Anal. Chem. 1996, 68, 3928-3933
Two-Dimensional Separations: Capillary Electrophoresis Coupled to Channel Gel Electrophoresis Yi-Ming Liu and Jonathan V. Sweedler*
Department of Chemistry, University of Illinois at UrbanasChampaign, 600 South Mathews Avenue, Urbana, Illinois 61801
Two-dimensional separations provide extremely high peak capacities. Coupling capillary zone electrophoresis with ultrathin channel gel electrophoresis offers a convenient and efficient way to perform such two-dimensional microseparations. By means of in situ polymerization, highconcentration (up to 50%T) polyacrylamide gels are prepared in 75 mm long, 25 mm wide, and 40 µm thick rectangular channels. By moving the outlet end of the capillary electrophoresis capillary across the entrance of the channel, both separations are completely preserved. Mixtures of peptides labeled by fluorescein isothiocyanate (FITC) are well resolved in less than 15 min, with theoretical plate numbers in the range of 20 000-50 000 for each independent separation. Significant enhancement in separation efficiency and peak capacity over onedimensional separations are demonstrated by this combination. The two-dimensional separations of a model mixture of peptides, a tryptic digest of trypsinogen, and 50% increase in comparison with free solution (peak capacity of 20).15 Before a new channel is used, it is “preelectrophoresed”. For a 40%T, 3.3%C gel-filled channel, the channel current (at 3.2 kV) decreases rapidly (in ∼20 min) from >1.5 mA to a stable 320 µA during the preelectrophoresis. Channel current varies with the concentration of the gel in the channel. A 5%T, 3.3%C gel-filled channel, for example, has a stable channel current of 860 mA at 3.2 kV. These gel-filled channels can be repeatedly used; for 15 repeated separations (voltage, 3.2 kV; run time, 10 min, the resolution between two FTC-amino acids tested, FTC-aspartic acid and FTC-proline, is reproducible (to within 5%). Channels with lower %T are more durable. Lastly, too high a voltage across the channel results in gas bubbles. A voltage of 5 kV may damage a 5%T, 3.3%C gel-filled channel, and 7.5 kV may damage a 40%T, 3.3%C gel-filled channel. The reproducibility of a series of gel(50) Giddings, J. C. Unified Separation Science; John Wiley & Sons: New York, 1991; Chapter 5.
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filled channels has been evaluated. For these experiments, three 40%T, 3.3%C gel-filled channels were prepared on three different days and used to separate proline and aspartic acid labeled by FITC, with four successive separations performed on each channel. The relative standard deviations (RSDs) are 7% for FTCaspartic acid, 9.3% for FTC-proline, and 3.5% for unreacted FITC. As previously stated, in the gel electrophoresis, analyte migration is hindered by the structure of the high-concentration and cross-linked polyacrylamide gels (sieving and/or interaction with the polymer matrix). Hence, molecular size plays a very important role in the separation process. For example, FITClabeled Leu-enkephalin and angiotensin II have similar mass-tocharge ratios but migrate at substantially different speeds in the gel. In CZE, separations are based on the differences in analyte mass and charge, which result in differences in electrophoretic mobility. The combination of a CZE separation with the ChGE separation should result in a two-dimensional separation method that provides very high separation efficiency and peak capacity while retaining many of the CZE advantages such as small sample volumes and fast separations. For a two-dimensional separation, the capillary outlet is scanned across the inlet of the channel. The output from the CCD detector is a series of time slices showing bands at the outlet end of the rectangular channel. However, samples are injected and start the second separation step as they elute from the CZE capillary, so that the time to reach the channel detection zone is a combination of CZE elution time and the channel elution time. To obtain “time-corrected” images that have axes representing true elution times for the two independent separations, the time that the outlet end of the CZE capillary injected each band is subtracted from each CCD column result. Figure 4A shows the resulting time-corrected electropherogram of the standard peptide mixture. Both the one-dimensional CZE and the ChGE electropherograms show five separated major peaks (Figure 5). Using standards, we find that the unreacted FITC coelutes with FITClabeled Val-Tyr-Val in the CZE separation and coelutes with FITClabeled Gly-Tyr in the ChGE separation, and neither separation resolves the pair of FTC-Leu-enkephalin/FTC-Met-enkephalin. However, complementary separation is achieved using the combination of the two methods, as can be seen in Figure 4A. As a demonstration of the sensitivity and resolving power of this method, a single B2 neuron of Aplysia californica is dissected and derivatized, and a portion (∼10 nL/30 µL) is injected onto the CZE capillary. A section of the resulting electropherogram is shown in Figure 4B. In this experiment, the second-dimensional ChGE separation sampled the effluent from the CZE separation between 12 and 18 min, just after the unreacted FITC eluted. By not injecting the much larger unreacted FITC peak into the ChGE separation channel, the later-eluting minor peaks from labeled peptides and other amine compounds are more easily detected and displayed. The peak capacity of a separation with unit resolution can be calculated by
Nc ) L/4σ where L is the time period over which peaks are distributed, and σ is the average standard deviation of these peaks.50 For the peptide mixture sample (Figure 4A), the peaks elute in the CZE axis from 5.2 to 7.2 min, and the average 4σ peak width is 0.11 3932
Analytical Chemistry, Vol. 68, No. 22, November 15, 1996
Figure 4. False color two-dimensional CZE-ChGE electropherograms, where red indicates the highest fluorescence intensity and blue the lowest. (A) Separation of a FITC-labeled model peptide mixture. (B) Analysis of a portion (
