the run buffer for 2 min at 10 psi (68.9 kPa) prior...

Methods and TheoryInstrumentation

CE and CECMicrofl uidics

Proteomics and 2-DE

ISSN 0173-0835 · ELCTDN 28 (3) 285-492 (2007) · Vol. 28 · No. 3 · February 2007

ELECTROPHORESISREPRINT

www.electrophoresis-journal.com

D 7995

Electrophoresis 2013, 34, 2585–2592 2585

Ashley L. MorrisChristopher R. Harrison

Department of Chemistry andBiochemistry, San Diego StateUniversity, San Diego, CA, USA

Received December 19, 2012Revised March 15, 2013Accepted May 5, 2013

Research Article

Adsorption of buffer ion pairs can alterlong-term electroosmotic flow stability

Dynamic capillary coatings have become widespread due to their efficacy in modifying theEOF in capillary electrophoretic separations and ability to limit unwanted analyte-surfaceinteractions. However, our understanding of exactly what types of interactions are takingplace at the surface of a capillary when these dynamic additives are present is limited.In this work, we have chosen a simple, small molecule additive, tetramethylammoniumto examine its influence on the EOF under typical separation conditions. What we haverevealed is that this simple compound does not interact with the capillary surface in a verysimple manner. Our initial hypothesis of a direct ionic interaction with the silanol surfacehas evolved with evidence of complex ion pairing between the silanols, the tetramethy-lammonium, and the buffer ions. This ion pairing can result in drastic changes in theEOF over time, and that the EOF can only be restored to initial levels with harsh rinsescontaining sodium hydroxide.

Keywords:

Dynamic coating / Electroosmotic flow / Ion pair / TetramethylammoniumDOI 10.1002/elps.201200687

� Additional supporting information may be found in the online version of thisarticle at the publisher’s web-site

1 Introduction

CE is a dynamic separation tool that can be used to analyzea great variety of analytes and has become a widely usedanalytical tool. However, one difficulty still plaguing CE isthe adsorption of analytes onto the capillary surface, whichamong other complications, can result in irreproducible EOFleading to inconsistent migration times and irreproducible re-sults [1]. The adsorption of analytes onto the capillary surfacecan be reduced by altering the silica surface with a protectivecoating [2–4], common approaches include the use of perma-nently derivatized coatings [5, 6] or dynamic coatings [7–9].Permanent coatings are achieved through chemical modifi-cations of the silica surface, wherein a less adsorptive surfaceis created by affixing appropriate compounds to the capillarysurface through covalent bonds between the compound andthe surface silanol groups [5,6]. However, the reaction condi-tions for these permanent coatings can be time-consumingand challenging to recreate, leading to difficulties in coating

Correspondence: Assistant Professor Christopher R. Harrison, De-partment of Chemistry and Biochemistry, San Diego State Univer-sity, 5500 Campanile Drive, San Diego, CA 92182, USAE-mail: [email protected]: +1-619-594-4634

Abbreviation: TMA, tetramethylammonium

reproducibility [9]. Dynamic coatings, on the other hand, relyon physical or electrostatic adsorption of the compound ontothe silica surface and can be generated by using a range ofprincipally cationic buffer additives [8–10]. These additivescan be adsorbed as a rinse, prior to separations, or they maybe included in the separation buffer to allow for continuousregeneration of the capillary coating.

As the magnitude of the EOF is principally controlledby the zeta potential [8, 11, 12] generated at the surface ofthe capillary, alterations to the surface charge through bufferpH changes [13, 14], or through the adsorption of cationiccharges to the surface [7–9, 15], will greatly impact the EOF.Cationic buffer additives are able to impact the zeta potentialby adsorbing to the silanol surface. This action can suppressthe anionic charge, in part or in whole; conversely, if anionsare adsorbed to the silica surface, the net anionic charge ofthe surface may be enhanced [2]. There are many classesof buffer additives that can be used in CE separations; onecommonality to most of the additives is the presence of acationic group within the molecule, often present for electro-static interactions with the surface or to alter the magnitudeof the surface charge [9, 16]. Some classes of buffer additivesthat can be used to modify the EOF include amino acids,organic acids surfactants, detergents, and amines [8, 16–18].Among these compounds, tetraalkylammoniums salts, sim-ple cationic compounds, have been widely used in separatingmetal complexes [19], proteins, and peptides [9, 20].

