Anal. Chem. 1997, 69, 333-337
Vacancy Capillary Zone Electrophoresis and Differential Capillary Zone Electrophoresis: Variations on a Well-Known Theme Frans E. P. Mikkers
Nederlandse Philips Bedrijven, Centre for Manufacturing Technology, SAQ 1766, P.O. Box 218, 5600 MD Eindhoven, The Netherlands
Two new variants on capillary zone electrophoresis (CZE) are described and experimentally investigated. In vacancy CZE the sample, diluted with a background electrolyte, fills the electrode vessels and the separation capillary. When pure background electrolyte is injected, the resulting electropherogram represents the composition of the sample. The electropherogram is almost identical with the result of a conventional CZE experiment. In differential CZE the sample again fills the electrode vessels and the separation compartment. After injection of a slightly different sample, a differential electropherogram is obtained that represents the differences between the two samples. The retention times of both new variants are comparable with conventional CZE, the separation efficiency, in terms of theoretical plates, is marginally lower, and both show good quantitative characteristics. The concept of vacancy electrophoresis explains the origin of unexpected system peaks in conventional CZE when multiple co-ions in the background electrolyte are used. For an adequate assessment of the structure or properties of a chemical substance, it is often of decisive importance that the substance can be isolated from its native environment. All branches of chemistry therefore require separation methods for analytical and preparative purposes. Most appropriately, the Dutch word for the entire science of chemistry, “Scheikunde”, means the art of separation. During the last sixty years, separation techniques have evolved in a rather spectacular way and have become a self-evident factor in analytical chemistry. Separation techniques always use at least one driving force for the achievement of separation, and all possible force fields and their respective combinations probably have been investigated in the past. Electrical forces1 have proven to be an extremely powerful agent for achieving separation, and again numerous variants have been investigated. They all can be covered by the term electrophoresis. Capillary zone electrophoresis (CZE) is a rapidly expanding analytical technique for the separation of ionic species in solution. Its relative simplicity, the analogy with chromatography, and its speed and separation efficiency are promising for application in the analytical field. In recent years the technique has gained a great deal of interest and the developmental efforts have resulted in the availability of several commercial available and wellperforming CZE instruments. The main reason for the develop(1) Giddings, J. C. Sep. Sci. 1969, 4, 181-187. S0003-2700(96)00781-0 CCC: $14.00
© 1997 American Chemical Society
mental boost2 seems to be the use of capillaries with small inner diameters in order to reduce the rather deleterious effects of ohmic heat dissipation. An obvious prerequisite for the lowvolume configuration is the availability of very sensitive and stable detection systems and the use of low-volume injection systems. Although originally conceived for the analysis of ionic solutes,3 it is nowadays also possible to analyze nonionics.4 The field of application of CE comprises organic and inorganic solutes, from low molecular weight up to extreme high molecular weight. The analytical capabilities are wide, ranging from small and simple, e.g., sodium ions, to large and complex, e.g., tobacco mosaic viruses and latex particles in the nanometer-size range. Miniaturization has gone down to analytical determinations in biological singlecell compartments5 and in instrumentation as small as on-chip instruments.6 It seems that capillary electrophoretic instrumentation is nearing a state of maturity. From the methodological point of view, considerable progress has been made in recent years. It is generally accepted that in CE multiple parameter interactions occur and as a result the relation between operating conditions and the derived experimental information is not a straightforward one. In this respect, computational simulations of electrophoretic separation systems have proven to be of great value. At the moment, however, they have two commonalities: mathematical intricacy and lack of transparency. It appears that as yet the experiment is faster than the computational simulations and often basic thinking gives faster and more reliable results. Nevertheless, methodological understanding of what happens during separation can help the interpretation of experimental results. Wang and Hartwick7 used a binary background electrolyte and always found a disturbance peak of either negative or positive UV response. They were not able to explain the reason for this disturbance peak. Beckers8 found so-called system peaks when background electrolytes are used with two co-ions and did put a considerable effort in the mathematical modeling of such systems but did not draw the major conclusion. Surprisingly the same phenomena have been known for quite some time in chromatography9 but as yet have (2) Jorgenson, J. W.; Lukacs, K. D. Anal. Chem. 1981, 53, 1298-1302. (3) Mikkers, F. E. P.; Everaerts, F. M.; Verheggen, T. P. E. M. J. Chromatogr. 1979, 169, 1-10. (4) Terabe, S.; Otsuka, K.; Ichikawa, K.; Tuchiya, S.; Ando, T. Anal. Chem. 1984, 56, 111-113. (5) Oleferowicz, T. M.; Ewing, A. G. Anal. Chem. 1990, 62, 1872-1876. (6) Fan, Z. H.; Harrison, D. J. Anal. Chem. 1992, 64, 1926-1932. (7) Wang, T.; Hartwick, R. A. J. Chromatogr. 1992, 589, 307-313. (8) Beckers, J. L. J. Chromatogr. 1995, 696, 285-294. (9) Zhukovitski, A. A. In Gas Chromatography 1964; Goldup A., Ed.; The Institute of Petroleum: London, 1965; p 25.
