Methods of detection
Equipment set up
Application of CE to toxin detection in past studies
Capillary Electrophoresis is a separation technique that presents scientists with lots of advantages over other separation methods. In this review, the various modes of capillary electrophoresis and different detectors coupled to it were discussed. Toxins commonly found in food is presented as well, with emphasis on natural toxins such as mycotoxins, bacterial toxins, and paralytic shellfish toxins. Finally, applications of capillary electrophoresis for toxin analysis in different studies were summarized.
Keywords: Capillary electrophoresis, toxins, detectors, natural toxins
Food analysis may be viewed as a two-step activity involving separation stage and detection step. The effectiveness of food analysis depends on the accuracy and precision of both steps. Food ingredients and food products are analyzed for several reasons including determination of nutrient composition, evaluation of quality attributes, and detection of hazardous substances such as toxins.
Certain substances of natural origin in food pose significant health risk when ingested. These substances are known as toxins. Processing and handling of foods can also result in accumulation of toxins in foods thus toxins may range from microbial sources such as mycotoxins, neurotoxins in shell fish to toxins that accumulates during growth such as glycoalkaloids in potatoes and in processing such as acrylamide.1 Rapid detection and quantification of toxins in food is important because of the health hazard associated with it. Methods that have been used in detection of food toxins have been reported and they include biological assays such as ELISA, bioluminescence assay and protein phosphatase inhibition assay, high performance capillary electrophoresis, chromatographic methods like HPLC and GC/FID.2 Amongst these analytical techniques, capillary electrophoresis can be regarded as a more efficient and fast method of toxins determination when coupled to a detection system.3
Capillary electrophoresis was introduced in the 1980s and started gaining popularity recently because of its versatility as a separation method. It is used in the analysis of multiple components of food, including ionic and non-ionic components of food.3
Several advantages associated with capillary electrophoresis over other separation methods include versatility of application. This implies that a wide range of analytes such large biomolecules like DNA, inorganic compounds and organic molecules can be separated using this method. Also, the possibility of employing different modes of separation on the same piece of equipment as well as its ability to interface with different detection systems have increased the use of capillary electrophoresis in analytical chemistry. Other advantages such as minimal use of sample, solvent and ruggedness of capillary electrophoresis have been reported. 4, 5
Although capillary electrophoresis presents analyst with high separation efficiency and fast separation, its main drawback is its poor sensitivity due to its low dimension. There will be a compromise with the resolution of CE if volume of injected sample is increased to improve sensitivity. Other methods of improving sensitivity of capillary electrophoresis which largely involve the pre-concentration of samples prior to separation with CE have been reported. These include solid phase extraction, sweeping and field amplified stacking.6
This articles gives a comprehensive review of the application of capillary electrophoresis in the separation and quantification of various toxins in foods.
Capillary electrophoresis is a collective term for methods used in separation of molecules through narrow tubes when electric field is applied. The difference in migration of the charged molecules results in separation of analytes in a sample.3 Based on this, capillary electrophoresis is largely used for the separation of chemical compounds with similar structure.7
Before the introduction of capillary electrophoresis, the traditional electrophoretic method was first described by Tiselius in 1937 and it was used to separate protein fractions in a sample placed in buffer solution with applied electric voltage. However, the separation was poor due to thermal convection effect. To mitigate this, slab gels were introduced to the electrophoretic system leading to the invention of gel electrophoresis with relatively higher separation efficiency when compared to the traditional electrophoresis but lower separation efficiency when compared to HPLC and GC. To improve the separation efficiency and analysis time of gel electrophoresis, narrow tubes were introduced to the electrophoresis system for rapid heat dispersion, thus capillary electrophoresis can be viewed as an improvement of the slab gel electrophoresis system.3
Different modes of separation are employed in capillary electrophoresis depending on the type of analytes. Capillary zone electrophoresis (CZE) is a separation mode that involves migration of charged molecules (positive and negative) to the terminal end of a fused silica capillary filled with electrolyte. This is caused by the action of electro-osmotic flow. The velocities at which the ionic species migrate within an electrophoretic system differs and can be calculated by mathematical relation. This velocity of migrating species is largely dependent on electrophoretic mobility and applied electric field. This implies that charged species tend to move faster at higher electric field. Also, the choice of buffer affects the efficiency of separation because pH and composition of buffer affect mobility of charged species.5 The various parts of capillary electrophoresis is shown in figure 1.
