Study of quantitative analysis of traces in low-conductivity samples using capillary electrophoresis with electrokinetic injection

doi:10.1016/j.chroma.2005.07.030

Journal of Chromatography A

Volume 1091, Issues 1–2, 14 October 2005, Pages 163-168

https://doi.org/10.1016/j.chroma.2005.07.030 Get rights and content

Abstract

In this work we tried to clarify the relation between the signal detected by indirect UV mode and the concentration of the analyte introduced by electrokinetic (EK) injection from samples having small but different conductivities. A simple external calibration procedure was proposed for quantitative determination of traces that are present in low-conductivity samples. It was found that measuring the current through the capillary filled with the samples provides useful correction factor for the external calibration.

Introduction

In capillary electrophoresis (CE) method there are two techniques to introduce a minute part of the sample into the separation capillary: hydrodynamic (HD, samples driven by a pressure) and electrokinetic (EK, samples driven by an electric field). In the majority of the published works in CE, hydrodynamic injection is preferred because its theoretical fundamentals are much more simple and more established, and it is more reliable and accurate for quantitative analysis. In EK injection the variations in conductivity of samples due to matrix effects result in differences in voltage drop and quantity loaded during the injection, therefore EK injection is generally not as reproducible as HD sample introduction. Despite of the known quantitative limitations, the practice of EK injection is very simple, it can provide much more sensitive determinations (field-amplified injection) than HD injection and its use in some fields (e.g. capillary gel electrophoresis, lab-on-a chip-technology) cannot be substituted.

There are numerous works dealing with the theory of EK injection [1], [2], [3], [4], [5], [6], [7], [8], [9]. Some of them separate the occuring problems as EK injection of samples with conductivities similar and different to that of the background electrolyte (BGE). In case of EK injection the sample enters the capillary by a combined effect of electroosmosis and electrophoresis, therefore the amount of the analyte ion injected depends on its mobility (“mobility bias”) and a larger amount will be introduced if the analytes co-migrates with the electroosmotic flow (EOF). Therefore, when two different analytes are present in the sample of the same concentration, different amounts will be injected resulting different responses. These variances can be disregarded using external calibration only if the conductivities of the samples are equal, however the conductivities of real samples are almost always different. Since the injected analytes also depend on the relationship of the conductivities of the sample solution and the BGE, the bias between different samples with different matrix (“matrix bias”) caused by EK injection cannot be corrected by means of common calibration procedures.

In EK injection since the capillary is almost completely filled with the BGE, the EOF is practically not influenced by the conductivity of the sample solution, however, the electromigration of the analytes depends on the conductivities of each sample. The total amount (n_a) of the analyte a injected is given by: $n_{a} = \frac{1}{4} π d^{2} (μ_{EOF} \pm \frac{k_{BGE}}{k_{s}} μ_{a}) \frac{U_{inj}}{L} t_{inj} c_{a}$ where the d is the inner diameter of the capillary, the μ_EOF is the mobility of the EOF, the μ_a is the electrophoretic mobility of the analyte, U_inj is the injection voltage, L is the total length of the capillary, t_inj is the injection time, c_a is the molar concentration of the analyte, k_BGE and k_S are the conductivities of the BGE and the sample solution, respectively.

The mobilities of analytes and the EOF can be determined experimentally on the basis of the migration times of the analyte (t_a) and a neutral, unretained compound (t_EOF), thus these mobilities can be expressed by: $μ_{EOF} = \frac{L_{eff} L}{U t_{EOF}} and μ_{a} = \frac{L_{eff} L}{U t_{a}}$ where the U is the separation voltage, and L_eff is the effective length of the capillary.

If the mobilities in Eq. (1) are substituted for expressions of Eq. (2) then the amount of the analyte a injected will be: $n_{a} = \frac{1}{4} π d^{2} (\frac{1}{t_{EOF}} \pm \frac{k_{BGE}}{k_{S}} (\frac{1}{t_{a}} - \frac{1}{t_{EOF}})) \frac{U_{inj} L_{eff}}{U} t_{inj} c_{a}$ from the Eq. (3) it is obvious that the injected amount of neutral components, (where t_eof and t_a are the same) does not depend on the matrix of the samples.