Analytical Chemistry, Vol. 69, No. 3, February 1, 1997 333
Table 1. Stock Solutionsa no.
cationic constituent
abbrev
concn (mmol/L)
conductivity (µS/cm)
1 2 3 4 5 6 7
imidazole 2-methylimidazole 1-benzylimidazole dimethylamine monoethanolamine diethylamine diethanolamine
IMI MEMI BEMI DMA MEOA DEA DEOA
5.05 5.15 5.07 2.00 5.12 5.09 3.81
380 359 303 195 389 353 252
a The anionic counterconstituent was acetic acid, and the pH was 4.75.
Figure 1. Zone electrophoretic separation schemes: (A) conventional zone electrophoresis; (B) vacancy CZE; (C) differential CZE.
not been mentioned or investigated for electrophoresis. In this respect, it is of importance to return to the basics of CZE. The generally accepted experiment design for CZE is shown in Figure 1A. A sample is injected into a background electrolyte and through the action of an externally applied field a separation is obtained. This separation is detected and represented in an electropherogram, either in a time or spatially based version. Almost always the electropherogram is used to deduce information on the sample, as the latter one is apparently deficient in information. On the other hand, it should be obvious that given the resulting electropherogram, a known sample, and background electrolyte another quantity, e.g., the applied voltage or the column length, could easily be determined, provided one of the two latter ones is known. Realizing that it is only the deficiency of information on the sample that is to be solved, Figure 1A cannot be the only solution to the problem. Thus in Figure 1B and C, two alternative schemes are shown. The scheme in Figure 1B differs from (A) only in the fact that the positions of the background electrolyte with respect to the sample have been changed. Thus when background electrolyte is injected in a CE system, where the capillary and the electrode vessels are loaded with the sample, an electropherogram should be obtained that again contains the desired information. In analogy with chromatography,9 such a separation scheme can be called vacancy electrophoresis. Ultimately when a sample is injected into a CE system where the capillary and the electrode vessels are filled with an electrolyte that differs only marginally from the sample, the resulting electropherogram should reveal the differences between the two electrolytes. Such a separation scheme, shown in Figure 1C, can be called differential electrophoresis. EXPERIMENTAL SECTION For the experiments, a HP3D CE instrument (Hewlett-Packard, Waldbronn, Germany) equipped with a fused-silica capillary was used. The total length of the 75 µm. i.d. capillary was 48.5 cm, resulting in a separation length of 40 cm. The operating temperature was 20 °C, and all experiments were performed at 30 kV unless stated otherwise. Samples were pressure injected, 25 mbar, and UV detection at 200 nm was applied. The applied chemicals were of analytical grade, and ultrapure water was used for electrolyte preparation. The electrolytes were prepared in the form of stock solutions, and all additional electrolytes could be made by appropriate mixing. An overview of the stock solutions is given in Table 1. 334 Analytical Chemistry, Vol. 69, No. 3, February 1, 1997
Figure 2. Separation of three imidazole compounds in the vacancy setup (A) and in the conventional setup (B). For conditions and explanation, see text. For abbreviations, see Table 1.