C© 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.electrophoresis-journal.com

2586 A. L. Morris and C. R. Harrison Electrophoresis 2013, 34, 2585–2592

Buffer additives are not the only means used to protectthe silica surface and yield reproducible EOFs. Several stud-ies have also shown that using capillary rinses in betweenseparations can improve EOF reproducibility and stability inlieu of using additives [21, 22]. Capillary rinses work, ide-ally, by desorbing any molecules from the silanol surface[9, 17, 21, 23, 24] and by preventing memory effects [22] fromresidual molecules on the capillary.

Despite the advantages of dynamic coatings, our experi-ence with the long-term reproducibility of the EOF of sepa-ration buffers containing tetramethylammonium (TMA) in-dicated that there is a change in the equilibrium betweenthe TMA and silanol surface over extended periods of time.These results contradicted what would be expected of a dy-namic coating with the TMA in the separation buffer, whereinthe free TMA would be capable of constantly regeneratingand equilibrating with the surface-adsorbed TMA. This dy-namic equilibrium is the prevailing theory on how such qua-ternary ammonium additives are able to influence the EOF[9,15,20,25]. However, in their study of how small quaternaryammonium cations, Steiner et al. [15] proposed that the for-mation of ion pairs, or a surface ion exchange process, couldaccount for the changes in migration times of anions whenseparated in the presence of these ammonium compounds.We hypothesize that the changes that we have seen in theEOF over time with TMA may in fact be the result of sim-ilar ion-pairing interactions taking place between the bufferions, TMA, and surface silanols. To investigate this further,we have undertaken a study to elucidate the nature of theinteractions between the silanol surface, TMA, and the otherbuffer ions. To probe the nature of the interactions, we havestudied the impact of various capillary rinsing scenarios aswell as numerous separation buffers consisting of anionic,cationic, and zwitterionic buffers.

2 Materials and methods

2.1 Chemicals

The buffer additive TMA bromide was purchased from AcrosOrganics (Fair Lawn, NJ, USA). Separation buffers preparedfrom sodium phosphate dibasic, sodium phosphate tribasicdodecahydrate, citric acid, sodium bicarbonate, and sodiumcarbonate anhydrous were purchased from EMD Chemicals(Billerica, MA, USA), phosphorous acid was purchased fromAlfa Aesar (Ward Hill, MA, USA), 1,3-bis[tris(hydroxymethyl)amino]propane 99%, triethanolamine hydrochloride 99+%(Acros Organics), ethylenediamine dihydrochloride 98+%(Acros Organics), ACES 99% extra pure (Acros Organics),HEPES free acid (EMD Chemicals), and tricine 98% for bio-chemistry (Acros Organics). Additional chemicals used in-clude the EOF marker, mesityl oxide (Acros Organics), andmethanol-certified ACS (Fisher Scientific), sodium hydrox-ide (EMD Chemicals), and hydrochloric acid 37% from BDH(Radnor, PA, USA).

2.2 Equipment

EOF measurements were completed on a Beckman CoulterP/ACE MDQ CE system (Fullerton, CA, USA), with a photodi-ode array detector, with measurements taken at 241 nm. Dataacquisition was accomplished by using 32 Karat

