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Experiences of modern science demonstrate that the combination of what seems to be the opposite features leads to the obtainment of new unexpected results. Mainly such a combination of the properties of water (aquosystems) and “concretion” (dioxides of silica and silicon which make up the capillary), allowed to create a new method of analysis which is called capillary electrophoresis (Skoog & Holler, 2007). Basically, electrophoresis, as well as capillary action, had been known for a long time, but only a few decades ago the scientists managed to develop a new analysis method that used these phenomena in order to divide assays of complex mixtures into separate building blocks. At the current moment, capillary electrophoresis is one of the most promising methods of analysis, as it develops dynamically and receives wider applications in various fields of analytical chemistry. Simplicity and accessibility of this method, as well as indisputable advantages, especially during measurements, give hope that its methodological backing shall have dynamic development and capillary electrophoresis will soon be included in the list of physicochemical methods of analysis frequently used in everyday laboratory practice (Lin, Nathan, Keating, 2000). This method employs the phase boundary surface area properties between the two phases – liquid and solid.
Groton Biosystems, as well as homemade capillary electrophoresis instruments, are used. Capillary consists of polyimide-coated fused silica (SiO2). UV absorption detector is used to measure the emerging electropherogram peaks. The chemicals needed in this experiment are Sodium phosphate buffer (0.01 M, pH=7.56), and a sample mixture in phosphate buffer Cyanocobalamin, Nicotinic acid, Thiamine hydrochloride. Cyanocobalamin should be used with caution, as it is considered a sensitizer and an irritant.
Various materials and equipment are required in order to conduct the experiment. We shall require a SiO2 capillary, a digital multimeter, a high-voltage power supply, an integrator, a plexiglass box with HV interlock, a platinum electrode assembly, a 1 kΩ resistor, a vial holder, and a digital timer.
The sample and buffer vials are picked up from the stockroom (two sets for each instrument). The samples and buffer vials are loaded into the Groton Biosystems capillary electrophoresis instrument and the system is flushed according to the instructions. Then it is necessary to run the hydrodynamic injection and separation of the vitamin sample, 3 replicates. Afterward, the electrokinetic injection and separation of the vitamin sample should be initiated, 3 replicates. The resulting data files are uploaded to the webspace in Excel format.
Working with the homemade capillary electrophoresis instrument, the samples and buffer vials are loaded, and the system is set up according to the instructions. After this, we run the electrokinetic injection and separation of the vitamin sample, 1 replicate. Following this, we conduct the peak width for an electrokinetic injection. The next step includes running the hydrodynamic injection and separation of the vitamin sample, 1 replicate.
Let me turn your attention to the processes that occur on the interphase boundary between the inner surface of the silica capillary and the water-electrolyte solution that fills up the capillary. The newly formed surface of the molten SiO2 contains mainly siloxane groups. Having contacted water or water solution vapors, the siloxane groups which possess olefinic bonds become unstable and acquire a molecule of water, forming silanole groups. During the contact of water with the SiO2 surface, the silanole groups dissociate with the loss of H+ ions. The degree of breakdown depends on the temperature as well as the contents of the aqua solution, in particular, on the level of pH. When pH is >2.5, dissociate silanole groups are emerging on the SiO2 surface and form a negative surface charge. Dissociated ions located on the SiO2 surface as well as in the electrolyte are hydrating. Due to the forces of electrostatic interaction, the oppositely charged hydrated ions on the surface and in the fluid get attracted. The reacting forces are so great that ions (some cations and remaining silanole groups) partially lose the hydrating water. As a result, the first layer of cations, directly adjacent to the surface loses mobility and bonds. As the “fluffy” hydrated cations are unable to fully settle in form of a monolayer and fully compensate the negative charge of the surface, some fraction of the cations that neutralize the negative charge moves back into the solution and forms a charge which is dispensed in the fluid volume adjacent to the phase boundary, and due to lower surface bonding energy has the ability to travel. Disregarding strong electrostatic interaction, the charge recombination does not occur. Resulting from this, the interacting systems of charges form a double electric layer that as if consists of two isolated from one another capacitor arms with each charged oppositely. One of the arms is formed by negatively charged scraps of silanole groups, whereas the other one consists of two parts – a motionless layer of cations directly adjacent to the silica surface, and a diffuse layer formed by the cations within the fluid volume. The distribution of cations between the motionless and diffuse layers, and consequently the thickness of the double electric layer depends primarily on the general electrolyte concentration in the solution. The higher is the concentration, the larger fraction of the diffuse layer’s positive charge moves into the motionless layer, and the thinner is the diffuse layer. With the concentration of binary monocharged electrolyte of 10-3…10-4 M, the double electric layer is 30-50 μm thick. Let us roll the examined surface into a tube with an inner diameter of 50-100 μm. In this case, almost all fluid that fills it will present a diffuse part of the double electric layer. A tube of such diameter is called a capillary. If we were to apply electric field along the capillary’s axis, there will be longitudinal movement of free electric charge carriers within the capillary in opposite directions, and as there is excessive ion concentration in the diffuse part of the double electric layer, the number of ions moving to cathode shall be significantly greater, and their movement will drag all the other fluid mass in the capillary. In this case, we are dealing with electro-osmotic volume flow directed at the cathode, which will perform passive diffusion of the solution inside the capillary. Consequentially, there is directed movement of fluid mass in the capillary caused by the voltage differences, at this all fluid moves at the same speed. This is an essential condition that allows obtaining high accuracy grade of the method.
Emphatically, in the framework of this experiment, we had to reject considering various techniques and methods developed in the chemico-analytical practice in terms of capillary electrophoresis. For example, in order to register signals during capillary electrophoresis, we could use linear photodiode array detectors, mass-spectrometer detectors, and so on. In terms of analytical methods, micellar electrokinetic chromatography, capillary electrochromatography, capillary gel electrophoresis, and capillary isoelectric focusing could be used for such experiments.
Skoog, D.A.; Holler, F.J.; Crouch, S.R “Principles of Instrumental Analysis” 6th ed. Thomson Brooks/Cole Publishing: Belmont, CA 2007.
Lin H.; Natan, M.; Keating, C. Anal. Chem. 2000, 72, 5348-5355.