ELECTROPHORESIS

Table of Contents
Table of Contents........................................................................................................ 2 INTRODUCTION:.......................................................................................................... 3 ELECTROOSMOTIC FLOW:............................................................................................3 BASIC PRINCIPLE AND OPERATION...............................................................................4 DIFFERENT FORMS OF ELECTROPHORESIS..................................................................4 Slab Electrophoresis.................................................................................................5 DNA Gel Electrophoresis..........................................................................................5 SDS-PAGE Gel Electrohpresis.......................................................................................7 Two-dimensional SDS-PAGE Gel Electrophoresis.......................................................8 CAPILLARY ELECTROPHORESIS....................................................................................9 a) Sample Introduction.............................................................................................9 b) Detection...........................................................................................................10 Capillary Zone Electrophoresis..................................................................................10 Capillary Gel Electrophoresis (CGE)...........................................................................10 Capillary Isotachophoresis (CITP)...............................................................................11 Capillary Isoelectric Focusing (CIEF)..........................................................................11 CAPILLARY ELECTROCHROMATOGRAPHY...................................................................11 SUMMARY.................................................................................................................. 12

Page 2 of 12

ELECTROPHORESIS

INTRODUCTION:
We know that positively charged ions (cations) move towards cathode and negatively charged ions (anions) move towards anode. Thus, charged particles can move towards respective electrodes and the direction is decided according to their charge. Electrophoresis is a separation technique based on the migration of ions in an electric field. It is one of the most important techniques in analytical chemistry. The positively charged ions migrate towards a negative electrode and the negatively charged ions migrate towards the positive electrode. Ions have different rates of migration depending on their total charge, size, and shape and can, therefore, be separated by this technique. This separation technique was developed by Swedish chemist Arne Tiselius and he was awarded Nobel prize in the year 1948 for his valuable contributions. This versatile technique is now-a-days applied to separate several species like drugs, inorganic ions, carbohydrates, amino acids, peptides, proteins, nucleic acids, etc. In fact, there have been innumerable applications of this technique in the biotechnological research and industry. In electrophoretic separation technique, a small amount of sample is allowed to flow through a paper or a semisolid gel media under a dc potential. These media could be porous and are immersed into an aqueous buffer solution. At a particular potential gradient, there is a differential movement of ions. The velocity of migration in the electric field is proportional to the charge-tosize ratio.

ELECTROOSMOTIC FLOW:
When a high potential is applied across a capillary tube made of silica containing buffer solution, the positively-charged ions migrate towards the negative electrode and carry solvent molecules in the same direction. As shown in Fig. 1, the electric double layer is developed on the interface of silica and solution. The surface of the silicate glass capillary contains negatively-charged functional groups (formed due to the presence of Si-O-H )

Fig. 1: A charge distribution in capillary during electroosmotic flow

Page 3 of 12

ELECTROPHORESIS
and they attract positively charged counterions. This overall solvent movement is called electroosmotic flow. During a separation, the uncharged molecules move at the same velocity as the electroosmotic flow (with very little separation). The positively-charged ions move faster and negatively-charged ions move slower.

BASIC PRINCIPLE AND OPERATION

From the foregoing discussion, it should be clear that electrophoresis is a separation method based on differential rate of migration of charged species in a buffer solution across which a dc electric field is applied. An electrophoresis separation is achieved by injection of a small volume of sample into an aqueous buffer solution which is contained on a porous support medium like paper or semisolid gel or a narrow tube. A simple schematic diagram of gel electrophoresis is shown in Fig. 2.

Fig. 2: A simple schematic diagram of gel electrophoresis

The potential applied causes the species to migrate towards the one or the other electrode. The rate of migration of a given species depends upon the charge and also the size. Thus, the separations are based upon the differences in charge to size ratios of the various species. The larger is this ratio, the faster an ion migrates under the influence of the electric field.

DIFFERENT FORMS OF ELECTROPHORESIS

Now-a-days electrophoresis is one of the most important techniques for molecular separation because this powerful technique is reasonably easy and inexpensive. Electrophoresis can be onedimensional (1D) meaning one plane of separation or two-dimensional (2D) meaning two planes of separation. One-dimensional electrophoresis is used for most routine protein and nucleic acid separations. Two-dimensional separation of proteins is used for finger printing, proteomics studies, etc. Broadly, different types of electrophoresis are categorised as follows: i) Slab electrophoresis ii) Capillary electrophoresis
Page 4 of 12

