Determination of the Isoelectric Point of the Capillary Wall Inc

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JOURNAL OFCHROMATOGRAPHY A

ELSEVIER Journal of Chromatography A, 662 (1994) 369-373

Determination of the isoelectric point of the capillary wall incapillary electrophoresis

Application to plastic capillariesVojtgch RohliEek, Zdengk Deyl, Ivan MikSik

Inst it ut e of Physiology, Videli skh 1083, Prague 4, Czech Republi c

(First received October 12th, 1993)

AbstractA refined method for measuring endoosmotic flow in capillaries based on weighing the amount of liquid

transferred is described. The method has been applied to the estimation of isoelectric points of two types of plasticcapillaries (polytetrafluoroethylene and polytetrafluoroethylene-polyhexafluoropropylene copolymer). Contrary toexpectation both plastic capillaries exhibited considerable electroosmotic flow and changes in hydrodynamic flowresistance without applied voltage. The nature of these phenomena is discussed.

1. Introduction

A crucial phenomenon in capillary electro-phoresis is electroosmotic flow. This flow origi-nates from the negative charges caused by thepresence of silanol groups on the inner surface ofthe fused-silica capillary. Though widely usedmainly because of their commercial availability,fused-silica capillaries exhibit some disadvan-tages caused mainly by sorption of solutes to thecapillary wall. In our attempts to apply capil-

laries made of other UV-transparent materialsthe need of estimating their isoelectric point hasarisen. In the present communication we de-scribe a simple and precise approach for estimat-ing isoelectric points.

Several methods have been described formeasuring the electroosmotic flow in capillaries[l]. By weighing the solution emerging from the

*Corresponding author.

capillary on an analytical microbalance the prob-lem of adsorbing a neutral marker on the capil-lary wall can be circumvented [2,3]. Anothermethod exploits the measurement of the electro-phoresis current when a buffer with differentionic strength is introduced [4]. It is also possibleto measure directly the [ potential of the capil-lary wall as reported by Van de Goor et al. [5].This measurement either uses weighing of theeffluent (see also Altria and Simpson [2,3]) orinvolves measuring of the streaming potential

when the solvent is pumped through the column.In this way the 4 potential and electroosmoticflow of polytetrafluoroethylene (PTFE) capil-laries was measured.

A number of papers have been published onmanipulating the electroosmotic flow [6,7]. Inthe simplest case the pH and the ionic strengthof the electrolyte can be adjusted to give theoptimum speed for a given separation. Anotherpossibility is to vary the electroosmotic flow by

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370 V. Rohli Eek et al. I J . Chr omatogr. A 662 (1994) 369-373

introducing additives to the background buffer.By using surface active agents or organic solventselectroosmotic flow speed can be changeddramatically or reversed [8,9]. No matter what

method of electroosmotic flow adjustment isused, the charge of the capillary wall plays animportant role in these considerations.

2. Experimental

2.1. M ateri al and methods

For capillary wall isoelectric point measure-ment a set of 5 mM disodium citrate buffers wasused. The rather low buffer concentration wasused to keep the current within reasonable limits(less than 80 PA) during the experiment. Buffercomponents were of analytical-reagent qualityand were obtained from Lachema (Brno, CzechRepublic). Runs with applied voltage were run at4.0 kV per 70 cm x 200 pm I.D. capillary. Theapplicability of the method was demonstratedwith PTFE (Norton Performance Plastics, Wil-lich, Germany) and polytetrafluoroethylene-polyhexafluoropropylene (Kablo, Vrchlabi ,Czech Republic) capillaries. When purchased theouter diameter of the tested capillaries was 1 mmwith an I.D. of 500 pm; before measurement thecapillaries were drawn to achieve I.D. of 200pm. This rather large I.D. was used in order toobtain easily measurable volumes of transferredelectrolyte. For stabilization after drawing thecapillaries were left to rest for at least one day.Stabilization time was also necessary after fillingthe capillary with the electrolyte, because evenmodest filling pressure made the inner diameterexpand and this causes additional flow upon

shrinking. A stabilization time of 1 h was foundsufficient.

2.2. Procedure

Estimation of the isoelectric point of thecapillary was based on measuring the electro-osmotic flow in a capillary inclined to give agravity flow around of 8 mg per 5 min. Theamounts of the background electrolyte that have

passed through the capillary were plotted againstpH and the isoelectric point of the capillary wasestimated as the intersection of lines obtainedwith and without applied voltage.