TMsoftware

(Beckman Coulter). The fused silica capillaries of 360 �mod and 50 �m id were obtained from Polymicro Technolo-gies (Phoenix, AZ, USA), and were cut to a length of 40 cm(32 cm to detector). New capillaries were prepared by the fol-lowing rinse procedure: methanol, 3 min at 10 psi (68.9 kPa),3 M hydrochloric acid, 3 min at 10 psi (68.9 kPa), 3 M sodiumhydroxide, 8 min at 10 psi (68.9 kPa); a distilled water rinse,2 min at 10 psi (68.9 kPa), was performed between each rinsesolution. The capillary was flushed with the run buffer for2 min at 10 psi (68.9 kPa) prior to each EOF measurement,unless stated otherwise. The neutral EOF marker, mesityl ox-ide, was introduced at the anode by applying a pressure of0.5 psi (3.4 kPa) for 7 s; a voltage of 10 kV was applied toperform each separation. The EOF stability was monitoredover 20 cycles, where a cycle consisted of three EOF mea-surements; thus, a total of 60 separations were measured foreach buffer system. Taking into account possible electrolysiseffects that can occur after repeated separations, [26] the sep-aration buffer vials were alternated based on cycle number tominimize the pH changes that could occur during the stud-ies. Two pairs of 2 mL buffer vials were used in alternationfor each cycle of the 20-cycle set; one pair assigned to evencycles, the other to odd cycles. This process did occasionallyresult in some systematic variability in the EOF between vialpairs, likely due to slight differences in buffer levels resultingin some hydrodynamic flow in the separation. Additionally,all rinses of the capillary with the separation buffer were donewith a separate buffer vial, not used for electrophoresis; therewas a unique rinse buffer vial for each separation buffer vialpair.

2.3 Buffer preparations

All buffer solutions were prepared using 18.2 M� water froma Milli-Q Academic water filtration system purchased fromMillipore (Billerica, MA, USA). The buffers were preparedto a final concentration of 20 mM TMA and 20 mM of therespective buffer. The pH was measured using an Accumentbasic pH meter system (Fisher Scientific) and adjusted byusing 3 or 0.5 M hydrochloric acid and sodium hydroxide asneeded.

2.4 Calculations

The average and SD of the EOF were calculated for each cycleusing Microsoft Excel

TMsoftware. A normalization of the EOF

values was calculated to better compare results between dif-ferent capillaries due to inherent variances that occur throughthe manufacturing processes, the data were normalized by di-viding each cycle average EOF by the average EOF in cycle 1.


Electrophoresis 2013, 34, 2585–2592 CE and CEC 2587

Figure 1. Comparison of av-erage phosphate buffer EOFvalues with TMA (�, 36min/cycle) without TMA (♦ 21min/cycle). Separation condi-tions: 20 mM phosphate or20 mM TMA and 20 mM phos-phate; pH 7.22; capillary 40 cm(effective length 32 cm); sepa-ration voltage 10 kV; detection,UV 241 nm; marker mesityl ox-ide; injection time 5 s.

Normalized EOF data were graphed (cycle number vs. nor-malized EOF) in order to compare different trials to inferany significant increases or decreases in the EOF over theexperiment duration. The graphical data represent the aver-age EOF measured for the three separations that comprise acycle; no error bars are shown on the graphs as the variancebetween EOF measurements in a cycle were smaller than canbe depicted on the scale of the graphs.

3 Results and discussion

3.1 TMA buffer additives

TMA was selected as a model buffer additive to study the in-teractions between itself, the silanol groups, and buffer ions.The selection was made in part due to the modest hydropho-bic characteristic of TMA in comparison to other quaternaryammonium compounds, thus the interactions are anticipatedto be principally ionic in nature. Knowing that buffer additivesform adsorbed layers on the capillary surface [7–9], a baselineEOF was needed to be established, and phosphate was cho-sen as the separation buffer to be used with and without theinclusion of TMA. In addition to acting as a baseline mea-surement for the EOF, this allowed us to gauge the potentialimpact of electrolysis on the buffer pH and consequently, theEOF. As can be seen in Fig. 1, over the period of 20 cycles ofEOF measurements (60 total measurements), the phosphatebuffer showed a modest change in the EOF, with a net in-crease in EOF of 4.2% from the initial to final measurement.With a phosphate-TMA separation buffer, a similar change inEOF over the same number of cycles was anticipated, thoughwith lower EOF magnitude, due to the silanol-TMA surfaceinteractions [15, 16]. However, a 34.2% increase in the EOFwas observed (Fig. 1) over the course of the 20 cycles, well be-yond the change that was seen with phosphate buffer alone.The EOF values for the phosphate-TMA buffer indicate aninitial suppression of the EOF but the suppression is notmaintained over the duration of the 20 cycles. This is indica-tive of a changing dynamic at the surface of the capillary,

wherein the adsorption of TMA is potentially decreased, orthat the cationic charge is otherwise suppressed, such thatthe magnitude of the anionic surface charge increases overthe series of separations. To better understand the relation-ship between the capillary surface and TMA, and what maybe causing the change in TMA adsorption, capillary rinsesand changes in buffering ion were examined.

