Pharmaceutical Analysis Blog

Detection in Capillary Electrophoresis (CE)

We went through the fundamentals, the separation medium BGE, the capillary and injection in capillary electrophoresis. In this article of CE Solutions we will reach the end of the system, the detector. We will discuss the most popular detection principles in use in industry and CE-related good working practice.

UV Detection
The usual detection techniques for liquid separation are also available for CE and UV detection is by far the most common. Modern equipment comes with build-in UV/VIS single wavelength or diode array detectors, specially designed to focus the beam of light into the capillary. Absorption is a function of the path length (Lambert-Beer’s law). As we detect on-column in CE, the path length is directly related to the capillary diameter. Compared to other liquid separation techniques, this means that the path length is rather short, which limits detection sensitivity. The loss in sensitivity from the path length is partly compensated for by the high efficiency in CE and by the possibility to use low UV wavelengths. High efficiency means reduced band broadening and a higher concentration in the sample bands. With the aqueous background electrolytes (BGEs) that we usually employ in CE, we can go down to 190–200 nm for UV detection. Such low wavelengths usually mean a significant gain in sensitivity and wider applicability. Thirdly, we learned in the past years a lot about injection stacking techniques. With stacking, low sample concentrations end up in higher detection concentrations (CE Solutions, Issue 5).

Indirect UV Detection
For compounds that do not exhibit UV absorption, indirect UV detection can be used. Indirect UV detection is universal and non-selective. In the BGE, an absorbing co-ion is dissolved, also called the UV-probe. The non-absorbing analyte band displaces the absorbing UV-probe. So at the position of the non-absorbing analyte band there is less absorption and the signal will show a negative peak. The amount of displacement depends on the charge of the UV-probe and the analyte ions and on their mobilities. A measure of the amount of displacement is called transfer ration. The highest transfer ratios are obtained when the mobility of the UV-probe matches the mobility of the analyte. Moreover, the mobilities of the analytes compared to the UV-probe will determine the shape of the peaks. If the mobilities are similar, symmetric peaks are obtained. If the mobilities differ from the mobility of the UV-probe, electromigration dispersion occurs and we observe the typical triangular peaks. Consequently, the UV-probe should match the analytes at hand.

   The advantages of indirect UV detection are its universal and non-selective applicability and the possibility to determine absorbing and non-absorbing analytes simultaneously. The disadvantages are that analytes can show up as positive and negative peaks and that you cannot use the diode array detector for additional identity confirmation. For identification of the analytes it is therefore important that the migration times are highly reproducible. To increase reproducibility, the use of an internal standard and comparing relative migration times or relative mobilities is recommended.

   There are several commercially available kits for the indirect UV detection of small cations and anions, organic acids and forensic anions, for example. Typical applications are drug counter ions, fermentation broths, carbohydrates, metal ions and explosive residues etc.

 CE Solutions #6: Detection in Capillary Electrophoresis

Fluorescence Detection
A fluorescence detector is almost as straight forward to use as a UV-detector. The fluorescence detector comes as a module that can be exchanged with the UV or DAD module within the instrument or you can add a module to your instrument. For a good detection signal, a strong light source is needed for excitation. For many years this has been a laser (laser induced fluorescence LIF), but now also LEDs are appearing on the market.

 CE Solutions #6: Detection in Capillary Electrophoresis-2
Figure 1: Schematic representation of the sheath flow and sheathless interface for coupling capillary electrophoresis to mass spectrometry.

   The advantages of LIF detection are its sensitivity and its selectivity. Its selectivity is not only an advantage, but can also be a disadvantage. Either the analytes of interest need to show native fluorescence, or labelling is required. Many labelling reagents have been developed over the years and are commercially available. Usually, labelling requires derivatization during the sample preparation.

 CE Solutions #6: Detection in Capillary Electrophoresis-3
Table 1: Changing the capillary diameter has a huge influence on the amount injected. In this table you see different settings for maintaining the same injected volume or injection plug length. For convenience, pressures are given both in mbar and psi.

Contactless Conductivity Detection
Contactless Conductivity Detection (CCD) is rapidly gaining foothold within industry. The CCD detector is now commercially available with a simple mechanical construction and is relatively cheap. Two cylindrical electrodes, the actuator and pick-up electrode, are placed around the capillary, without the need to remove the polyimide coating. The capillary does not need to be fused silica, but can be of any non-conducting material, such as PEEK®, Teflon® etc. Also rather small internal diameters, such as 10 µm, can be used. The detector measures the difference in conductivity between the BGE and the analyte bands. For optimal sensitivity, the difference in conductivity between BGE and analyte should be as high as possible. So for CCD, zwitterionic buffers with low conductivity, such as a MES-Histidine buffer, are frequently used. Alternatively you can apply indirect conductivity detection if the conductivity of the analytes is low. Contactless conductivity detectors can easily be used in parallel to other detectors such as UV/DAD, LIF or MS.

 

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