RESULTS AND DISCUSSION Vacancy Capillary Zone Electrophoresis. In the vacancy variant, the electrode vessels as well as the separation capillary are filled with the sample to be analyzed or with the sample diluted with the background electrolyte. The background electrolyte is injected as a short sample pulse. The separation of some imidazole compounds was chosen as a model system. Imidazole, 2-methylimidazole, 1-benzylimidazole, and monoethanolamine, stock solutions 1-3 and 5, respectively, of Table 1, were mixed in a 1:1:1:1 volume ratio and the mixture was used to fill the electrode vessels and the capillary. Stock solution 5, monoethanolamine, was used for injection during 1 s at a pressure of 25 mbar. From the resulting electropherogram (Figure 2A), it follows that the injection of monoethanolamine caused three UV “peaks” and it was tempting to assign them to the imidazole compounds. Obviously these imidazole compounds easily can be separated in a conventional CZE setup. For this, monoethanolamine was used for filling the electrode vessels and the separation compartment, and now the imidazole mixture was injected for 1 s. The experimental result is shown in Figure 2B. Except for some minor differences both electropherograms look the same: (1) the retention times are almost the same; (2) in the conventional CZE experiment the imidazole compounds give positive UV peaks, whereas in the vacancy variant negative peaks are present; (3) in the conventional CZE experiment the peak shape is leading for imidazole and tailing for 2-methylimidazole and 1-benzylimidazole,
whereas in the vacancy variant imidazole is tailing and the two other imidazoles have a leading distribution. The individual peak positions of the vacancy experiment were verified by using 1:1 volume mixtures of the respective imidazoles and monoethanolamine: the retention times of the different imidazoles proved to be nearly the same for the vacancy and the conventional experiments. In this respect it should be mentioned that at the pH used for the electrolyte system some electroosmotic flow is still present. This osmotic flow may well show minor differences as the electrolyte systems, as applied, had small differences in electrical conductivity. The UV absorbance behavior seems to be straightforward. In the conventional setup the direct UV detection mode could be used, whereas in the vacancy variant the indirect UV detection mode had to be used. The relative differences in peak heights can be explained by the fact that in CZE the magnitude of the UV signal is governed by ionic mobilities, molar absorptivities, and locally generated concentrations.10 The difference in peak shape is perhaps more striking. In the conventional experiment the peak shapes conform theory:3 Sample constituents with an ionic mobility larger than the co-ion of the background electrolyte should show a leading distribution whereas constituents with an ionic mobility smaller than the co-ion of the background electrolyte should show a tailing distribution. Recently it has been shown that this general rule becomes more complex for weak electrolytes where pH effects become dominant.10 In fact the form of the distribution depends on whether a stable moving boundary can be formed on either the front side or the rear side of the distribution.11,12 In our case, however, the electrolyte systems are well buffered at pH 4.75 and all cationogenic constituents behave like strong ions. Thus in the vacancy experiment the ethanolamine has a mobility smaller than imidazole and the ethanolamine-induced vacancy should migrate with a tailing distribution. The mobility of ethanolamine is larger than the ionic mobilities of the two other imidazoles and thus their induced vacancies should migrate with a leading distribution. As mentioned in the introduction, the information that is obtained from an electrophoretic experiment depends on how the experiment is performed. Electrophoresis in this respect is WYDIWYG: What You Do Is What You Get. In conventional zone electrophoresis, the injected amount of a sample constituent preferably should be linearly related to the detected peak area. The same should be true for the vacancy variant. Thus, applying the separation scheme of Figure 1B, the sample load of monoethanolamine was increased by enlarging the sampling time: 0.5, 1, and 1.5 s. The results from Figure 3 show that the monoethanolamine-induced vacancies increase with the sample load of monoethanolamine; moreover, the relation proved to be fairly linear. Apparently in this way the signal to noise ratio (S/N) can be improved. Although interesting, these experiments are only of secondary importance as not the background electrolyte but the sample is the subject of interest: in vacancy zone electrophoresis the injected volume should be kept constant and the capillary column contents should be varied. To investigate the relation between peak and sample concentration, a new model system was chosen. The stock solutions dimethylamine, mono(10) Beckers, J. L. J. Chromatogr. 1994, 679, 153-165. (11) Mikkers, F. E. P.; Everaerts, F. M. In Analytical Isotachophoresis; Everaerts, F. M., Ed.; Elsevier Scientific Publishing Co.: New York, 1980; pp 1-17. (12) Hjerten, S. Electrophoresis 1990, 11, 665-690.
Figure 3. Influence of the injection time in the vacancy setup. For conditions see text. Injection time: (A) 0.5, (B) 1.0, and (C) 1.5 s. Table 2. Mixing Scheme for Amine Mixtures stock solution (Table 1) constituent mix 1 2 3 4 5 6 7
4 DMA
5 MEOA
6 DEA
7 DEOA
2 MEMI
1 1 1 1 1 1 1
1 0.5 0.25 0 1.5 1.75 2
1 1 1 1 1 1 1
1 1.5 1.75 2 0.5 0.25 0
5 5 5 5 5 5 5
Figure 4. Separation of four amine compounds in the conventional setup (A) and in the vacancy setup (B): (A) capillary, electrolyte solution 2 of Table 1; inject, electrolyte solution no 1 of Table 2; (B) capillary, electrolyte solution 1 of Table 2; inject, electrolyte solution 2 of Table 1. For abbreviations, see Table 1.
ethanolamine, diethylamine, and diethanolamine of Table 1 were mixed with the 2-methylimidazole stock solution, accordingly to the scheme of Table 2. In the vacancy setup, the resulting mixtures were used for filling the electrode vessels and the separation capillary, whereas the 2-methylimidazole stock solution of Table 1 was used for injection. Again the amines easily can be analyzed in a conventional CZE setup applying 2-methylimidazole stock solution as the background electrolyte. For both the vacancy setup and the conventional setup the conditions were 20 kV, 25 mbar, and 2 s. A typical result is shown in Figure 4. Analytical Chemistry, Vol. 69, No. 3, February 1, 1997
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Figure 5. Comparison of the peak areas obtained with the vacancy and the conventional setup. For explanation see text: (9) monoethanolamine; (0) diethanolamine.