3.2 Capillary rinses

The use of capillary rinses between runs has been shown toimprove the stability and reproducibility of the EOF [21, 27]by renewing the surface and removing any adsorbed ana-lytes or impurities left behind after prior separations. Thereare several common rinse procedures that have been inves-tigated, which include flushing the capillary with run buffer[21, 22, 28], sodium hydroxide [22], methanol [21, 22], and hy-drochloric acid [29]. Buffer rinses have been shown to onlyrinse the capillary and not chemically alter the surface of thecapillary [21], whereas methanol and sodium hydroxide havethe ability to remove analytes or any molecules that may haveadsorbed onto the silica surface. In particular, methanol hasthe advantage of removing organic solutes that adsorb to thesurface as it is capable of disrupting the hydrophobic interac-tions that may occur between the silica surface and adsorbedcompounds [21,22]. Sodium hydroxide rinses are caustic andas a result can remove adsorbed layers and reveal a fresh silicasurface for the buffer additives to adsorb to during the nextseparation [21]. Consequently, sodium hydroxide has the abil-ity to disrupt ionic interactions between silanol groups andadsorbed compounds. By studying the four rinse methodsand their impact on the reproducibility of the EOF, the in-teraction between the silanols, TMA, and buffer ions may beinferred.

As can be seen in Fig. 2, rinsing the capillary with bufferonly (buffer rinse method) leads to a continual increase inEOF, which implies a net loss of positive charges adsorbedto the capillary surface, and alludes to the fact that TMA canno longer readily reestablish the initial equilibrium with the



Figure 2. Comparison of normalized, average, EOF values for allfour rinse protocols, buffer only (♦), methanol followed by waterrinse (�), 0.5 M sodium hydroxide followed by water rinse (�),full rinse: methanol, sodium hydroxide then water (*). Separationconditions: buffer 20 mM phosphate and 20 mM TMA; pH 7.22;capillary 40 cm (effective length 32 cm); separation voltage 10 kV;detection, UV 241 nm; marker mesityl oxide; injection 0.5 psi for5 s, where one cycle consists of 36 min.

silanol surface after a period of time. A nearly identical in-crease in the EOF is observed for the methanol rinse method,indicating that the limitations on the equilibrium at the sur-face of the capillary are not the result of hydrophobic interac-tions. On the other hand, the sodium hydroxide rinse method,as well as sequential rinses with methanol then sodium hy-droxide (full rinse method), yields a much more consistentEOF. After an initial modest decrease in the EOF, there was agradual change in the average EOF, such that after 20 cyclesthe EOF was comparable to the average EOF measurementsfrom the first cycle. The fact that the EOF can be kept mostconsistent when sodium hydroxide rinses are used, points to-ward an ionic interaction as being the principle interferencein the reestablishment of the initial surface charge that arisesfrom the adsorption of TMA to the silanol surface.

Although they did not investigate the impact of TMA onthe EOF, Smith et al. [21] also witnessed stabilization in themigration time of their buffering system when using a com-bined rinse of methanol and sodium hydroxide. This alsosheds some light on the potential cause for the consistentincrease in the EOF that was observed when a sodium hy-droxide rinse was not used. There are two likely reasons foran increase in the EOF under the given buffer conditions:either there is a decrease in the adsorption of TMA to thesilanol surface, or there is an increase in anionic charges atthe surface of the capillary. Given that it is unlikely that anyof the separation buffer components, or the mesityl oxideEOF marker, could adsorb to the silica surface and interferewith the TMA equilibrium between the surface and buffersolution; it is likely that a change in the surface charge isresponsible for the altered EOF values. An increase in theanionic charge due to the silanols is unlikely given the pHof the separation buffer is well above the pKa for silanols. Itwould seem more likely that the anionic buffer, phosphoricacid, is being adsorbed to the surface of the capillary. The di-

rect adsorption of phosphate anions is unlikely due to chargerepulsion between the phosphate and silanol; however, theassociation of phosphate with surface-adsorbed TMA cationsmay be possible. Steiner et al. [15] showed that the forma-tion of ion pairs between quaternary ammonium buffer ad-ditives and buffer anions was possible. It is not inconceivablethat such an ion pair could form between an adsorbed TMAcation and a free phosphate anion. This would explain thenet increase in anionic surface charge over the course of theseparations, a trend that would only be mitigated with themost caustic rinses, which will effectively refresh the silanolsurface as well as disrupt any ionic interactions that exist atthe surface. To further examine this possibility, tests were un-dertaken with a range of anionic, cationic, and zwitterionicbuffering ions.

