Zak K. Shihabi

Department of Pathology,Wake Forest UniversitySchool of Medicine,Winston-Salem, NC, USA

Stacking by electroinjection with discontinuousbuffers in capillary zone electrophoresis

The work presented here demonstrates that electroinjection can be performed usingdiscontinuous buffers, which can result in better stacking than that obtained by hydro-dynamic injection. The sample can be concentrated at the tip of the capillary leavingpractically the whole capillary for sample separation. This results in several advan-tages, such as better sample concentration, higher plate number and shorter time ofstacking. However, sample introduction by electromigration is suited for samples freeor low in salt content. Samples, which are high in salt content, are better introduced bythe hydrodynamic injection for stacking by the discontinuous buffers. Different simplemethods to introduce the discontinuity in the buffer for electroinjection are discussed.

Keywords: Acetonitrile / Discontinuous buffers / Electroinjection / Isotachophoresis / StackingEL 5000

1 Introduction

A major problem in capillary electrophoresis is the poorsensitivity of detection. As the dimensions of the capil-laries or the separation channels on the chip decreaseboth the separation speed and efficiency improve greatly.However, the sensitivity of detection worsens. Specialdetectors, such as laser-induced fluorescence and elec-trochemical, are one solution for this problem. However,another simple alternative is sample stacking. It is veryconvenient to utilize the electrical current not just forseparation but also for sample concentration directly onthe capillary. In simple terms, stacking can be viewed asinjecting a large volume of sample followed by inducingthe two edges of the sample plug to migrate at differentvelocities towards each other leading to sample concen-tration. Large sample volume can be introduced byhydrodynamic injection or electromigration (electroinjec-tion) [1, 2]. Isotachophoresis (ITP) and transient ITP areoften used to introduce large volume of sample for stack-ing but usually in the hydrodynamic mode [3–7]. As thesample volume increases in the hydrodynamic mode,the capillary volume (length) for the separation partdecreases. Consequently, this leads to some decrease inplate number and resolution. Field-amplified injection,and head-column field-amplified injection are used forconcentration by the electromigration with high degreeof stacking [8, 9]. The electromigration injection offers afew advantages over the hydrodynamic injection in stack-

ing [10, 11]. In this case, the sample is concentrated in thetip of the capillary, leaving the whole capillary for separa-tion resulting in a better plate number [10]. Furthermore,stacking by hydrodynamic injection requires a long timefor sample transfer, which can exceed the separationtime especially for instruments that utilize height differ-ence for sample introduction [11]. Discontinuity in the buf-fer is the main common basis for most of the differentmethods of stacking [1, 2, 12].

The aim of this work is to demonstrate that discontinuousbuffers for transient ITP can be applied for stacking in theelectroinjection mode by their inclusion directly at theelectrode, in the sample, or both. As a consequence, arapid and high degree of concentration, theoreticallyequivalent to filling the whole capillary volume with sam-ple can be achieved, meanwhile, leaving essentially thewhole capillary volume for separation. Thus, the overallanalysis with this type of injection is rapid, with high platenumber, a high degree of concentration, and with verygood resolution. However, major differences are observedbetween electroinjection and the hydrodynamic injectionin the transient ITP.

2 Materials and methods

Instrument: A Model 2000 CE (Beckman, Fullerton, CA,USA) was set at 6 kV with detection at 280 nm. The sam-ples were injected either by electromigration for 30 s at3–5 kV, or hydrodynamically under low pressure, for 30 s,as specified in each figure. In the hydrodynamic injectionmode, the sample volume corresponded to filling 20% ofthe capillary volume (to the detector). Separation buffer:The 200 mM electrophoresis buffer was either borate,pH 7.8 or phosphate (pH 7.6) as specified. In someexperiments the buffer was switched to 200 mM triethano-

Correspondence: Prof. Z. K. Shihabi, Department of Pathology,Wake Forest University School of Medicine, Winston-Salem,NC 27157, USAE-mail: zshihabi@wfubmc.eduFax: �336-716-9944

Abbreviation: TEA, triethanolamine

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WILEY-VCH Verlag GmbH, 69451 Weinheim, 2002 0173-0835/02/1508–2394 $17.50�.50/0

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lamine (TEA) (pH 7.8) as specified. A short untreatedcapillary, 50 �m ID � 26 cm, effective length 17.5 cm(Polymicro Technologies, Scottsdale, AZ, USA), wasused to achieve fast analysis. Standards: stock solutionsof tyramine (1 mg/mL) and normetanephrine (1 mg/mL)were dissolved in water.

3 Results and discussion

In practice, ITP is a very demanding technique. Since itrequiresproperchoice ofseveral conditions. However, tran-sient ITP is easier to perform since it can be accomplishedusing conventional CZE instruments, producing the familiarGaussian peak shape [3–7]. It can be used with several buf-fers, provided a proper terminating ion is present.

