Concentration By Capillary Stacking

As mentioned earlier in this chapter, electrophoretic techniques offer a relatively simple means to concentrate the sample directly on the capillary by injecting a large volume of sample. Under nonstacking conditions, the sample volume is kept below 1% of the capillary volume. However this volume can be increased to 10-30% of the capillary volume using stacking conditions. The drug "stacks" at the interface of the injection buffer and the run buffer (see also Chapter 2 for a more detailed discussion). This helps to

Fig. 2. Analysis of serum free phenytoin (P) from a patient (1.8 mg /L) using a Beckman CE Model 2000. Serum was filtered in a Centrifree filter (Amicon, Beverly, MA), 50 ^L of the filtrate was removed, mixed with 100 ^L acetonitrile containing 2 mg/L isobutyl methylxanthine (X) as an internal standard, and injected on a capillary: 50 ^m x 40 cm at 9 kV (N, neutral molecules).The separation buffer, borate 300 mM, pH 8.7. (A) The patient at loading of 1%, 214 nm. (B) The patient at 10%. (C) The patient at 10% loading spike with 2.5 mg/L. (Note that injecting the traditional small volume of sample <1% of the capillary does not give enough sensitivity to detect the free phenytoin. Increasing the sample volume to 10% of the capillary gives good sensitivity provided acetonitrile is present in the sample. In the absence of acetonitrile (18) the peaks are short, distorted, and overlap, not shown.)

Fig. 2. Analysis of serum free phenytoin (P) from a patient (1.8 mg /L) using a Beckman CE Model 2000. Serum was filtered in a Centrifree filter (Amicon, Beverly, MA), 50 ^L of the filtrate was removed, mixed with 100 ^L acetonitrile containing 2 mg/L isobutyl methylxanthine (X) as an internal standard, and injected on a capillary: 50 ^m x 40 cm at 9 kV (N, neutral molecules).The separation buffer, borate 300 mM, pH 8.7. (A) The patient at loading of 1%, 214 nm. (B) The patient at 10%. (C) The patient at 10% loading spike with 2.5 mg/L. (Note that injecting the traditional small volume of sample <1% of the capillary does not give enough sensitivity to detect the free phenytoin. Increasing the sample volume to 10% of the capillary gives good sensitivity provided acetonitrile is present in the sample. In the absence of acetonitrile (18) the peaks are short, distorted, and overlap, not shown.)

alleviate the poor detection limits of CE. Stacking, however, requires careful planning and an understanding of the method. For example, the field strength should be very high in the injected sample relative to the electrophoresis buffer so the drugs migrate rapidly to the solvent interface before the separation step takes place. Buffers, that do not generate much Joule heating, such as, e.g., borate, become important for this step (20). Methods have been described for stacking by: low ionic strength buffer in the sample, stacking by inclusion of acetonitrile in the sample, and isotachophoresis. Stacking in MEKC is more difficult but a few methods have been described recently.

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