The determination of the isoelectric point ofthe capillary wall was done by weighing. Theexperimental set-up is shown in Fig. 1. Therewere two reasons for not using levelled electrodejars: (1) constant flow allowed for more preciseweighing of the liquid that has passed throughthe capillary and (2) the constant flow throughthe capillary cooled the system and avoidedbubble formation inside the capillary column.The capillary was for most of its part placed intoa tube filled with circulating paraffin oil forcooling and temperature stabilization. Thecathode jar was placed on a balance tray of adigital balance (Sartorius 2004 MP6; Sartorius,Gottingen, Germany). Because it was demandedthat the accuracy of weighing was 0.1 mg atleast, any influence on the jar mass had to beeliminated. There were four possible sources oferror foreseen, namely (i) transmission of thesmall changes of the position of the capillaryassembly, (ii) friction between the contact andthe negative terminal of the high-potentialsource (HTS), (iii) electrostatic forces inside thebalance room and (iv) evaporation of the fluid inthe jar.

Fig. 2 shows this part of the arrangement indetail. At the passage of the capillary throughthe end of the paraffin oil bath the capillary isextended with a highly elastic silicon rubbertube. This tube passes tightly through the upperpart of the jar. A thin Pt wire is placed into thistube (the path of the current is short-circuited in

Paraffne oil bath \\ r+

Cathode jar

Balance tray

Fig. 1. Schematic representation of the experimental ap-paratus.


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L ? Rohli Eek et al. I J . Chr omatogr. A 662 (1994) 369-373 371

Fig. 2. Detailed representation of the cathode end arrange-ment. For details see Experimental.

this way) to abolish any electro-endoosmoticforces in it.

The connection to the negative terminal of theHTS was performed by a fluid contact material-

ized by wire A tightly passing the upper part ofthe jar. The other end of this wire was immersedin a vessel outside the balance tray filled with a1% NaCl solution. Wire B connects the fluid inthis vessel with the negative terminal of theI-ITS. An auxiliary wire C connects the metallicbalance tray with the fluid contact. Because themetallic case of the balance was also connectedwith the negative I-ITS output any electrostaticfield inside the balance was eliminated.

A small (0.4 mm) hole was drilled in the upperpart of the jar to eliminate the influence of airpressure changes caused by the changing liquidheight in the jar as well as possible changes inthe atmospheric pressure. The diameter of thejar was 30 mm; in this way transferring of 10 ~1of the fluid during one measurement caused afluid height difference in the jar of only 0.03 mmwhich can be neglected.

The upper electrode jar was connected to thepositive output of the HTS source. Because of itselevated position hydrostatic flow from this jarthrough the capillary was generated. This ar-

rangement was preferred to the zero flowmode (equilibrated jar levels) as in the set-upused no reversed flow due to pH changes couldoccur. Finally the ratio between the flow withand without applied potential was evaluated andthe pH at which this ratio = 1 defined the isoelec-tric point. The measurements were done over a5min period at 4 kV and 0.1 mA. The meanvalue of 10 pairs of measurements was used forconstructing the graph.

3. Results and discussion

As demonstrated in Fig. 3 both the BTFE andpolytetrafluoroethylene-polyhexafluoro-

propylene capillaries exhibited distinct electro-osmotic flow with isoelectric points at 3.25 and3.0, respectively. The reliability of these mea-surements is evaluated in Table 1. Estimation ofelectroosmotic flow by the weighing method hasbeen shown to be precise and uncomplicatedconfirming the previous results of Altria andSimpson [2,3]. There are, however, two phe-nomena which need some discussion. First, asthe capillaries are made of tetrafluoroethyleneand tetrafluoropropylene-hexafluoropropylene,

respectively, they should be devoid of anychargeable functional groups on their inner sur-face. Consequently, from the strictly theoreticalpoint of view they should be also devoid of anyelectroosmotic flow, which, however, is not thecase. Investigations at the producers of thesecapillaries confirmed that no additives (softeners)of any kind that may be responsible for the innersurface charge are used during manufacturing ofthese products. As also dipole-caused chargesare unlikely to occur in these capillaries, it isfeasible to assume that charged buffer compo-nents may be sorbed on the capillary wall load-ing it with some (reproducible) charge thatcauses the electroosmotic flow when the capillaryis attached to a high-voltage source. This conclu-sion is supported by the fact that electroosmoticflow at very low pH (below 2.5) ceases and thedata obtained in this range are poorly reproduc-ible. The decrease of the electroosmotic flow atvery low pH values with imposed voltage ascompared to the flow without voltage could beascribed to the properties of the electric double

layer.The other phenomenon seen in Fig. 3 is the

incline of the line corresponding to the flow at noimposed voltage. It has to be emphasized thatpractically no differences in buffer specific den-sities were observed (5 mM buffers were used)and therefore the phenomenon observed cannotbe explained on this basis. Theoretically thisdependence should be represented by a horizon-tal line parallel to the x-axis. However, if one