3.3 Buffering ions

Previous studies on buffer additives [8–10] have typicallyshown that the inclusion of buffer additives stabilizes theEOF without the need of capillary rinses. In particular, Co-hen and Grushka’s [16] examination of amine, amino acid,and acidic buffer additives has shown a more consistent EOFover time and ultimately improvement in precision and re-producibility. To better understand the impact of TMA andthe buffer ion on the EOF stability, three different types ofbuffering ions were examined: anionic, cationic, and zwitter-ionic buffers. These three types of buffer ions should impactthe long-term stability of the EOF differently if there is in factthe formation of an ion pair at the surface of the capillary.The anionic buffers are anticipated to form ion pairs withthe TMA, increasing the EOF over time. Additionally, it isanticipated that the strength of the ion pair will be propor-tional to the magnitude of the EOF change. Cationic bufferson the other hand should not ion pair with TMA, due tocharge repulsion, thus stable long-term EOF values are antic-ipated. Similarly, zwitterionic buffers are anticipated not toinfluence the long-term EOF values, however, they may becapable of ion pairing with TMA. In doing so, there will be nonet change in the capillary surface charge due to the cationicand anionic nature of zwitterionic buffers.

Twenty cycles, consisting of three runs each, were com-pleted for each of the buffers tested, a rinse with the sep-aration buffer was implemented between each run, and allseparation conditions were kept the same. Additionally, vialswere alternated based on odd/even cycle numbers in order toslow the effects of electrolysis [26]. The switching of vials canresult in some slight variations in the EOF between cycleswhen minor hydrodynamic flows occur to different extentsbetween vial pairs; this results in a sawtooth-like appearancein some of the figures, however, the overall trends remainthe same. It should be noted that the error in the replicateEOF measurements for each cycle was calculated to be, onaverage, 0.9% RSD. This error is too small to be visible on thescale of the figures presented in this work.



Figure 3. Comparison normalized, average, EOF values foranionic buffers: phosphorous acid (♦, pH 7.22, 36 min/cycle), citric acid (�, pH 6.48, 42 min/cycle), bicarbonate (�,pH 6.59, 30 min/cycle), and phosphate (×, pH 7.22, 36 min/cycle).Separation conditions: 20 mM buffer and 20 mM TMA; capillary40 cm (effective length 32 cm); separation voltage 10 kV; detec-tion, UV 241 nm; marker mesityl oxide; injection 0.5 psi for 5 s.

3.3.1 Anionic buffer ions

Four anionic buffers were studied for their interactions withTMA over long-term EOF measurements: phosphoric acid,citric acid, carbonic acid, and phosphorous acid. The separa-tion buffers were adjusted to pH values between 6.5 and 7.5in order to provide adequate buffering capacity by the respec-tive buffer. As it can be seen in Fig. 3, all four of the anionicbuffers yielded increasing EOF values over the course of the20 cycles studied. This trend is anticipated if the bufferinganions are ion pairing with the TMA and no sodium hydrox-ide rinses are used to disrupt these ion pairs or regenerate afresh silanol surface.

The magnitude of the increase in the EOF is not identicalfor each of the anionic buffers, the percentage increase in theEOF, over the 20 cycles, for each of the buffers was calculatedto be: phosphorous acid 78.9%, bicarbonate 61.7%, citric acid53.3%, and phosphate 34.2%. The observed difference in themagnitude of the EOF change may be explained by the abilityof each anion to form an ion pair with TMA. It is knownthat the more hydrophobic the anion, the more effectively itcan ion pair with a cation [30]. In comparing a composite ofknown Hofmeister series containing three of the four anionsstudied [31], we can see that the most hydrophilic anions(e.g. HPO4

2−) had the least impact on the change in theEOF:

CO2−3 > SO2−

4 > PO3−4 > HPO2−

4 > CH3COO−> citrate3−

> tartrate2−> HCO−3 > OH−> Cl−> Br−> CrO−

4

> NO−3 > ClO−

4 > SCN−

For each of the buffers examined, the counterion tothe buffer was the sodium cation. As cations are knownto have a significantly weaker influence on the Hofmeisterseries, it is not anticipated that the nature or concentrationof the counterion will influence the stability of the EOF.