ELECTROPHORESIS

Slab Electrophoresis
In slab electrophoresis, separations are carried out on a thin flat layer (slab) of porous semi-solid gel containing an aqueous buffer. In general, slab has dimensions of a few centimeter on a side and is capable of separating more than one sample simultaneously. Samples, as small as few L, are introduced as bands or spots on the slab and finally the separated species are detected by staining. Typical examples are DNA gel electrophoresis, SDS-PAGE gel electrophoresis and two-dimensional SDSPAGE gel electrophoresis, etc. You will now learn about their principle, operational aspects and applications.
DNA Gel Electrophoresis In this technique, DNA samples are run through agarose gel with the help of a potential and differential mobility of different DNA samples is responsible for their separation. The following instruments and reagents are important for the preparation and running of DNA gel electrophoresis: Gel casting trays: These are available in a variety of sizes and are composed of U-shaped UV-transparent plastic. The open ends of the trays are closed with tape while the gel is being cast in between the A and B walls. The tape is removed before electrophoresis.

Fig. 3: Gel casting tray

These are plastics combs around which molten agarose is poured to form sample wells in the gel. Once the gel is cast, comb is easily removed and the wells are made in the gel-slab. The size of the comb determines the volume of the well produced. Electrophoresis buffer: The buffer which is used for the DNA gel electrophoresis usually is Tris-acetate-EDTA (TAE) or Tris-borate-EDTA (TBE). Loading buffer: This buffer is important not only for the loading of the samples in the wells of the DNA gel but also to impart a color for the tracking of the samples. The components of the loading buffer are glycerol and dye. Glycerol helps to allow the sample to go into the sample wells and dyes help visual monitoring of the sample and the extent the electrophoresis has proceeded. Ethidium bromide: This is used for the detection of the DNA inside the gel. Ethidium bromide is a fluorescent dye giving fluorescence when intercalated inside the DNA and is used for staining nucleic
Page 5 of 12

Combs:

ELECTROPHORESIS
acids. Ethidium bromide is carcinogenic and should be handled as a hazardous chemical and one should wear gloves while handling it. Transilluminator: Transilluminator is an ultraviolet light source which is used to visualize ethidium bromidestained DNA in gels.

After DNA gel electrophoresis is run, the whole slab is kept over transilluminator. The spots of DNA in the agarose slab can easily be found by the fluorescence of intercalated ethidium bromide (Fig. 4).

Fig. 4: Slab electrophoresis separating DNA samples. Rectangle boxes are the wells generated by comb in agarose slab. (a) and (b) represent the gel-slab before and after run, respectively. Well M contains standard marker to examine the mass of the DNA.

Among linear and supercoiled DNA, the later will move faster in agarose slab as compared to the former because of the difference of surface area. Hence, a plasmid DNA could easily be examined whether it is linear or supercoiled or a mixture of two. In fact, it is easily understandable that a nicked plasmid will move slower in DNA gel than the native one. Pulse-Field Gel Electrophoresis (PFGE) allows investigators to separate much larger pieces of DNA than conventional agarose gel electrophoresis. In conventional gels, the current is applied in a single direction (from top to bottom). However, in PFGE, the direction of the current is altered at regular intervals. Initially, the agarose gel is prepared and samples are loaded into the wells after mixing with blue loading buffer. Then the current is turned on and the direction of the current is changed in a regular pattern. This is repeated until the loading dye reaches near the end of the gel. The gel is then soaked in a solution containing ethidium bromide which fluoresces orange when bound to DNA.

Page 6 of 12

ELECTROPHORESIS
SDS-PAGE Gel Electrohpresis
In this technique, protein samples are run through page-gel with the help of a potential and differential mobility of different protein samples is responsible for their separation according to the mass of the protein. Polyacrylamide gels are formed from the polymerization of two compounds viz. acrylamide and N,N-methylene- bisacrylamide. Bis acrylamide is a cross-linking agent for the gels. The polymerization is initiated by the addition of ammonium persulphate along with eitherdimethylamino-propionitrile (DMAP) or N,N,N,N,- tetramethylethylenediamine (TEMED). Gels are caste in between two glass-plates and combs are also used to prepare the wells. Like DNA-gel casting, combs are removed after polymerisation of the polymer. The gels are neutral, hydrophillic, three-dimensional networks of long hydrocarbons crosslinked by methylene groups. Acrylamide (CH2=CH-CO-NH2) and methylenebisacrylamide (CH2=CH-CO-NH-)2CH2 react in presence of ammonium persulphate (APS), (NH4)2S2O8. APS is the sulphate free radical generator and crossed linked polymeric slab is produced.

