Technical Notes

Stacking from the Sample Stream in CZE Using aPneumatically Driven Computerized Sampler

Ruth Kuldvee and Mihkel Kaljurand*

Department of Chemistry, Tallinn Technical University, Ehitajate tee 5, Tallinn EE0026, Estonia

It is demonstrated that a pneumatically driven computer-ized sampling device for capillary electrophoresis facili-tates sample stacking by the head-column field amplifi-cation (HCFA) technique. This device utilizes a rapidexchange between buffer and sample in a narrow channelat the separation capillary inlet and makes possible thecombination of two classical injection modessthe elec-trokinetic and hydrodynamic modes. Detection limitsobtained were about 9 nM for alkylbenzylamine cationswith common UV detection.

Recent developments in nonconventional sample introductionin capillary electrophoresis have focused on the possibility offorcing the sample stream to pass the separation capillary inlet.The advantages of such input devices are the absence of a voltagerise/drop time during sampling, ease of operation because no vialmanipulations are involved, and ease of automation and comput-erization. Such an input system has been described by Verheggenet al.1 and Bushey and Jorgenson.2 Recently, Kuban and co-workers3,4 and Fang et al.5 developed an inlet system for usingCE as a detector for a flow injection analysis technique. Theauthors of this note developed a pneumatically driven computer-ized sampler for CE.6 Liu and Dasgupta described an ingeniousfalling drop sample introduction system.7

In all these systems, the sample is believed to be introducedby the well-known electrokinetic phenomenon during the timewhen sample is present or passes the capillary inlet. However,since sampling requires periodic rinsing of the input channels bybuffer and sample solution, sample could well be introducedhydrodynamically by pressure applied to the sample solution. Inmany samplers cited above, the amount of the sample introducedby the latter mechanism is probably negligible.

In this note, we report that a certain amount of the sample isintroduced into the capillary head during the rinse and demon-strate that this facilitates head-column stacking of sample from

the stream flowing past the capillary inlet if the sample solutionconductivity is much lower than the running buffer conductivity.Head-column stacking was demonstrated to be a powerful andsimple way to reduce detection limits in CE.8-11 Analyte detectionlimits can be improved by more than 2 orders of magnitudecompared to the common electrokinetic sampling. In this work,the detection limits achieved for two cations were 250 ppb forthe latter method, when for the first one it was 2.5 ppb (9 nM)using common UV detection.

EXPERIMENTAL SECTIONEquipment. The CE system consisted of a homemade

autosampler, a homemade high-voltage supply delivering 18 kV,an 80-cm full length (50 cm to detector), 50-µm-i.d. capillary(Polymicro Technologies, Phoenix, AZ), and an Isco CV4 UVdetector. Detector signal was digitized and transferred to the 486-type computer via a Keithley ADC-16 analog-to-digital board. Thesame board delivered digital signals to the solenoid valvescontrolling the autosampler. The autosampler schematics isshown in Figure 1. It operates on the principle of rapidly changingbetween buffer and sample in the input channel of the autosamplerblock by pressure applied either to the buffer or to the samplervessel. Total dimensions of the plexiglass sampler body are 35× 25 × 25 mm3. Input channel has length of 45 mm, with 1.5mm i.d. Thus, the total input channel volume is about 80 µL.Assuming that the input channel and capillary are filled withrunning buffer and high voltage is applied, the sampling isperformed according to the sequence of pressures applied asshown in Table 1. It is evident from Table 1 and Figure 1 that, toexecute the sampling process, pressures must satisfy the followingrelationship: P1 ) P2 < P3 ) P4. The pressures are deliveredby two solenoid valves. A thorough description of the sampler isgiven in ref 6.

Chemicals. Each analysis was performed with sodiumphosphate buffer as background electrolyte at pH ) 6.7 and ionicstrength I ) 0.1. All solutions were prepared by dissolvingchemicals of analytical grade in deionized water. Benzyltriethy-lammonium chloride (BTEA) and benzyltributylammonium chlo-ride (BTBA), both purchased from Merck, were used as samples.

(1) Verheggen, Th. P. E. M.; Beckers, J. L.; Everaerts, F. M. J. Chromatogr.1988, 452, 615.

(2) Bushey, M. M.; Jorgenson, J. W. Anal. Chem. 1990, 62, 978.(3) Kuban, P.; Karlberg, B. Anal. Chem. 1997, 69, 1169.(4) Kuban, P.; Engstrom, A.; Olsson, J. C.; Thorsen, G.; Tryzell, R.; Karlberg,

B.; Anal. Chim. Acta 1997, 337, 117.(5) Fang, Z.-L.; Liu, Z.-S.; Shen, Q. Anal. Chim. Acta 1997, 346, 135.(6) Kaljurand, M.; Ebber, A.; Somer, T. J. High Resolut. Chromatogr. 1995,

18, 263.(7) Liu, H.; Daskupta, P. K. Anal. Chem. 1997, 69, 1211.