For our specific tests, there were no correlation betweenthe concentration of the sodium ion and the stability of theEOF.

We can further see the impact of the hydrophilic/hydrophobic nature of the anion on the stability of the EOFin observing two of the anionic buffers at different pHs. Car-bonate and phosphate are both polyprotic acids, for whichthe Hofmeister rankings are known for their various states ofprotonation. Given buffer pH values of 10.22 and 6.59, a car-bonic acid buffer will be present in solution predominantlyas the carbonate and bicarbonate ions, respectively. Bufferswith these pHs were tested in the same manner as previousbuffer tests and the change in the EOF over the 20 cycles wasfound to be 61.7% at pH 6.59 and 5% at pH 10.22 (Support-ing Information Fig. 1). Similarly, for the phosphate bufferat pH 12.38, where the phosphate anion predominates, theEOF change over the series of runs was 3% compared to achange of 34.2% at pH 7.22 where monohydrogen phosphateis the principal form of the anion.

To determine if the observed ion-pairing trends weredependent upon the buffer concentration of the separationsystem, 20, 40, and 80 mM bicarbonate buffers were exam-ined under the same separation conditions as previously de-scribed. The change in EOF over the 20 cycles was 61.7, 63.4,and 63.4% for these respective concentrations (SupportingInformation Fig. 2). The similar percent changes and slopesfor the bicarbonate systems suggest that the buffer concen-tration has no bearing on the ability to form the ion pairs butis in fact governed by the TMA cations and their ability toform ion pairs.

As the counterion to TMA may have some influence onthe stability of the EOF, tests were performed with TMA-Cl.Compared to the bromide ion, chloride is ranked as more hy-drophilic on the Hofmeister series. To compare the two coun-terions, a series of separations was performed with TMA-Cland a 20 mM phosphate buffer at pH 7.23. With TMA-Cl,an 11.0% increase in the EOF was observed as compared toa 34.2% increase with the TMA-Br pairing (Supporting In-formation Fig. 3). It is clear that the bromide counterion,which is ranked as more hydrophobic on the Hofmeister se-ries, does have an increased influence on the EOF stabilityas compared to chloride. Though this inevitably had someimpact on the other anionic buffer measurements, the trendin the influence of the buffers would be the same regardlessof the TMA counterion. As the ion pairing of hydrophobicanions may be in large part due to their hydrophobic nature,we examined how the use of a methanol rinse might be ableto have a greater impact on the disruption of the ion pair, orits removal from the capillary surface, and further stabilizethe EOF similar to the impact of sodium hydroxide rinses.The use of a methanol rinse was shown earlier to have noimpact on the change in the EOF for the highly hydrophilicphosphate anion (Fig. 2). However, when used as a rinsefor a TMA–phosphorous acid separation buffer the methanolrinse maintained a long-term EOF that was comparable toboth a sodium hydroxide rinse and full rinse protocol (Sup-porting Information Fig. 4). This would indicate that very



Figure 4. Representation ofthe potential adsorption of (A)TMA to the capillary surface,(B) TMA in the presence of ananionic buffer, (C) TMA in thepresence of a cationic buffer,and (D) TMA in the presenceof a zwitterionic buffer.

hydrophobic ion pairs can be disrupted, or removed from thesilica surface, with a rinse with methanol.

It appears that a potentially strong ion pair associationbetween the TMA cations and the anionic buffering ions isoccurring at the surface of the capillary. Seemingly, this ionpairing is occurring specifically at the surface of the capillaryas the TMA and buffer is mixed and at equilibrium well beforeintroduction into the capillary. We hypothesize that the TMAinitially adsorbs to the silica surface as a free cation (Fig. 4A),which interacts with the surface silanols, suppressing someof the negative charge of the capillary surface. The resultantincrease in the anionic surface charges on the capillary can beattributed to two possible adsorption schemes: the first wouldbe the ability for anions in the separation buffer to ion pairwith adsorbed surface TMA; and second, ion pairs from theseparation buffer are able to displace unpaired TMA cationsfrom the capillary surface (Fig. 4B) over the duration of 20cycles.