Although the separation efficiency of CZE is high, however, it has the drawback not being able to separate neutral molecules. To overcome this, Micellar electro kinetic chromatography (MEKC) was developed. Chromatography principle is employed for the separation of neutral molecules using a pseudo-stationary phase by adding surfactant such as sodium dodecyl sulfate to the background electrolyte thus MEKC is used for separating both neutral and charged molecules. Other surfactants that can be used include bile salts and quaternary ammonium salts. 3,5
Capillary electrochromatography is a less common CE mode although high separation efficiency is achievable through this mode. It is unique because of the striking similarities it shares with a regular chromatographic method in that it uses a silica packed column as stationary phase with electro-osmosis being responsible for the mobile phase flow.8
Capillary Isotachorphoresis (CITP) is another peculiar CE separation mode in which ionic compounds are distributed in discrete zones between a leading electrolyte and terminating electrolyte with different flow velocity while the discrete zones move at the same velocity. Steps are used to denote analytes concentrations on an isotachopherogram in capillary isotachorphoresis mode as oppose to CZE mode where analytes intensities are represented by peaks on electropherogram. 3,8
The possibility of separating molecules based on their Isoelectric point is the principle employed by Capillary Isoelectric focusing mode of CE.it is based on the principle that when a capillary is filled with protein sample along with a solution that can create pH gradient. The instrumental set up is such that the anodic end of the capillary is inserted into an anolyte while at the same time the cathodic end is immersed in catholyte. The protein sample becomes separated after migration to a zone in the capillary where it has neutral pH.8
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Figure 1. Diagrammatic representation of capillary electrophoresis
Methods of detection
Since CE is essentially a separation method, it cannot be used to determine the concentration of an analytes, thus it is usually coupled to a detector that can quantify the amount of analyte in a sample. CE has the drawback of tiny peak volumes because of the very small amount of sample injected in to the capillary, thus a sensitive detection method of detection is required to give accurate concentration of analyte.4
Several methods of detection are available to CE but UV-VIS detector is the most commonly used. These techniques include laser induced fluorescence, electrochemiluminiscence, conductometric detection, refractive index detection, and amperometric detection.
UV-Vis detector can be considered as in column detector because they are coupled directly to the CE equipment and detection is done by creating a window space on the column by scraping off the polyimide coating on a section of the CE. The observed drawback of UV-vis detector is obvious because of low dimension of capillary. This results in low concentration sensitivity as well as short linear range. The use of z-shaped capillary and bubble cells have been suggested as means of improving the path length in UV-vis detector6
Laser induced fluorescence (LIF) is a very sensitive in column detector coupled to CE, with advantage of selectivity. High energy excitation is possible with Laser induced fluorescence leading to high radiation output. This is the reason behind the sensitivity of LIF. However, the instrumentation is usually complex, expensive and it requires appropriate sources of laser. 2,6
Micellar electrokinetic chromatography (MEKC) coupled to fluorescence detection has been used successfully to detect paralytic shellfish poisoning toxins in shellfish. In the same vein, detection of tetramine, a toxin found in whelks was detected with Electron Spray Ionization-Mass Spectroscopy (ESI-MS) after separation with capillary electrophoresis7
Electrochemiluminiscence detector have also been coupled to CE for detection of analytes. However, they are mostly applicable in analytes containing tertiary amine group. At the electrode surface, analytes in the CE effluent reacts with Ru(bpy)32+ and give rise to an electropherogram based on the characteristics of the analytes. 8 Capillary electrophoresis coupled to electrochemiluminiscence detector is shown in figure2.
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Figure 2. Capillary electrophoresis coupled to electrochemiluminiscence detector
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7. Keyon, A.S.; Guijt, R.M.; Gaspar, A.; Kazarian, A.A.; Nesterenko, P.N.; Bolch, C.J.; Breadmore, M.C. Capillary electrophoresis for the analysis of paralytic shellfish poisoning toxins in shellfish: comparison of detection methods. Electrophoresis 2014, 35, 1496-1503.
8. Li, S.F.Y. Capillary electrophoresis: principles, practice and applications. Elsevier: 1992; Vol. 52,
- Quote paper
- Oladimeji Adewusi (Author), 2017, Application of Capillary Electrophoresis in quantification of toxins in food, Munich, GRIN Verlag, https://www.grin.com/document/367200