In general, the use of internal standards offers better precision than without standards, some one- and two-internal standards methods have been described [4], [10]. Lee and Yeung improved the quantitative precision of CE with EK injection by monitoring the electrophoretic current during injection [4]. They showed that the effects of sample conductivities on the amounts of analytes introduced with EK injections could be nullified by proper corrections with the measured conductivities of sample and BGE together with the migration times of analyte and the EOF. They achieved 0–5% level of accuracy with the exception of low conductivity samples (smaller than 10 mM phosphate in sample) [4]. However, in a review on EK injection it is stated that “in high-purity water (matrix-free conditions) external calibration can also be used” [8]. Some authors found that standard addition can be well applicable for analysis of samples including large amount of matrix material [5], however thus the number of the measurements should be at least doubled or tripled. Most studies on the topic establish that it is important to further understand EK injection and to explore new ways to improve its quantitative capability [4], [8].

The indirect UV detection is a popular and well-applicable detection mode in CE for analysis of inorganic anions, which are often UV non-active ones, e.g. [11]. It was observed that the slopes of calibration plots for alkali metal and alkaline earth metal cations are identical for the monovalent cations on one hand and for the divalent cations on the other hand, and the values of the slopes for the divalent analytes are different from those for the monovalent analytes by exactly a factor of two [6]. The EK injection seems to have compensated the differences in transfer ratio between the higher- and lower mobility ions, that is the transfer ratio is the same for all analyte ions having the same valency [6].

Although the EK injection is generally not suggested for quantitative determinations with the statement that different conductivities of samples cause false results, there is a large number of application of EK injection trying to utilize the simply attainable high sensitivity. The aim of our work is to try to clarify the relation between the analytical response and the concentration of the analyte introduced by EK injection from samples having small but different conductivities. We propose a simple external calibration procedure for accurate quantitative determination of traces present in low-conductivity samples using EK injection. All our present work is restricted for the analysis of highly diluted (low-conductivity) samples.

Section snippets

Instrumentation

The capillary electrophoresis instrument was a HP ^3DCE model (Agilent, Waldbronn, Germany). The sample solutions were introduced by EK injection (−2.5 kV for 15 s) at the cathodic end of the capillary. Separations were performed using fused-silica capillaries of 64.5 cm × 50 μm I.D. (effective length: 56 cm) (CS-Chromatographie, Langerwehe, Germany). The applied voltage was −25 kV (reversed polarity). The detection was carried out by on-column photometric measurement (detecting wavelength: 450 nm,

Results and discussion

While in case of hydrodynamic injection the response (peak area) depends linearly on the concentration of the analyte in a large concentration range (5–100 μM) (Fig. 1a), the graphs are hyperbola-shaped when EK injection is applied (Fig. 1b). After a very short initial linear part the graphs will be curved. The probable reason of this shape is that increasing the concentration of the standards (in pure water), the total ion content of the sample solution will be higher and more comparable to the

Conclusion

The main advantage of the use of EK injection is that the attainable sensitivity can be much better than using HD injection, however EK suffers from the poor reliability in quantification. In our work it was proved that the combination of EK injection of larger amount of sample (field-amplifield stacking) can provide a very simple, cheap, rapid and effective tool for reliable quantitative analysis of traces of inorganic anions in low conductivity samples using the proposed external calibration

Acknowledgement

Our work was supported by the Hungarian Scientific Research Fund (F040468) and the GVOP (3.2.1.-2004-04-0032/3.0).

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Study of quantitative analysis of traces in low-conductivity samples using capillary electrophoresis with electrokinetic injection

Abstract

Introduction

Section snippets

Instrumentation

Results and discussion

Conclusion

Acknowledgement

Anal. Chem.

J. Chromatogr.

J. Chromatogr.

J. Chromatogr. A

J. Chromatogr. A

J. Chromatogr.