The structure of the polymer The instruments and reagents important for PAGE-Gel are gel casting apparatus, loading buffer and running buffer. Casting of gel is done in such a way that the whole slab will have two portions. One portion (at the top) is called stacking gel and the rest part (the bottom one) is called the running gel, (see Fig. 5(a)). When the detergent SDS (sodium dodecyl sulphate) is added to the proteins during the sample preparation, proteins become negatively charged by their attachment to the SDS anions. When separated on a polyacrylamide gel, the procedure is abbreviated as SDS-PAGE (for Sodium Dodecyl Sulphate PolyAcrylamide Gel Electrophoresis). These negatively charged proteins are allowed to run through the polyacrylamide gel towards positive electrode. The technique is a powerful tool for the identification of the protein as well as determination of mass of a protein. A known marker protein mixture is used to compare and determine the mass of a protein.

Page 7 of 12

ELECTROPHORESIS

Fig. 5: (a) Schematic diagram of PAGE-gel electrophoresis and (b) A typical PAGE gel after Coomassie staining

Two-dimensional SDS-PAGE Gel Electrophoresis Each amino acid has its own isoelectric point i.e., pI. pI is the pH at which the mobility of the amino acid is zero. Hence, proteins which comprise amino acids, do have a characteristic pI value. In two-dimensional gel electrophoresis, proteins are separated according to their pI and then they are allowed to mix with SDS. Denatured proteins are then allowed to enter a polyacrylamide gel. The passage of the proteins by potential ultimately separates the proteins according to their masses.

Page 8 of 12

ELECTROPHORESIS

Fig. 6: Spots found after staining in 2D gel electrophoresis

CAPILLARY ELECTROPHORESIS
Capillary electrophoresis is a more sensitive technique and with the similar principle, it could detect samples as small as few nL. Besides being more sensitive, this separation technique has high-speed and high resolution. Instead of staining of the samples as in slab electrophoresis, there is one quantitative detector at the end of the capillary. The capillary can also be filled with a gel, which eliminates the electroosmotic flow (vide infra). Thus, the separation is accomplished as in conventional gel electrophoresis but the capillary allows higher resolution, greater sensitivity and on-line detection. A buffer filled fused silica capillary that is typically 10 to 100 m in internal diameter and 40 to 100 cm long, extends between two buffer reservoirs that also hold platinum electrodes. Sample introduction is performed at one end and the detection at the other. The polarity of the high-voltage power supply can be as indicated in Fig. 7 or can be reversed to allow rapid separation of anions. Although the instrumentation is conceptually simple, there are significant experimental difficulties in the sample introduction and detection due to the very small volume involved. Since the volume of a normal capillary is 4-5 L, injection and detection volumes must be of the order of a few nano litres or less. Let us now study more about sample introduction and detection.

Fig. 7: A schematic representation for capillary electrophoresis

a) Sample Introduction The most common sample introduction methods are as follows: Electrokinetic injection Pressure injection

Page 9 of 12

ELECTROPHORESIS
With electrokinetic injection, one end of the capillary and its electrodes are removed from their buffer compartment and placed in a small cup containing the sample. A potential is then applied for a period of time causing the sample to enter the capillary by a combination of ionic migration and electroosmotic flow. The capillary end and electrode are then placed back into the regular buffer solution for the duration of the separation. This injection technique discriminates by injecting larger amounts of more mobile ions relative to the slower moving ions. With pressure injection, the sample introduction end of the capillary is also placed momentarily into a small cup containing the sample and a pressure difference is then used to drive the sample solution into the capillary. The pressure difference can come from applying a vacuum at the detector end by pressurizing the sample or by elevating the sample end. Pressure injection does not discriminate due to ion mobility but cannot be used in gel field capillary. For both the injection procedures, the volume injected is controlled by the duration of the injection. Injections of 5-50 nL are common, and the injection of volumes below 100 pL has been reported. For a buffer with density and viscosity near the values for water, a height differential of 5 cm for 10 s injects about 6 nL with a 75 m inside diameter capillary. Microinjection tips constructed from capillaries pulled down to very small diameters allow sampling from picolitres environments such as single cells or substructures within single cells. This technique has been employed to study amino acids and neurotransmitters from single cells. b) Detection There are several ways one can detect the samples after capillary electrophoresis. Examples are absorbance methods, fluorescence methods, mass spectrometry, conductivity measurements, potentiometry, amperometry, radiometry, electrochemical detection, etc.

Capillary Zone Electrophoresis Capillary Zone Electrophoresis also known as Free-Solution Capillary Electrophoresis (FSCE), is the simplest form of Capillary Electrophoresis. It can be used to separate ionic species by their charge and frictional forces. The separation mechanism is based on differences in the charge-tomass ratio of the analytes. In traditional electrophoresis, electrically charged analytes move in a conductive liquid medium under the influence of an electric field. Fundamental to CZE are homogeneity of the buffer solution and constant field strength throughout the length of the capillary. Introduced in the 1960s, the technique of capillary electrophoresis (CE) is designed to separate species based on their size to charge ratio in the interior of a small capillary filled with an electrolyte. This technique is used for the separation of small ions as well as for the separation of molecular species. Following are the applications of this technique.