(8) Chien, R. L.; Burgi, D. S. Anal. Chem. 1992, 64, 489A.(9) Burgi, D. S. Anal. Chem. 1993, 65, 3726.

(10) Martinez, D.; Borrull, F.; Calull, M. J. Chromatogr. A 1997, 788, 185.(11) Zhang, C.-X.; Thormann, W. Anal. Chem. 1996, 68, 2523.

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S0003-2700(98)00111-5 CCC: $15.00 © 1998 American Chemical Society Analytical Chemistry, Vol. 70, No. 17, September 1, 1998 3695Published on Web 07/03/1998

Sodium hydroxide was obtained from Chempol and phosphoricacid from YA-Kemia. Solutions were filtered through 0.45-µmMillipore filters.

RESULTS AND DISCUSSIONIt follows from the description of the operation of the sampler

that the time spent in step 2 determines the electrokineticsampling time of the autosampler and that steps 1 and 3 shouldbe as short as possible to enable pure electrokinetic sampling.Optimization of the operating parameters of the autosampler hasbeen studied and will be published in a separate paper. Only themain results will be given here. The pressure actuating thebuffer/sample stream should be within (0.3-0.85) × 105 Pa. Ifthe pressure is outside these limits, severe reduction of theseparation efficiency results, together with large baseline ir-regularities. This is probably due to overloading of the capillarywith sample if the pressure is higher than 0.85 × 105 Pa; sample-to-buffer exchange does not occur with the required speed if thepressure is lower than 0.3 × 105 Pa. This pressure is determinedby the length and diameter of the sampler channels and connect-ing tubing, thus being specific for the particular sampler design.

If the pressure is within the limits, sample-to-buffer (or vice versa)replacement (or rinse time) requires about 0.125 s.

The separation efficiency (expressed, e.g., in theoretical platenumbers) decreases with increasing rinse time. But if the sampleconcentration is low enough (1 ppm and lower) and the sampleis dissolved in water, then it is possible to increase the rinse timeto 10 s without serious loss of efficiency. In this case, samplestacking could be expected by the field-amplified effect. Thesituation is illustrated in Figure 2, where three different sampleintroduction techniques have been compared. Different concen-trations for comparisons were used deliberately, to have measur-able sample component peaks at least in two of three electro-pherograms. First, the sample was introduced hydrodynamicallyonly by forcing the sample stream to flow through the inputchannel while the high voltage was switched off. The durationof steps 1 and 3 was 2.5 s, and the duration of step 2 was 0.25 s.After sampling, the high voltage was switched on again. In thiscase, the sample was introduced only by pressure applied to thesample stream. The electropherogram (Figure 2a) consists of awater peak only; no BTEA and BTBA peaks can be detected sincetheir concentrations are only about 125 ppb. The same procedurewas repeated with the same durations for sampling steps 1-3,with high voltage now applied. The electropherogram (Figure2b) now consists of a water peak with the same size as in theformer case plus two cation peaks present with very good signal-to-noise ratio, despite the fact that the concentrations of theanalytes are much lower (15 ppb). The fact that water peaks arethe same size in both cases demonstrates that the sample isintroduced, indeed, hydrodynamically during the rinse time andthe contribution of electroosmosis is negligible. In the firstexperiment, with high voltage switched off, the stacking occursonly within the sample zone introduced into the column. In thesecond case, when high voltage is switched on during thesampling, sample probably stacks to the column head from thewhole volume around the capillary inlet. A third experiment wasperformed using short (0.25 s) rinse time (steps 1 and 3) but with

Figure 1. Pneumatic autosampler: (A) sample rinse, (B) bufferrinse.

Table 1. Sampling Sequence Logic

step action P1 P2 P3 P4 duration

1 sample rinse off on on off 0.125-10 s2 electrokinetic sampling off off on on 0.25-10 s3 buffer rinse on off off on 0.125-0.25 s4 pherogram run off off on on 5 min-1 h

Figure 2. Illustration of the effect of stacking from the samplestream. (a) Rinse time 2.5 s, sample kept in input channel for 0.25 s,high voltage switched off; (b) rinse time 2.5 s, electrokinetic samplingtime 0.25 s; (c) rinse time 0.25 s, electrokinetic sampling time 2.5 s.Peaks: 1, BTEA; 2, BTBA; 3, water; ?, unknown. Note the differencesin sample concentrations: in a, b, and c, the sample concentrationswere 125, 15, and 250 ppb, correspondingly.

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sample in the input channel for 2.5 s, and regular electrokineticsampling took place. The electropherogram has much smallerwater peak than in the previous two cases and two low-intensitycation peaks, despite the fact that the total duration of the samplingsteps 1 and 2 is equal in the second and third experiments.Concentrations of the analytes are 250 ppb.

