In this installment, guest authors and capillary electrophoresis (CE) experts Breadmore and Sänger-van de Griend bring a practical
perspective to enhancing the sensitivity of CE. They compare stacking and sweeping injection methods and provide practical
suggestions about how to enhance sensitivity for charged and neutral compounds.
Table I: In-capillary sample concentration in CE
One of the most frequently cited concerns in capillary electrophoresis (CE) is that the concentration limits of detection
are inferior to what can be achieved with liquid chromatography (LC) (1). Extensive research has been carried out to overcome
this limitation and now there are many different ways to increase sensitivity by controlling the way the sample is injected,
how it is prepared, and how the targets are separated. After all, the concentration in the sample vial does not need to be
the same as the concentration needed for detection if we can concentrate on-line. Early researchers in the field diluted samples
in the electrolyte (parallel to dissolving or diluting in eluent in LC); however, doing so dilutes the target analytes, further
reducing sensitivity. A better approach is to use the sample straight, which means that the composition of the sample plug
is different from the composition of the electrolyte. Although intuitively this may not seem like a smart idea, depending
on the exact analyte and conditions there may be a significant difference in their migration behavior in the sample zone compared
to their migration behavior in the electrolyte. Very early researchers realized that this approach could enhance sensitivity.
A brief search of the literature yields many excellent research papers and reviews (1–5), with different kinds of concentration
mechanisms occurring because of different analyte, matrix, and electrolyte properties. Table I gives an overview of the most
frequently used concentration techniques; as can be seen there are quite a number of them. It is worthwhile noting that these
are the most common single concentration approaches used. It is possible to combine multiple mechanisms to produce even more
powerful approaches; however, these are more complex. Most reviews discuss the various techniques from the mechanistic point
of view. Here, we'd like to consider these options from a practical point of view and ask the question: I have XYZ in my sample;
how do I improve my sensitivity? After all, in real life one usually cannot pick all conditions and sample matrices to suit
a theoretically useful technique; instead, one has to work from the situation at hand.
Figure 1: Concentrating in CE. Creating velocity differences of the analyte in the sample zone versus the velocity of the
analyte in the BGE. (a) Initial situation after injection and before applying the voltage. (b) Stacking, the analyte has a
higher velocity in the sample zone than in the BGE. (c) Sweeping, the analyte has a lower velocity in the sample zone than
in the BGE.
First of all, the fundamental premise of concentration in CE comes back to creating a different velocity of the analytes of
interest in the sample zone compared to their velocity in the background electrolyte (BGE) (6). There are many different techniques
based on a change in velocity. Each technique has its own abbreviation, but the only difference may be a change in direction
of the electroosmotic flow (EOF). These techniques fall into two groups: stacking techniques and sweeping techniques. If the
velocity in the sample zone is higher than in the BGE (Figure 1b), the technique is generally called stacking (7). When stacking, the analytes quickly migrate through the sample zone to the electrolyte boundary where they slow down
and "stack" together on the boundary with an increase in concentration and, thus, sensitivity (Figure 1b). In so-called sweeping techniques, in contrast, the velocity in the sample zone is slower than the velocity in the electrolyte. As the boundary
between the electrolyte and sample moves through the sample matrix, analytes are concentrated around this moving boundary
and are considered to be "swept" together with an increase in concentration and, thus, sensitivity (Figure 1c) (8).
Perspectives in Modern HPLC: Michael W. Dong is a senior scientist in Small Molecule Drug Discovery at Genentech in South San Francisco, California. He is responsible for new technologies, automation, and supporting late-stage research projects in small molecule analytical chemistry and QC of small molecule pharmaceutical sciences. LATEST: Seven Common Faux Pas in Modern HPLC