We examined how the EOF would change for a capil-lary if the capillary was left, filled, with the separation bufferover an extended period of time, allowing more time for anykinetically hindered equilibria to be established. The TMA-phosphorous acid separation buffer was examined, whereintwo cycles (six separations) were performed, on a freshly pre-pared capillary, which was then stored for 24 h, filled with theseparation buffer. After 24 h of rest, the EOF was once againmeasured over two cycles, then left to rest, filled with the sepa-ration buffer, for another 24 h. This process was repeated for atotal of four times, resulting in ten full cycles of EOF measure-ments. The data from these tests can be found in SupportingInformation Fig. 5, where the cycle EOFs are plotted for boththe resting capillary and a typical TMA-phosphorous acid sep-

Figure 5. Comparison of normalized, average, EOF values forcationic buffers: triethanolamine (*, pH 7.22, 45 min/cycle); sepa-ration conditions: 20 mM triethanolamine and 20 mM TMA; cap-illary 40 cm (effective length 32 cm); separation voltage 10 kV;detection, UV 241 nm; marker mesityl oxide; injection time 5 s;bis-tris (�, pH 6.76, 42 min/cycle), ethylenediamine (◦, pH 7.05,42 min/cycle); separation conditions: 20 mM buffer and 20 mMTMA; capillary 40 cm (effective length 8 cm); separation volt-age 10 kV; detection, UV 241 nm; marker mesityl oxide; injection0.5 psi for 5 s.

aration buffer tested over 20 cycles with no rest. The resultsshow a more rapid decline in the EOF for the resting capil-lary, with a slope of 0.068 versus 0.049 for the continuouslytested separation buffer. The changes in slopes of the rest-ing and continuously tested phosphorous acid-TMA systemswould suggest that time plays a role in the adsorption of theion pairs to the surface of the capillary but based on the timescale of an average separation, the time-dependent kineticshas a reduced influence on the adsorption of ion pairs on thesurface.



3.3.2 Cationic buffer ions

The use of a cationic buffer was anticipated to prevent thechanges in the EOF observed for the anionic buffers, as thecationic buffers will be unable to ion pair with the TMA, thusunable to suppress the cationic surface charge. As can beseen in Fig. 5, triethanolamine, bis-tris, and ethylenediamine,when used as a separation buffer with TMA, decreased inEOF, over the course of 20 separation cycles, by 13.8, 18.1,and 31.7%, respectively. The magnitude of the EOF change isnot as significant as seen for the anionic buffers; however,it is clearly not a stable EOF of the course of the cycles.We hypothesize that the cationic buffer ions are capable ofadsorbing to the capillary surface, alongside the TMA cations(Fig. 4C).

The synergistic adsorption of both the TMA and thebuffer cations may be the result of the buffer ions beingable to adsorb to the capillary surface in the spaces betweenTMA cations, potentially with some overlap of the hydropho-bic portions of each molecule favoring the adsorption. Inobserving the extent of the EOF change over the 20 cycles, itis apparent that the cationic buffers do not impact the EOFequally. When the average charge of the buffers is examined,based on the pH and pKa(s) of the three buffers, it can beseen that the buffer with the greatest impact on the EOFalso had the lowest average charge; the average charge fortriethanolamine, bis-tris, and ethylenediamine were +0.78,+0.48, and +0.46, respectively. Although one would antic-ipate the buffer of highest cationic charge to be the mostreadily adsorbed, the greater electrostatic repulsion betweenthe cation and the permanently +1-charged TMA may limitthe adsorption further. In all instances, the counterion to thecationic buffers was chloride. As indicated in Section 3.3.1,the chloride has a minor impact on the EOF stability as com-pared to the bromide counterion to TMA, thus the counterionto the cationic buffers is not anticipated to have had a signif-icant impact on the stability of the EOF.