The relationship between peak area and rinse time is linearup to the rinse time of 10 s, with a correlation coefficient of 0.9989.The reproducibility of peak areas is about 2.3% if the sample isstacked from the sample stream. If the rinse time is longer than10 s, the current through the capillary drops significantly (by 95%)during the rinse time, and the corresponding electropherogramhas an irregular baseline with deteriorated peak shapes. Figure3 presents an electropherogram of a 2.5 ppb sample (about 9 nMof both analytes), thus exhibiting the detection limit achieved inpresent work. If we compare this detection limit with thosereached with electrokinetic injections250 ppb (see Figure 2)s

the detection limit is 2 orders lower in the first case.Thus, a sample plug can be introduced to the column head by

three different means: (1) from the flowing sample stream bypressure with high voltage applied simultaneously, (2) from theflowing sample stream by pressure with high voltage switchedoff, and (3) by electroosmosis during electrokinetic sampling. Themechanism responsible for the improved detection limits obtainedin the first case compared to other cases is probably thefollowing: by applying pressure, a small sample plug is appliedto the capillary head, and, if high voltage is applied as well,stacking is likely to occur from the whole volume of the inputchannel. In pure hydrodynamic sampling, the sample plug is alsointroduced at the capillary head; however, since the next samplingstep is to replace the capillary inlet channel with buffer, the sampleamount in this plug is too small to be stacked and forms anobservable peak on the electropherogram. If the sample solutionis introduced electroosmotically, stacking also applies to the wholesample channel; however, the electroosmotically introduced low-conductivity plug at the column head is too small to ensure anobservable stacked amount of sample in the plug.

To understand the mechanism of the injections with long rinsemore clearly, we studied the sampling in two cases: either thesample is flowing past the capillary inlet or standing still. In thecontext of this paper, it would be convenient to call the stackingfrom the stagnant sample static stacking and the stacking fromthe sample stream dynamic stacking. To establish the possibledifference between the two techniques, the following two experi-ments were performed. In the first experiment, sampling con-sisted of a long rinse time (step 1 in Table 1), and electrokineticsampling time (step 2 in Table 1) with the two steps having equalduration. High voltage was on during the whole samplingprocedure. In the second experiment, rinse time was equal tothat in the first experiment, but electrokinetic sampling time wastwice as long as in the first experiment. In the second experiment,high voltage was switched off during the rinse time. In the secondcase, the contribution of the pure hydrodynamic sample introduc-tion remains, but if the concentration is low the contribution canbe ignored.

If the stacking from the flow does not involve any peculiaritycompared to the static stacking, the peak areas of cations mustbe the same in the first and the second experiments when thehigh voltage was switched off or on because the head-columnHCFA phenomenon applies in both cases. At a sample concentra-tion of 50 ppb, the cations peak area ratio was about 2 timesbetween dynamic and static stacking, and the increase in the rinsetime did not influence the results significantly. At a lower sampleconcentration (10 ppb), increasing the rinse time by a factor of 2increased further the peak areas ratio approximately 2.5 times.Figure 4 illustrates the results. A possible explanation is that, inthe case of long static sampling, the sample channel can bedrained empty of cations: i.e., the predominant part of the samplewas expelled and sample stacking took place only from the sampleportion remaining in the input channel, which was rapidly drainedoff the sample. Independent studies by Zhang and Thormann alsoindicate that input vessels can be drained empty if the HCFA

Figure 3. Electropherogram of BTEA and BTBA. Sample concen-tration 2.5 ppb. Peaks: 1, BTEA; 2, BTBA; 3, water.

Figure 4. Electropherogram of the BTEA and BTBA. Sampleconcentration 10 ppb. Rinse time for electropherograms a and b was2.5 s, electrokinetic sampling time was 2.5 and 5.0 s, accordingly.Rinse time for electropherograms c and d was 5 s, electrokineticsampling time was 5 and 10 s. (a,c) High voltage switched on duringrinse time; (b,d) high voltage switched off during rinse time. Peaks:1, BTEA; 2, BTBA.

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occurs.12 In experiment 1, where high voltage was on during thesample rinse, the total sample volume from which the stackingoccurs is larger (due to the flow of fresh sample into inputchannel) than in experiment 2. Thus, this experiment confirmsour hypothesis that, during rinse time, a water plug penetratesthe capillary and ensures head-column field amplification and thatthere exist some differences between dynamic and static stacking.Such differences increase with decreasing concentration. To give

a satisfactory explanation needs further studies. Another advan-tage of the dynamic stacking should be mentioned. In ref 11,the authors speculate about possible electrolysis degradation andoverheating of the sample. Stacking from the stream should avoidsuch disadvantages.

Received for review February 2, 1998. Accepted May 22,1998.

AC9801115(12) Zhang, C.-X.; Thormann, W. Anal. Chem. 1998, 70, 540.

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