25 Capillary Electrophoresis

Dr. Varinder Kaur

Objectives: To study the basics of capillary electrophoresis and know the following questions.

  1. What is capillary electrophoresis?
  2. How it works?
  3. What type of detectors is used in capillary electrophoresis?
  4. What are the main applications of capillary electrophoresis?

1. Description

The capillary electrophoresis (CE) technique was introduced in late 1800’s employing experimentation with the help of glass U tubes. Later, it was explored by Arnes Tiselius (1930) for the separation of proteins, Hjerten (1960’s) for the use of capillary and Jorgenson and Lukacs for the separations of both inorganic and organic compounds. The replacement of U tube with capillary having thin dimensions enhanced the surface to volume ratio and increased the efficiency making the separation better. This led to exhaustive use of this technique for the separation of components in various fields. The capillary electrophoresis uses an electric field for the separation of components of a mixture within the narrow dimensions of a tube.

Principle

It is based upon the separation of charged molecules under the influence of electric field. The principle can be illustrated in more simple way as given below.

It is well known that ionic molecules can have positive or negative charge and in the absence of electric field, the charge is cancels to form an uncharged molecule (A). The uncharged molecules move randomly in the solution phase. However, under the influence of electric field, the charges are attracted by the oppositely charged electrodes and they start moving. This decreases the randomness and leads to the separation of charges on the two opposite poles (B). The positively charged ions move towards the cathode (-vely charged electode) and negatively charged ions move towards the anode (+vely charged electrode). In this way, the oppositely charged components can be separated by electrophoresis. Secondly, the size of the cationic and anionic components helps in the separation of similarly charged species. In other words, capillary electrophoresis offers separation of species on the basis of their electrophoretic mobility under the influence of applied voltage. The separation in capillary electrophoresis depends upon various factors such charge on the species, viscosity, applied field and size of the species. The charge and size of the species affect the electrokinetic mobility like more charge and smaller size of the species enhances the migration rate of species. Similarly, high field strength increases the electrokinetic mobility.

2. Instrumentation

The instrumentation of capillary electrophoresis system consists of various components like a high-voltage power supply, a capillary tube, a detector and an output device. The high voltage power supply is connected to the electrodes, which induce an electric field to initiate the migration of the sample from the anode to the cathode through the capillary tube.

The CE capillary is made up of fused silica (with polyimide coating) having circular cross-section with 10-100 m inner diameter and 40-100 cm length. The two ends of the capillary tube are dipped into different solvent reservoirs containing platinum electrodes. The sample is introduced into the first reservoir which enters into the capillary from one end. For this, it is dipped into the sample vessel and then the sample is forced to enter into the capillary either by pressure at the first end or applying vacuum at the detector end. Alternatively, sample is introduced by electroosmotic flow. Firstly, the capillary is flushed with desired buffer solution and then, the sample is introduced. When a high voltage is applied across the fused silica capillary, solvent migrates towards the cathode. This flow is attributed to the formation of electrical double layer at the silica-solution interface. Under weakly acidic conditions (above pH 3), the silanol groups (Si-OH) ionize to form negatively charged groups (Si-O-) which interact the positively charged buffer species. Out of the positively charged species collected at the capillary surface, the outer layer cations are attracted towards the negatively charged cathode. This drags the solvent (because these species are solvated) towards the cathode and leads to the introduction of sample with electroosmotic flow. The electrophoretic movement of the species is used for the separation and then detection by a suitable detector.

3.  Detectors used in Capillary Electrophoresis 3.1 UV-visible absorption detection

The UV-Vis absorption detection is one of the highly suitable techniques for the detection of analytes in capillary electrophoresis. In UV-based detection, aTeflon coated or polyamide coated capillary acts as reference cell. Furthermore, to improve the path length of the reference cell, its shape can be modified to form a bubble shape cell tube or additional tube can be introduced at the point of detection.

 

3.2 Fluorescence emission detection

Fluorescence detection of the analytes separated in capillary electrophoresis provides some benefits fast response and improved limits of detection. However, it can be used only for the fluorescent analytes and the non-fluorescent analytes require pre-derivatization in order to detect the separation. The set-up for fluorescence detection in a capillary electrophoresis system is complicated.

3.3 Mass spectroscopy detection

Nowadays, CE instruments use mass spectrometry as a detection tool because of their high sensitivity and much lower detection limits. The best ionization method is electrospray ionization for capillary electrophoresis coupled with mass analyzers. It has been found that small-diameter (i.e., 5-mm internal diameter.) separation capillaries provided a 25-50-fold sensitivity as compared to larger ones due to the decrease in mass flow of buffer and improved ionization efficiency in smaller capillaries. In addition, cetyltrimethylammonium chloride in the buffer also enhances the separations. In MS detection, outlet end of the CE capillary is inserted into the electrospray interface. The liquid emerging from the capillary mixes with the flowing gas stream and nebulized into spray. The vaporized and ionized sample is delivered to the MS detector. The sample is scanned by selecting a range of mass values and components are detected. Usually, volatile buffer systems are used with MS like ammonium formate.

3.4 Other detector methods

Other detection systems include refractive index detectors, amerometry, conductometry and potentiometry.

4. Modes of separation in capillary electrophoresis

The various modes to use capillary electrophoresis are summarized below.