Capillary Gel Electrophoresis (CGE) Capillary gel electrophoresis is generally performed in a porous gel polymer matrix, the pores of which contain a buffer mixture in which the separation is carried out. In early slab electrophoresis studies, the primary purpose of the polymeric medium was to reduce analyte dispersion by convection and diffusion and to provide a medium that could be conveniently handled for detection
Page 10 of 12

ELECTROPHORESIS
and scanning. It subsequently developed that this type of medium could provide a molecular sieving action that retarded the migration of analyte species to various extents depending upon the pore size of the polymer and the size of the analyte ions. This type of sieving action is particularly helpful in separating macromolecules such as proteins, DNA fragments, and oligonucleotides that have substantially the same charge but differ in size. Currently, most macroscale electrophoresis separations are carried on a gel slab. Some capillary electrophoretic separations of species that differ in size are also performed in gels contained in capillary tubes. The most common type of gel used in electrophoresis is a polyacrylamide polymer formed by polymerizing acrylamide ( -CH2 = CH CO NH2) in the presence of a cross linking agent (vide infra). The pore size of the polymer depends upon the ratio of monomer to cross linking agent. An increase in the amount of cross linking agent results in smaller pore size. Other gels that have been used for capillary gel electrophoresis include agarose, a polysaccharide extracted from a marine alga, methyl cellulose and polyethylene glycol.

Capillary Isotachophoresis (CITP) Isotachophoresis (ITP) is a focusing technique based on the migration of the sample components between leading and terminating electrolytes. Solutes having mobilities intermediate to those of the leading and terminating electrolytes stack into sharp, focused zones. Although it is used as a mode of separation, transient ITP has been used primarily as a sample concentration technique.

Capillary Isoelectric Focusing (CIEF) This technique allows amphoteric molecules, such as proteins, to be separated by electrophoresis in a pH gradient generated between the cathode and anode. A solute will migrate to a point where its net charge is zero. At the solutes isoelectric point (pI), migration stops and the sample is focused into a tight zone. In CIEF, once a solute has focused at its pI, the zone is mobilized past the detector by either pressure or chemical means. This technique is commonly employed in protein characterization as a mechanism to determine a protein's isoelectric point.

CAPILLARY ELECTROCHROMATOGRAPHY Capillary Electrochromatography (CEC) is a hybrid separation method that couples the high separation efficiency of CZE with HPLC and uses an electric field rather than hydraulic pressure to propel the mobile phase through a packed bed. Because there is minimal backpressure, it is possible to use small-diameter packings and achieve very high efficiencies. Its most useful application appears to be in the form of on-line analyte concentration that can be used to concentrate a given sample prior to separation by CZE. Electrochromatography, a hybrid of capillary electrophoresis and HPLC, offers some of the best features of each of the two techniques. Two types of capillary electrochromatography have been developed since the early 1980s: packed column and micellar electrokinetic capillary. To date, the later has found more widespread applications. Capillary electrochromatography appears to offer several advantages over either of the parent techniques. First, like HPLC, it is applicable to the separation of uncharged species. Second, like
Page 11 of 12

ELECTROPHORESIS
capillary electrophoresis, it provides highly efficient separations of microvolumes of sample solution without the need for a high- pressure pumping system. In electrochromatography, a mobile phase is transported through a stationary phase by electroosmotic-flow pumping rather than by mechanical pumping; thus, simplifying the system significantly. In addition, electroosmotic pumping leads to the plug flow profile rather than the hydrodynamic profile. The flat face of plug flow leads to less band broadening than the hydrodynamic profile. This results in greater separation efficiency.

SUMMARY Electrophoresis is an important technique in analytical chemistry. The basic process is to run a charged macromolecular species against a potential through a thick semi-solid media called gel. The species are separated according to their mass, size and charge. There are mainly two types of electrophoresis techniques used now-a-days and they are slab electrophoresis and capillary electrophoresis. DNA electrophoresis, SDSPAGE gel electrophoresis and two-dimensional SDSPAGE gel electrophoresis are typical examples of slab electrophoresis whereas Capillary Zone Electrophoresis (CZE), Capillary Gel Electrophoresis (CGE), Capillary Isotachophoresis (CITP), Capillary Isoelectric Focusing (CIEF) are the applications of capillary electrophoresis.Both the techniques are used for the separation of DNA proteins and other macromolecules.

Page 12 of 12