Tests of the impact of the rinsing process, namelythe sodium hydroxide and full rinse protocols, on the sta-bility of the EOF and the surface-adsorbed cations wereconducted with ethylenediamine (Supporting InformationFig. 6). The rinses revealed that, much as was seen with theTMA-phosphoric acid separation buffer, the use of a sodiumhydroxide rinse, or a full rinse method can effectively main-tain the magnitude of the long-term EOF at a value muchcloser to the original EOF than can be achieved without arinse protocol, within 5% of the original EOF.

3.3.3 Zwitterionic buffer ions

Zwitterions are unique buffers in that they possess a positiveand negative charge on the same molecule. They can be usedfor the prevention of protein adsorption onto the capillarysurface [32], for the complexation of inorganic dianions [33],and are common buffer additives because of their ability toimprove a separation without increasing the ionic strength

Figure 6. Comparison of normalized, average, EOF values forzwitterionic buffers: ACES (�, pH 6.75, 36 min/cycle), HEPES (♦,pH 7.24, 30 min/cycle), and tricine (�, pH 7.44, 30 min/cycle).Separation conditions: 20 mM buffer and 20 mM TMA; capillary40 cm (effective length 32 cm); separation voltage 10 kV; detec-tion, UV 241 nm; marker mesityl oxide; injection 0.5 psi for 5 s.

of the separation buffer [34, 35]. Additionally, zwitterionicbuffers can be beneficial to use over other buffers becausethey can endure the effects of prolonged electrolysis moreeffectively [26]. As can be seen in Fig. 6, the magnitude of theEOF over the course of the 20 cycles studied shows minimal(±5%) change with all of the three zwitterionic buffers tested:ACES, HEPES, and tricine. The stability in the EOF obtainedwith these buffers is comparable to that which was achievedwith extensive rinse cycles with either cationic or anionicbuffers.

One reason that the zwitterionic buffers may not influ-ence the EOF significantly over the long term may be dueto their paired charges. If a zwitterionic buffer should forman ion pair with a TMA cation, via its anionic charge, andadsorbs onto the surface of the capillary, there will be no netchange in the charge of the surface due to the accompanyingcationic charge. Similarly, if the cationic charge adsorbs to thecapillary surface, the accompanying anionic charge negatesany change in the zeta potential, provided the zwitterion doesnot displace a TMA cation (Fig. 4D). No rinse protocols wereattempted with the TMA-zwitterionic separation buffers as itis anticipated that no significant difference would be observ-able, given that the reproducibility of the EOFs achieved withthese buffers is as good as any system that involved thoroughrinse procedures.

4 Concluding remarks

Buffer additives are commonly used to suppress the EOF andprevent analyte adsorption while maintaining EOF stability.Our work has shown that the use of the small quaternaryammonium additive, TMA, is capable of suppressing theEOF, yet it is also prone to the formation of ion pairs at thesurface of the capillary impacting the long-term stability ofthe EOF. If an anionic buffer is used with TMA additives, theEOF will be subject to a gradual increase over an extended



number of separations. We have shown that the magnitudeof this increase appears to be correlated to the hydrophobicnature of the buffer anion, with more hydrophilic anionsyielding a smaller increase in the EOF. In cases where ananionic buffer is used with TMA in the separation buffer theoriginal EOF can be recovered by including a rinse procedurebetween runs, either consisting of sodium hydroxide ora multistep rinse including both sodium hydroxide andmethanol.

Cationic buffers, when used with TMA also influencethe EOF over time, however, they typically tend to reducethe EOF further, presumably through coadsorption to thesilica surface, increasing the cationic surface charge of thecapillary. The optimal choice for long-term separations witha stable EOF, without the need to rinse the capillary, appearsto be zwitterionic buffers. We found that the magnitude ofchange in the EOF over 60 separations was, with zwitterionicbuffers, on par with the most extensive rinse protocols usedfor other separation buffers.

It appears from this work that the formation of ionpairs between the buffer additive, TMA, and buffer ionsis not only likely, but may result in ion pairs of surprisingstability that can adsorb onto the capillary surface altering theEOF.

The authors wish to thank San Diego State University forfunding and support for this research.

The authors have declared no conflict of interest.

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