4.1 Capillary Zone Electrophoresis (CZE)

Capillary zone electrophoresis follows the same principle, working and the details discussed above. In simple words, it deals with the use of open capillaries and relatively low viscosity buffer systems. The components move from one end of the capillary to the other as driven by the contributory effect of electrophoresis and electroosmotic mobility.

4.2 Capillary Gel Electrophoresis (CGE)

Capillary gel electrophoresis (CGE) is based upon the same principle but it involves the separation based on viscous drag. In this mode, a gel or viscous solution is filled in the capillary. This prevents the electroosmotic flow and the components are separated on the basis of electrophoresis only. The viscosity of the gel hinders the movement of larger molecules as compared to smaller molecules therefore, the components are separated on the basis of their size. This method is used for the separation of DNA molecules. The DNA molecules in CZE move with similar velocities irrespective of the size, however, in capillary gel electrophoresis the longer chains move slowly and get separated from the shorter chains.

4.3 Capillary Isoelectric Focusing (CIEF)

Normal ionic compounds consist of equal number of positively and negatively charged groups at a specific pH. This point is called as isoelectric pH or pI. At this point, the molecule behaves as a neutral species due to the cancellation of charge. Therefore, the molecules are unable to move in the influence of electric charge. In this mode of CE, special reagents called ampholytes are added to create a pH gradient within the capillary. Generally, the ampholytes are mixtures of buffers with a range of pKa values and they arrange themselves in the electric field according to their pKa value. Thus, the analytes introduced into this gradient move towards the point where pH is equal to pI and the movement of neutral species ceases until the pH gradient is stable.

4.4 Capillary Isotachophoresis (CITP)

It involves the introduction of sample plug between two different buffers having similar charge. On buffer has high mobility and acts as leading electrolyte however another buffer has low mobility and acts as trailing electrolyte. Under the high voltage, sample ions form discrete zones. The concentration of the analyte can be calculated from the length of the zone which is proportional to the concentration within the zone. This kode is generally used with conductance detection.

4.5 Micellar electrokinetic capillary electrophoresis (MEKC)

In order to analyze the un-charged molecules by electrophoresis, some agents are added in the buffer to facilitate the movement of un-charged species. In MEKC, a charged detergent (such as SDS) is added to the separation buffer, which allows the formation of micelles. The micelles are orientations of molecules in which the inner core is formed by hydrophobic ends and outer core is formed by hydrophilic ends. Therefore, the analytes interact with the micelle and migrate in the capillary with the migration rate of micelle. The time spent by the analyte in a micelle helps in the separation of the analytes. This mode is used for the separation of drugs, pesticides, food additives, etc.

4.6 Chiral Electrophoresis

This mode is used to separate the stereo-specific forms of a molecule i.e. enantiomers. The enantiomers have identical molecular weight and chemical formula but differ in the arrangement of the atoms in space. In this mode, a chiral selector is incorporated into the CE buffer, which interacts differently with the enantiomers and forms complex. The complex of the analyte and the selector migrates at a different rate depending upon the extent of their complexation. This leads to the separation of the two enantiomers.

4.7 Non-aqueous Electrophoresis

The modes discussed above employ aqueous media for separation, however, the electrophoresis performed by using non-aqueous systems (such solvents as acetonitrile, methanol, formamide, and dimethylformamide) added with the small amounts of acids or buffer salts can also be used. Some analytes are difficult to solubilize in aqueous systems but dissolve readily in organic solvents, thus non-aqueous CE offers an alternative for analytes that are difficult to separate under aqueous conditions. The analytes separated by this mode include drugs, dyes, preservatives, surfactants, and inorganic ions.

4.8 Capillary Electrochromatography (CEC)

It is a combination of liquid chromatography and electrophoresis. It involves partitioning of components between a stationary and a moving phase. In this mode, capillaries are packed with particles similar to HPLC columns and electroosmotic flow is used to drive the sample and mobile phase down the column. This improves the separation efficiency over that of the laminar flow of pressure driven systems. This mode is used to separate polyaromatic hydrocarbons.

5.  Applications of CE

5.1 Inorganic Analysis

Capillary electrophoresis has been successfully used for the analysis of inorganic cations and anions. The CE wall coatings facilitate the inorganic analysis. Especially, bonded phases improve the control of electro-osmotic flow with buffer additives. Most popular method for inorganic analysis involves derivatization of ions using organic chelators like cyanide, 8-hydroxyquinoline-5-sulfonic acid, 4-(2-pyridylazo)resorcinol, lactate, EDTA, and a-hydroxyisobutyric acid.

5.2 Organic Analysis

CE can be used to analyze small organic molecules. Numerous applications of CE in organic analysis have been listed in the past. For example, analysis of two retinoic acid isomers and their degradation products, dihydroxybenzoic acids, aromatic sulfonic acids, catecholamine metabolites, homovanillic acid and vanillylmandelic acid, mixture of oxalate, tartrate, malate, succinate, lactate, acetate, propionate, butyrate, and caprylate anions.

5.3 Surfactant separation

The quaternary ammonium compounds have been analyzed using CZE-UV absorbance detection. The separation can be improved by the addition of some additives.

5.4 Dye Analysis

CE has been successfully used for the analyses of anionic dyes and cationic intermediates with the help of borate-SDS buffers. For example; basic dyes, reactive dyes, acid dyes, and disperse dyes and pigments.

5.5 Food and Agriculture analysis

CE has found enormous applications in the food and agriculture chemical analysis. For example, analysis of essential oil extracts, pungent food components,etc.

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