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372 K Rohlitek et al. I J. Chromatogr. A 662 (1594) 369-373

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1.4

1. 3 I 1

3.0 3.5 4. 0 45 5.0 5. 5

2. s

23

1. s

I1. 5 I I I

: 30 3.5 4.0 4.5 5.0PH

PH

Fig. 3. Electroosmotic (dotted lines) and gravity (solid lines) flow vs. pH with (A) the PTFEpolytetrafluoroethylene-polyhexafluoropropylene copolymer capillary. x = 0 V; 0 = 4 kV.

capillary and (B) the

Table 1Parameters of the regression lines (flow vs. pH) in Fig. 3

OkV 4kV

Polytetrafluoroethylene-polyhexafluoropropylene

OkV 4kV

Intercept 1.180273 0.684799 1.392216 0.426285Slope 0.057819 0.209398 0.102162 0.426857RZ 0.990419 0.998017 0.977752 0.996776


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V. Rohl iEek et al. I J. Chrom at ogr. A 662 (1994) 369-373 373

admits that the buffer components are sorbed onthe inner capillary wall, then its properties arelikely to change bringing about changed con-ditions for the hydrodynamic flow. From the

nature of the experimental arrangement it isfeasible to assume that the forces causing buffercomponents sorption should be hydrophobic bynature and rather independent on the pH of therunning buffer used. Consequently the changesin the amount of the liquid flowing through thecapillary should reflect its hydrodynamic flowresistance, for instance if the sorbed entities aremore charged at higher pH being acidic bynature, then at higher pH the capillary should bemore easily wettable, exhibit lower hydro-

dynamic resistance and offer a higher flow-rate.In this case, however one would expect thatbeyond a certain pH where practically all func-tional groups sorbed to the wall are charged, theline of the pH verSuS flow-rate dependence willbent to become parallel to the x-axis. Such aneffect was, however, not observed. On the otherhand it is necessary to keep in mind that as withfused-silica capillaries the sorbed charged entitieswill act in a similar way as the ionized silicic acidgroups, i.e. they will be neutralized by hydro-nium ions (hydrated cations) forming a compactlayer overlaid (due to the thermal motion) by aloosely held layer termed usually diffuse layer.Combination of these effects may lead to thelinear dependences seen in Fig. 3. As a matter offact the difference of the investigated capillariesto the commonly used fused silica may be thatwhile the charged silanol groups in fused-silicacapillaries are covalently bound to the capillarysurface, with the plastic-type capillaries thecharge observed stems from hydrophobic sorp-

tion forces. The flow-rate changes observed atdifferent pH values are also unlikely to becaused by slow changes in capillary dimensionsduring use; both systematic experiments and

random measurements were done with the sameresult.

Application of capillaries manufactured fromother material than fused silica may broaden thepossibilities of capillary electrophoresis and mayhelp to overcome some well known problemse.g. irreproducible sorption of biopolymers tothe capillary wall.

4. Acknowledgement

This work was supported in part by the CzechMinistry of Education, Youth and Sports, grantNo. 0711.

5. References

[l] S.F.Y. Li, Capil lar y Elect rophor esb (Journal of Chroma-tography Library, Vol. 52), Elsevier, Amsterdam, 1992.

[2] K.D. Altria and C.F. Simpson, Anal. Proc., 23 (1986)453.

[3] K.D. Altria and C.F. Simpson, Chromatographia, 24(1987) 527.

141 X. Huang, M.J. Gordon and R.N. Zare, Anal . Chem.,60 (1988) 1837.

[5] A. van de Goor, B. Wanders and F. Everaerts, J.Chrom at ogr., 470 (1989) 95.

[6] H.H. Lauer and D. McManigill, Anal . Chem., 58 (1986)166.

[7] S. Fujiwara and S. Honda, An al . Chem., 58 (1986) 1811.[8] S. Terabe, K. Otsuka, K. Ichikawa, A. Tsuchiya and T.

Ando, An al . Chem., 56 (1984) 111.[9] X. Huang, J.A. Lucker, M.J. Gordon and R.N. Zare,

An al . Chem., 61 (1989) 766.

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Determination of the Isoelectric Point of the Capillary Wall Inc