Figure 6. Separations of four amines in the differential setup: capillary, electrolyte solution 1 of Table 2: (A) inject, electrolyte solution 4 of Table 2; (B) inject, electrolyte solution 1 of Table 2; (C) inject, electrolyte solution 5 of Table 2. For abbreviations, see Table 1.
In the conventional setup, the amines appear as negative peaks and only the dimethylamine has a leading distribution. Again this is in agreement with theory. In the vacancy setup, the amines appear as positive peaks and the distributions of dimethylamine, diethylamine, and diethanolamine are in agreement with the vacancy model. Monoethanolamine, however, has a tailing distribution. This probably is caused by the fact that whether a stable boundary can be formed at the front side or the rear side is not only determined by the ionic mobilities but also by the concentrations of the electrolytic constituents.11,12 From Table 2 it follows that the monoethanolamine and diethanolamine concentrations are the variables. Their respective peak areas were determined in both electrophoretic setups. A plot of the concentrations versus the peak areas should give straight lines, and even more a plot of the peak areas measured in the vacancy setup versus the peak areas in the conventional setup should give a straight line. From Figure 5 it can be seen that the result is quite satisfactory. Moreover, the responses are almost equal for the monoethanolamine and differ by a factor of 2 for diethanolamine. Again this is in agreement with the vacancy model. 336 Analytical Chemistry, Vol. 69, No. 3, February 1, 1997
Figure 7. Relation between peak area and sample concentration for the differential setup: (9) monoethanolamine, value × 0.569 mmol/ L; (0) diethanolamine, value × 0.423 mmol/L.
Figure 8. Influence of the injected sample in the vacancy/differential setup: capillary, electrolyte solution 1 of Table 2; injection of (A) imidazoleacetate or (B) ammonium acetate. For abbreviations, see Table 1.
Differential Capillary Zone Electrophoresis. In the differential setup, the electrode vessels and the separation capillary are filled with the sample. If the same sample is used for injection, no electropherogram should be obtained. Any electropherogram will reflect the differences between the capillary contents and the injected sample. To test this hypothesis the amine mixtures of Table 2 can again be used. The CE system was filled with amine mixture 1 and all mixtures of Table 2 were analyzed. For this we used a column voltage of 30 kV, and the samples were injected during 2 s at 25 mbar. Figure 6 shows some examples of such differential separations. Again the retention times were consistent, and depending on the concentrations in the sample, positive or negative peaks were obtained. Moreover, the differential mode proved to be quantitative. The measured peak areas are shown as a function of the sample concentration in Figure 7. Whenever the sample contains a higher concentration the UV peak will be positive and a negative peak indicates that the concentration in the sample is lower. The relation proved to be linear. The next important question is, what is the sensitivity of the differential setup? A plot of the S/N proved to be perfectly linear. Taking the S/N value of 3 as a limiting value, it followed that the monoethanolamine, at a concentration level of 0.569 mmol/L, should deviate at least 7%. For the diethanolamine, at a concentra-
tion level of 0.423 mmol/L, the deviation should be more than 13%. Enhancement of the sensitivity of the method will be a subject of future investigation. Returning to the experiments of Wang7 and Beckers,8 now the occurrence of their so-called system peaks should be clear. If we consider the amine mixture 1 of Table 2 as a background electrolyte, it is obvious that in cationic separations a background electrolyte with five co-ions is present. If a sample that deviates from the background electrolyte is injected, not only the sample cations will be detected but also the co-ions of the background electrolyte. The relative peak shapes, leading/tailing, magnitude, peak, or dip, will depend on the background electrolyte as well as on the sample. To illustrate the effects imidazoleacetate and ammonium acetate were injected in the amine mixture a of Table 2. Now the only species that the capillary contents and the sample have in common is the counter constituent. The results are shown in Figure 8 and confirm the suggested mechanism: The co-ions of the capillary contents as well as the cation from the sample give peaks. From the experiments shown, it must be concluded that electrolyte systems with multiple co-ions must be very carefully chosen and results should be very carefully interpreted.
CONCLUSIONS In CZE there are three alternative schemes to obtain information about a sample: the conventional, the vacancy, and the differential setup. Retention behavior in all three setups is consistent with electrophoretic theory and all setups can be used for quantitative purposes. System peaks that occur in conventional zone electrophoresis when multiple co-ions are used can be explained by the vacancy concept. The work presented here, once again, is a strong reemphasis of the close analogy of chromatography and electrophoresis. In contrast to analytical chromatography, electrophoresis as still is working at sample overload conditions: we either try to improve detection systems or learn to take full advantage of the “disadvantage”.
Received for review August 2, 1996. Accepted November 4, 1996.X AC960781F X
Abstract published in Advance ACS Abstracts, December 15, 1996.
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