Analytical Chemiluminescence/Chemiluminescence detection in capillary electrophoresis
D11. Chemiluminescence detection in capillary electrophoresis edit
Capillary electrophoresis has outstanding resolving power for extremely small samples, but this poses a challenge for detectors. Compared with other candidates, chemiluminescence detection has the advantages of being highly sensitive and requiring inexpensive equipment of simple design. In addition it is not affected by the high voltage used in the separation system, a particular problem for electrochemical detection, which is also highly sensitive. Ultra-violet absorbance detectors also have low cost and are widely used, but narrow capillaries make it difficult to arrange for long enough optical path lengths. Laser induced fluorescence has high sensitivity, but the equipment is costly and pre- or post-column derivatization of nonfluorescent analytes is necessary.[1]
Figure D11.1 – On-column coaxial flow interface between capillary electrophoresis and chemiluminescence detection (adapted from reference D9.1).
As with HPLC, there is an inherent problem of compatibility between the conditions needed for separation and those needed for chemiluminescence. Additionally, there is a potential problem with the stability of chemiluminescence reagents. Both of these are addressed by using the post-column mode rather than pre-column. Post-column interfaces are devices for mixing the eluent with the chemiluminescence reagents and for this purpose designs may make use of merging flow, coaxial flow or reservoir mixing. Interfaces may also be classified as off-, on- or end-column, depending on the site of detection and on whether this is isolated from the high voltage supply used for capillary electrophoresis.
The simplest interface, off-column and merging flow, did not find widespread application. Buffer flowed from a reservoir through the separation capillary and merged with the reagent at a four-way connector at the end of the column. The outlet arm carried the mixture to the chemiluminescence reaction coil and flow cell adjacent to a photomultiplier, while the fourth arm connected through a semi-permeable membrane to a second buffer reservoir (containing the ground electrode) immediately downstream of the merging point. This arrangement isolates the high voltage from the detection zone.
In contrast, an on-column coaxial flow interface has proved to be effective for a large number of applications.[2] Figure D11.1 shows the detector is located at the capillary outlet tip, so detection is “on-column”. The ground electrode is located in the effluent reservoir so that detection takes place within the high voltage zone. The separation capillary outlet is inserted coaxially into the reaction tube, giving rise to minimum turbulence and reproducible mixing.