Figure 1A shows the peak height and shape for the ana-lyte tyramine as dissolved and separated in the sameelectrophoresis buffer, 200 mM borate, pH 7.8, i.e., undernonstacking conditions, using electroinjection for 30 s. Inthis case, the buffer at the electrode and anode are bothborate, too. The peak shape reflects overloading becauseof the large sample volume. However, if the buffer at theanode is switched to 200 mM TEA buffer pH 7.8, immedi-ately before the voltage is turned on, the peak becomesmuch sharper and increases in height about 20-fold(Fig. 1C). This sharp peak is also observed when otherorganic buffers with amine groups such as Tris (Fig. 1D, orBis-Tris, TEA, 2-(N-cyclohexylamino)ethanesulfonic acid(CHES), or N-(2-hydroxyethyl)piperazine-2’-(2-ethane-sulfonic acid) (HEPES) are used. These buffers share incommon that they can act, under these conditions, asterminating ions, allowing transient ITP to occur. Thesebuffers are less stringent when used in transient ITP.For example, these different buffers gave good stackingover a wide pH range of 6.4–8.8 without the need for acoated capillary. If the buffer at the anode lacks such anamine group, e.g., borate or phosphate, the ITP step doesnot take place. In transient ITP, two steps occur: stackingand separation. The first step is the ITP which is very brief.During this step the leading ion (sodium) migrates rapidlyand the sample analytes concentrates behind as a“stack” of coins without separation provided a properterminating ion (lower mobility) is provided. As soon asthe leading ions enter the separation buffer they move faraway from the analytes zone thus the ITP stops and theseparation step, which is a longer one, starts. The sampleseparates by CZE into discrete or separate “zones”. Onlycompounds with intermediate mobility and with low con-centration concentrate in this method.

Figure 2 compares stacking by electromigration to thatby hydrodynamic injection (filling 20% of the capillaryvolume with sample), both under transient ITP conditions.

Figure 1. Importance of transient ITP on stacking byelectroinjection. The sample was injected by electromigra-tion at 3 kV for 30 s: (A) 30 mg/L tyramine dissolved in thesame 200 mM electrophoresis buffer, pH 7.8 (continuousbuffer conditions). (B) Water with no tyramine (blank). Thecapillary and the cathode contain 200 mM borate buffer,pH 7.8, while the anode contains 200 mM TEA, pH 7.8(transient-ITP). (C) As in (B) but the sample contained30 mg/L tyramine (transient ITP). (D) As in (C) but theanode contained 200 mM Tris buffer, pH 8.0 (transientITP).

For the same amount of time of injection, 30 s, the elec-tromigration gives at least five times better peak heightcompared to the hydrodynamic injection (note the differ-ence in the absorbance scale in the figure). In order toincrease the peak height by the hydrodynamic injection,the time for this process has to be increased greatly. How-ever, for the same peak height, the electromigration gaveslightly higher N compared to that by hydrodynamic injec-tion (N = 490 000 vs. 430 000, respectively). In general,stacking was faster by the electromigration compared tothat by the hydrodynamic injection.

CE

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2396 Z. K. Shihabi Electrophoresis 2002, 23, 2394–2398

Figure 2. Comparison of hydrodynamic vs. electroinjec-tion for stacking. Tyramine (30 mg/L) and normetane-phrine (60 mg/L) were dissolved in water and injectedfor 30 s. The anode contained a buffer of 200 mM TEA.(A) Hydrodynamic injection for 30 s (equivalent to 20% ofthe capillary volume); (B) electroinjection for 30 s at 3 kV.(Note the five times difference in the absorbance scale.)

The peak height in the hydrodynamic injection is de-pendant on the injection time (sample volume) and thesample salt content. At low sample volume, salts in thesample do not affect greatly peak height in transient ITP.However, as the sample size increase further, saltsimprove the peak height (Fig. 3). The leading ion usuallyaffects the degree of concentration. Thus, with large sam-ple size there is a need for higher sodium ion concentra-tion. In the electromigration method, the peak heightis affected also by the injection time and sample salt

Figure 3. Effect of sample size (seconds, s) and salt con-tent (0.5% NaCl) on sample peak height (mA) in the hydro-dynamic injection mode. The sample contained 100 mg/Ltyramine.

Figure 4. Effect of sample size in seconds (30 and 10 s)0% NaCl injection, voltage (kV), and salt content (0.5%NaCl, 10 s) on sample peak height (mA) in the electromi-gration. The sample contained 100 mg/L tyramine.

