with the sample solution and the analyte stacked into a sharp zone. Figure 4a illustrates an experi ment where 35 cm of the column was loaded with the sample solution. A great enhancement in detection sen sitivity is achieved compared with the conventional sample stacking technique in Figure 4b, where only 1 cm of sample was loaded into the column. Note that no separation will occur if the sample buffer is not re moved from the column (Figure 4c). In reality, it is sometimes easier to fill the whole column with the sam ple solution and then insert the col umn into the high-concentration support buffer reservoir to perform sample stacking. At the beginning of the run, there is no field enhance ment because the whole column is filled with sample solution. Conse quently, some of the analytes will be carried out of the column by the electroosmotic flow. However, as the high-concentration support buffer slowly replaces the sample buffer in side the column, the electric field in the remaining sample buffer region
will begin to increase rapidly, as de scribed in Equation 2. As the length of the sample buffer reaches the maximum fill length, given by Equa tion 17, the analytes can then mi grate opposite to the electroosmotic flow and stack at the rear end of the sample buffer. This technique for whole-column sample stacking is simple and requires no special tools.
Newfangled? Old Fashioned?
Sample stacking in electroinjection
Field-amplified sample injection. The concepts of sample stacking and field amplification can also be ap plied to electroinjection (26-29). Electroinjection of samples (prepared in a low-concentration buffer or wa ter) into a column filled with a higher concentration of the same buffer re sults in an electric field at the injec tion point that is much stronger than the electric field in the column ac cording to Equation 5. For a short in jection time such that χ « 1 and yx « 1, the electric field in the col umn changes very little from the original uniform field, and the elec-
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- Negative species
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Migration time (min)
Figure 4. Electropherograms showing improvement in sample stacking of an extremely large injection volume. (a) A 35-cm-long sample is loaded into the column and the sample buffer removed, (b) Conventional sample stacking using a 1 -cm-long sample, (c) The same as (a) but without the sample buffer removed. The negative species peak contains both A and B. The sample is a mixture of (A) phenylhydantoin (PTH) and aspartic acid (4 χ 1CT5 M each) and (B) PTH and glutamic acid (3.4 χ 1CT5 M each) prepared in water. Column: 50-μΓη i.d., 100-cm-long, untreated fused-silica capillary; buffer: 100 mM 2-(N-morpholino)ethanesulfonic acid (MES)/100 mM histidine at pH 6.2.
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ANALYTICAL CHEMISTRY, VOL. 64, NO. 8, APRIL 15, 1992 · 493 A
