Applying Capillary Electrophoresis Efficiently In The Lab: Part II
Written By: Jeff Chapman and John Hobbs, Beckman Coulter Inc., Fullerton, CA
Although capillary electrophoresis (CE) is used extensively in analytical laboratories, users still have many questions about how to employ the method and prepare samples. To answer these, representatives of Beckman Coulter (Fullerton, CA; 714-871-4848), have created a two-part report of real-world questions and answers to help users understand the benefits and methodology of CE applications. This is part II of the report. To read part I, click here.
Q: How easy is it to develop a CE method from scratch?
Q: Can HPCE be used in a "preparative mode"?
Q: How Sensitive is HPCE?
Q: Is capillary temperature control really necessary?
Q: Does HPCE require extensive sample preparation?
Q: How easy is it to develop a CE method from scratch? (Back to Top)
As with any analytical tool, the greater the flexibility in methods design, the more potentially complex the methods development can appear. In CE, users have the flexibility to define the separation "environment," so the task of developing methodology can appear quite daunting. On the surface, one must evaluate capillary temperature, buffer pH, buffer salt composition, buffer concentration, capillary surface, applied field strength, capillary length and sample injection volume. Of course, all of this must take into consideration the solubility characteristics of the analyte and the composition of the matrix with which it can be found.
As with HPLC and GC, experience simplifies the degree of methods development. The user's accumulated experience often narrows the methods-development strategy to a very small subset of conditions. In reality, most successfully implemented CE analyses require very little "methods development," as the core methodology for these applications need only to be "tweaked." A good example of this can be found with Beckman Coulter's chiral methods development strategy using highly sulfated cyclodextrins. Here, we essentially use one method to screen three different cyclodextrins for resolution of the enantiomers of interest. So far, we have achieved significant resolution of the enantiomers in 131 of 134 compounds that we have analyzed.
Q: Can HPCE be used in a "preparative mode"? (Back to Top)
The answer to this question is usually qualified by the questioner's definition of "preparative." CE as a technology is by its very definition an analytical technique. The use of the capillary format results in reduced joule heating and diffusion. This makes it possible to analyze in solution using high field strengths. The opposing side to this is that very small volumes and mass loads are introduced; limiting CE to an analytical tool. When pushed, this technique has been described as "micro-preparative;" when larger column dimensions (200 um) are used, enough mass can be collected for further analysis. This process has been routinely automated with fraction-collection algorithms that allow collection by time or detection gates.
Q: How Sensitive is HPCE? (Back to Top)
Although the use of nanolitre injection volumes is listed by many as an advantage, this same attribute becomes a liability with regards to assay sensitivity. By limiting the volume of sample introduced, you also limit the mass load required for detection. For this reason, assay sensitivity becomes a function of maximizing mass load and concentrating the separation zone. An assay's sensitivity can be impacted by more than 100-fold by simply loading larger sample volumes and concentrating the zones during a pre-separation phase using isotachophoresis. Additional strategies used to improve detection sensitivity have included the manipulation of capillary path length using bubble cells and Z-Cell arrangements.
For many routine protein applications, CE is typically operated in the ug-mg/ml concentration range. Basic pharmaceutical analysis is being accomplished at the 10 ng/ml range when incorporating extraction protocols and using selective electrokinetic injection techniques. For DNA analysis, pg-ng/ml concentrations are typically seen when using intercalator dyes and laser-induced fluorescence detection.
Q: Is capillary temperature control really necessary? (Back to Top)
The answer here is an emphatic yes! The first step to successful CE is in the definition of the actual set temperature, as this will influence viscosity impacting sample load and electroosmotic flow. The heat in the system will also impact the kinetics of any potential interaction that you are evaluating. The second step is in controlling the capillary at that defined temperature to maintain the reproducibility of migration behavior and injection volume.
Q: Does HPCE require extensive sample preparation? (Back to Top)
This is another very subjective question. As a general rule of thumb, sample preparation should be approached in the same manner as HPLC. This may involve solid phase extraction protocols to remove protein or excipients, dialysis to remove interfering salts, or buffer exchange for compatibility and solubility purposes.
Although CE appears to use much smaller tubes than traditional HPLC, the actual "pore" is much larger. In combination with the use of selective injection techniques (electrokinetic) one can quite often "get away" with more complex sample matrices than traditional chromatographic approaches. However, in the interest of assay robustness and operation in a routine environment, the cleaner the sample matrix, the simpler the methods required.
In summary, high-performance capillary electrophoresis is a valuable tool that is now finding its place as a workhorse in the analytical laboratory. Particularly for resolving highly charged polar analytes or resolve enantiomers of compounds with chiral centers, CE should be the first analytical separation technique that is used.
Additional CE questions were answered in part I of this two-part report.
For more information, contact Jeff Chapman, Beckman Coulter, 4300 N. Harbor Blvd., PO Box 3100, Fullerton, CA 92834-3100. Tel: 714-871-4848. Fax: 714-443-8283.