content, in addition to the injection voltage (Fig. 4). As theelectromigration voltage increase, so does the peakheight. Thus, by loading the sample at higher voltagesthe injection time can be shortened. Contrary to thehydrodynamic injection, salts in the ITP mode, decreasethe peak height for cations introduced by electromigration(Fig. 4). However, this effect can be compensated tosome extent by a longer injection time or higher injectionvoltage (Fig. 4). Salts have faster mobility and thus, con-centrate ahead of the other analytes at the tip of the cap-illary; thus decreasing the field strength and consequentlydecreasing the amount of the analyte being injected.However, the peak shape remains sharp indicating thatstacking due to the ITP step remains applicable regard-less of the decrease in peak height (Fig. 5C). The salteffect in this case is also different from that observed forthe stacking of the anionic compounds [3]. Leadinganions such as Cl migrate away from the electrode andfrom the capillary tip so they do not interfere in the stack-ing of anions. In addition to dissolving the sample inwater, stacking by electromigration can be accomplishedusing samples dissolved in organic solvents miscible inwater such as acetonitrile (Fig. 5A). The organic solventsare useful for dissolving some compounds not solublein aqueous solutions. In practice, acetonitrile has theadvantage of removing the protein from the sample. Thestacking for tyramine dissolved in acetonitrile (Fig. 5A) issimilar to that in water (Fig. 1C) requiring the transientITP step. In the absence of TEA at the anode (continuousbuffer) the tyramine peak is wide (Fig. 5B). Thus, stackingby transient ITP can be obtained in the electromigrationregardless if the sample was dissolved in acetonitrile or

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Figure 5. Effect of dissolving the sample in acetonitrile onstacking by electromigration under transient ITP condi-tions. The anode contains TEA while the separation bufferis 200 mM borate, pH 7.8. The sample was introduced byelectroinjection for 30 s at 5 kV: (A) Sample in 66% acet-onitrile; (B) sample as in (A), but the anode has a 200 mM

borate buffer, pH 7.8 (continuous buffer); (C) as in (A) �0.2% NaCl (final concentration).

water. Furthermore, the effect of salt in the sample onpeak height was similar regardless if the sample was dis-solved in acetonitrile (Fig. 5C) or water.

Buffer discontinuity can be introduced by altering the buf-fer in the electrode (anode) reservoir as well by altering thebuffer in the sample, or both. If TEA is added to the sam-ple (Fig. 6B) stacking can take place. However, the peakheight is less than that if TEA was present in both the sam-ple and the anode (Fig. 6A). Also, the peak is much shar-per in the first case. This may be due to the limitedamount of TEA, acting as a terminator, which can be intro-duced in the sample by electroinjection. Since someinstruments are not flexible enough to accommodatemultiple buffers, addition of the terminator buffer in thesample would be a simple option.

Figure 6. Effect of the buffer discontinuity in the samplevs. that at the anode: (A) 30 mg/L tyramine is dissolved inthe TEA buffer and the anode contained TEA buffer also;(B) 30 mg/L tyramine is dissolved in TEA while the anodehas 200 mM borate buffer, pH 7.8. (Note the two times dif-ference in the absorbance scale).

In transient ITP, different types of buffers can be chosenfor the separation step. Figure 7 illustrates that with elec-troinjection very good separation can be obtained usingeither borate buffer (200 mM, pH 7.8) or phosphate buffer(200 mM, pH 7.6). However, as illustrated in Fig. 7, thenormetanephrine peak shape is different in these twobuffers.

Increasing the time for the electromigration to 60 s (at5 kV) gives a concentration of about 120 times greaterthan that obtained by hydrodynamic injection under non-stacking conditions (continuous buffer at 1% of the capil-lary volume) (Fig. 8). Thus, theoretically the capillary canbe filled, at the tip, with the equivalent of more than 100%of its volume with sample in about 1 min, leaving thewhole capillary for separation. Palmer et al. [11] haveshown similar striking effects with electroinjection inMEKC.

4 Concluding remarks

This work demonstrates, (i), that discontinuous buffersand transient ITP can be used in the electromigration.Stacking can take place if the buffer in the sample or at

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Figure 7. Separation of tyramine (30 mg/L) and normeta-nephrine (60 mg/L) by transient ITP: Separation buffer in(A) 200 mM borate pH 7.8; and in (B) 200 mM phosphatebuffer, pH 7.6. Sample introduced by electroinjection for30 s at 3 kV. The anode contained TEA buffer.

Figure 8. Concentration by electromigration: (A) electro-migration for 60 s, 5 kV, of the sample final concentrationof 16 mg/L tyramine; and (B) direct injection filling 1% ofthe capillary; 1000 mg/L tyramine final concentration. Theseparation buffer is 200 mM borate, pH 7.8 and at theanode is TEA buffer. (Note the difference in sample con-centration is 62.5 times).

the anode is different from that in the capillary. (ii) Thecombination of electromigration with discontinuous buf-fers allows high sample concentration not possible withhydrodynamic injection. In addition to that, the long timerequired for large volume injection in the hydrodynamicmode, which sometimes exceeds that for the separation[11], can be avoided with electromigration. (iii) Salts in thesample improve stacking in the transient ITP by thehydrodynamic injection. On the contrary, sample salts inelectromigration decrease the peak height. Thus, in thetransient ITP, sample introduction by electromigration ismore suited for samples which are free or have low saltcontent, while the hydrodynamic injection is more suita-ble for samples containing salts.

The author would like to thank Dr. Christa L. Colyer,Department of Chemistry, Wake Forest University, forher comments.

Received September 20, 2001

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