Capillary electrophoresis of samples with a high concentration often leads to peak broadening due to sample destacking at the start of the separation. Using a ...
Destacking loading conditions on a CE chip for measuring samples with a high matrix concentration E.X. Vrouwe and A. van den Berg MESA’ Research Institute,
University oj’Twente, P.O. Box 217, 7500 AE Enschede, I?ethe&znds
The
Abstract Capillary electrophoresis of samples with a high concentration often leads to peak broadening due to sample destacking at the start of the separation. Using a typical CE chip under the right conditions a sample can be diluted during the sample loading step so that destacking during the separation is minimized.
Keywords: conductivity detection, electrophoresis, sample injection, sample matrix 1. Introduction
In order to obtain a good separation with capillary zone electrophoresis it is important that the ionic strength of the sample is lower than that of the background electrolyte (BGE). For the analysis of for example lithium in blood plasma this is a problem as the sodium concentration is approximately 140 n&I. Increasing the BGE concentration to match the sample would result in an increased Joule heating and, when using conductivity detection, a decrease in sensitivity. Therefore it is usually necessary to dilute the sample first. On chip sample dilution has also been shown possible by merging a sample stream with a BGE [l]. A close look at the sample loading procedure on a typical CE chip shows that sample dilution takes place during the loading, which could be used to inject dilute sample into the separation channel without the need of a special chip layout. 2. Theory
Consider a typical CE chip in which a pinched sample loading is used to form a plug in the The sample cross. electrokinetic sample loading is a combination of electroosmotic flow (EOF) and electromigration. Looking at a plain sample containing sodium and lithium immediately after starting the sample loading, four zones can be distinguished (Fig. 1). The undiluted sample (zone a) moves from the sample compartment into the channel with the
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Figure 1. Formation of zones during electrokinetic sample loading.
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speed of the EOF. Leading this zone are two more zones, containing only sodium (c) or both sodium and lithium (b). In essence the sample loading can be seen as a frontal mode separation [2]. Depending on how long the sample loading is continued any of these three zones can be injected into the separation charmel. The concentration of sodium and lithium in the trailing zone is equal to that in the sample compartment. For the two leading zones the Kohlrausch regulating function (1) can be used to calculate the concentration in each zone:
i
Pi
Where c, z and p are the concentration, charge and electrophoretic mobility of each ion in a zone. In the starting situation before any electric field is applied all channels are filled with BGE and only two zones are present. Everywhere in the channels the KRF is o2 and in the sample compartment the !SRF is wl. One property of the KRF is that its value does not change when ions migrate into and out of a zone. When the sample loading is started ions migrate out of the sample and into the BGE. To keep the value w1 in the channels unchanged the concentration of the sample ions entering the BGE has to change. For samples with a concentration higher that the BGE this means that a dilution occurs upon crossing the boundary between sample and BGE. Conversely dilute samples are stacked at the interface. There is one issue that makes the situation more complex and that is caused by the relative mobility of cations from the sample and BGE coions. In the case that the mobility of the sample cation is higher than that of the BGE co-ion the boundary between the zones is not sharply defined but diffuse instead. Compare this to the triangular peak shape that sometimes can be observed in electropherograms due to electromigration dispersion. For the sample loading this will result in a Figure 2. Photograph of the electrophoresis gradual increase in the concentration of chip (dimensions 3 x 1.5 cm) and a close-up sodium and lithium in the cross during of the end of the channel with the the pinching step. conductivity detection electrodes. The actual length of the separation channel is 2 cm.
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3. Experimental For all experiments a glass chip with integrated thin film conductivity detection electrodes was used as presented before (Fig. 2) [3]. The length of the separation channel was 2 cm and a double-T injector geometry was used. The separation buffer consisted of 50 mM 2-(N-mo~holino)ethanesulfonic acid (MES) and 50 mM histidine with 0.01% hydroxypropyl methyl cellulose (HPMC) to suppress the EOF. A computer controlled high voltage power supply (Ibis 411, IBIS Technologies, The Netherlands) was used together with a homemade conductivity detector. 4. Results and discussion The resolution between 5 mM lithium and 150 mM sodium increased considerably when not the EOF was used for sample loading but only the electromigration of sodium and lithium (Fig. 3). Although there was some residual EOF the HPMC suppressed it sufficiently. Without the HPMC, the zones would be so close together that timing the duration of the sample loading step would prove difficult. A consequence of diluting the sample is that the peaks were smaller then when using EOF assisted sample loading. Also there is an unmistakable effect of the sample loading time on the measured peak area (Fig. 4). This is the result of the low mobility of the histidine co-ion compared to sodium and lithium. It is expected that when a co-ion like potassium with a high mobility is used the peak areas will be independent of the loading time. For quantitative analysis of lithium it is important to use an internal standard to correct for variations in the injected amount of sample. Figure 4 shows that the ratio between sodium and lithium is independent of the injection time. If the concentration of lithium is constant and the amount of sodium is increased, more and more dilution occurs
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Figure 3. Separation of 5 mM lithium and 150 mM sodium. BGE 50 mM MESHis I) with 0.01% HPMC in the BGE and II) without HPMC.
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Figure 4. Measured peak area as function of the sample loading time. Sample consisted of 5 mM lithium and 150 mM sodium.
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, 0.4 0.35 0.3 0.25 0.2 0.15 0.1 0.05 0
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Figure 5. Peak area of sodium and lithium as function of the amount of sodium in the sample. The lithium concentration is 3 mM in all samples.
Figure 6. Calibration curve for lithium in 150 mM Na matrix. The dashed lines indicate the 95% confidence interval.
during the loading shown by the decreasing lithium peak area (Fig. 5). Still the ratio between the peak area of sodium and lithium does not change. The calibration curve demonstrates that it is possible to measure in the clinical relevant range between 0 and I mM lithium using the sodium peak as internal standard (Fig. 6). 5. Conelusions During a pinched sample loading stacking or destacking of the sample takes place, which can be used to dilute concentrated samples and prevent peak broadening during the separation. This made direct measurement of lithium concentrations below 0.5 mM possible in a sample containing 150 mM sodium chloride. Also in situations where the EOF is suppressed for other reasons the stacking and destacking effects during sample loading need to be considered. The next step will be to use the same procedure to measure lithium in blood plasma samples from patients on lithium therapy. Acknowledgements The authors gratefully acknowledge Foundation STW (TET 5370).
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References 1. 2. 3.
S.C. Jacobson, T.E. M&night, J.M. Ramsey, Anal. Chem. 71,4455-4459 (1999) C. J. Backhouse, H. J. Crabtree, D. M. Glerum; Analyst 127, 1169-l 175 (2002) E. Vrouwe, R. Luttge, A. van den Berg, Proc. Micro Total Analysis Systems 2002, Kluwer Academic Publisher, Dordrecht, The Netherlands, pp. 178-l 80 (2002)
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