Introduction to Capillary Electrophoresis
Capillary Electrophoresis (CE) is one of the possible methods to analyse complex samples. In High Performance Liquid Chromatography (HPLC) and Gas Chromatography (GC) the separating force is the difference in affinity of the sample components to a stationary phase, and or difference in boiling point. With both techniques the most important factor is the polarity of a sample component. In CE the separating force is the difference in charge to size ratio. Not a flow through the column, but the electric field will do the separation.
In Capillary Electrophoresis a capillary is filled with a conductive fluid at a certain pH value. This is the buffer solution in which the sample will be separated. A sample is introduced in the capillary, either by pressure injection or by electrokinetic injection. A high voltage is generated over the capillary and due to this electric field (up to more then 300 V/cm) the sample components move (migrate) through the capillary at different speeds. Positive components migrate to the negative electrode, negative components migrate to the positive electrode. When you look at the capillary at a certain place with a detector you will first see the fast components pass, and later on the slower components.
Figure 1: Principle of Capillary Electrophoresis
Mobility
As mentioned before the speed of a component (the mobility) is dependable on size and charge. The size is a combination of the sample component and the shield of water that is bound to the component. Even a small ion (as Fluoride, F-) can be big due to a large water shield. In general, the bigger the component, the slower it will migrate through the buffer.
The charge of ions can be strongly dependent on pH value. That is the reason why a buffer at certain pH is used for separations. For example Acetic Acid (pK value 4.756) will be almost completely negative charged at pH 7. The mobility (speed) of the acetic ions will be big. At pH 3, where about 80 % of the acetic acid is neutral, the mobility will be much lower. By changing the pH of a buffer system, the mobilities of the different components can be altered to achieve the best separation. In general the best pH for a separation is between the pK values of the sample components.
Endo Osmotic Flow (EOF)
In most applications the capillary that is being used is made of bare fused silica. This material has at its surface silanol groups (Si - O - H). These groups are slightly acidic. In buffers at higher pH value there are a lot of negative charges at the capillary wall (Si – O-). In the buffer fluid positive charges will be present because of the law of electrical neutrality. When a high voltage is generated over the capillary, these positive charges will start to migrate through the capillary towards the negative electrode. They will drag along the buffer fluid with them. This flow is called the (Electro) Endo Osmotic Flow (EOF). It is well possible to calculate a mobility of this EOF. The higher the pH, the more negative charges on the capillary wall and the more positive charges in the fluid. This will generate a stronger EOF.
Because the positive charges are all located close to the capillary wall, and there is no pressure force in the middle of the capillary, the flow profile of the EOF is completely flat. This will cause no peak broadening like the parabolic flow profile in HPLC and GC, and that is one of the reasons why such a high resolution can be achieved in CE. As mentioned before the EOF is towards the negative electrode. This flow drags along neutral components (which would not migrate without any fluid flow), and even positive components, whose mobility is lower than the mobility of the EOF, will migrate towards the negative electrode. In this way in one run negative, neutral and (slow) positive components can be separated and detected.
Different modes of Capillary Electrophoresis
In the paragraphs below some general used modes of capillary electrophoresis are explained.
Capillary Zone Electrophoresis (CZE or FSCE)
Capillary Zone Electrophoresis (CZE), also known as free-solution CE, is the most standard form of CE. Buffer is flushed through the capillary by pressure, sample is injected and high voltage is applied. Dependable on the polarity the EOF is towards the inlet or the outlet. Each sample component will migrate through the capillary at its own speed. Only the difference in mobility will cause the separation.
Capillary Gel Electrophoresis (CGE)
With this technique there is a gel matrix inside the capillary. Components with different size but the same mobility are separated with this technique. Components of bigger size will be slowed down more by the gel, and will migrate later through the capillary. Especially with protein and DNA separations this technique is frequently used.
Micellar ElectoKinetic Chromatography (MECC or MEKC)
In this way of electrophoresis micelles are generated in the buffer. These micelles have a non polar inside, and a polar (or charged) surface. Sample components in the buffer solution will be divided over the micelles and the buffer solution, dependable on the affinity to the micelles. Just like in HPLC and GC, there will be a certain stable diversion between buffer and micelles. When the migration speed of the buffer differs from the speed of the micelles, it is possible to separate different components on the fact that there is a different affinity for the micelles. In this way, there is a lot of synergy with HPLC and GC.
Non-Aqueous Capillary Electrophoresis (NACE)
With this technique components that are insoluble in water are separated, mainly depending on the use of organic solvents. The viscosity and dielectric constants of organic solvents affect both sample ion mobility and the level of electroosmotic flow.
Iso Electric Focussing (IEF)
When a pH gradient is applied across the capillary, and a voltage is applied from positive voltage at low pH to negative voltage at high pH, components will migrate to the pH value that equals their pI value. At lower pH value, the components are positively charged, at higher pH values the components are negatively charged. In this way, each component migrates to a different position in the capillary. When pressure is applied, the complete pH gradient moves through the capillary, and subsequently the components will pass the detection window.
Capillary Electro Chromatography (CEC)
With this technique a capillary is partly packed with silica based particles with a stationary phase. When high voltage is applied over the capillary, the buffer fluid will start to migrate due to the EOF that is present because of the silica. The sample will have, just as in HPLC, more or less affinity for the stationary phase. This is the separating force in this technique. The only difference between HPLC and CEC is that not a pressure pump is being used to force the mobile phase through the packed bed (HPLC), but a high voltage is used for this purpose.
Electro Chromatography (EKC)
With this technique there is a differential interaction of enantiomers with the cyclodextrins, which allows the separation of chiral compounds. This enantiomer analysis is used for the analyses of natural products, such as pharmaceutical/herbal products, toxicology compounds, food and food contaminants, forensic, fingerprinting and many more.
Capillary IsoTechoPhoresis (ITP)
With this technique two kinds of buffers are used. One buffer with high mobility as a leading buffer and one buffer with very low mobility as a terminating buffer. The mobility of the sample components must be between the leading and terminating. In the stable situation, all components migrate through the capillary at the same velocity (hence the name itsotacho=same speed). In the figure, two components A and B are positioned between L(eading) and T(erminating). For the mobilities: m(L)> m(A)> m(B)> m(T).
Because all components have the same speed, there will be different electric fields in each zone, as the law v=m*E is everywhere valid. The electric field in the Terminating zone will be highest. These differences in electric field result in the self-correcting behaviour. If a component (say B) is for some reason (diffusion) in zone A, it will be under a higher electric field giving it a higher speed. The result is that the ion migrates back into it's own zone. The same if it would be in the Leading zone, where it is in a lower electric field. The velocity is lower, and it will be caught by the quicker moving zone of B.
In addition, because of electrical laws, the concentrations are related to the concentration of the Leading electrolyte. For this reason, small concentration samples are strongly concentrated into very narrow zones. Especially this effect is used quite often to concentrate large volume injections.