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SUBMITTED TO : MR . HARSH KUMAR
( LEC, LPU)
NAME KUMAR SEC ROLL NO :- VIVEK :- B :-R109X53
REG NO : 1040070160
S. NO. 1. 2. 3 5. 6. 7. 8. 9. 10.
TOPIC ACKNOWLADGEMENT INTRODUCTION THEORY APPLICATION INRO- HISTORY OF GEL ELECTROPHRESIS WORKING PROCESS APPLICAION OF GEL ELECTROPHORESIS
PAGE NO. 3 4-5 6 6-14 6-7 8-9 7-10 11-17 18
REFRENCE AND BIBLIOGRAPHY
I gratefully acknowledge my indebtedness to my respected teacher MR. PRABHOJOT JASSAL who is always a source of inspiration for me, for providing this opportunity. Under his able guidance, I have learned how to overcome the odds and trying circumstances. I am also very thankful to my colleagues with whom I could discuss and give the final shape to this term paper. At the same time, I am also thankful to Dr. HARSH KUMAR who gave us an immense help in the completion of this project. Any constructive comments, suggestions, criticism from students and colleagues will be highly appreciated and gratefully acknowledged. THANK ING YOU
Electrophoresis is the most well-known electrokinetic phenomenon. It was discovered by Reuss in 1807. He observed that clay particles dispersed in water migrate under influence of an applied electric field. There are detailed descriptions of Electrophoresis in many books on Colloid and Interface Science.There is an IUPAC Technical Report prepared by a group of well known experts on the electrokinetic phenomena. Generally, electrophoresis is the motion of dispersed particles relative to a fluid under the influence of an electric field that is space uniform. Alternatively, similar motion in a space non-uniform electric field is called dielectrophoresis.
Electrophoresis occurs because particles dispersed in a fluid almost always carry an electric surface charge. An electric field exerts electrostatic Coulomb force on the particles through these charges. Recent molecular dynamics simulations, though, suggest that surface charge is not always necessary for electrophoresis and that even neutral particles can show electrophoresis due to the specific molecular structure of waterer at the interface. The electrostatic Coulomb force exerted on a surface charge is reduced by an opposing force which is electrostatic as well. According to double layer theory, all surface charges in fluids are screened by a diffuse layer. This diffuse layer has the same absolute charge value, but
with opposite sign from the surface charge. The electric field induces force on the diffuse layer, as well as on the surface charge. The total value of this force equals to the first mentioned force, but it is oppositely directed. However, only part of this force is applied to the particle. It is actually applied to the ions in the diffuse layer. These ions are at some distance from the particle surface. They transfer part of this electrostatic force to the particle surface through viscous stress. This part of the force that is applied to the particle body is called electrophoretic retardation force. There is one more electric force, which is associated with deviation of the double layer from spherical symmetry and surface conductivity due to the excess ions in the diffuse layer. This force is called the electrophoretic relaxation force. All these forces are balanced with hydrodynamic friction, which affects all bodies moving in viscous fluids with low Reynolds number. The speed of this motion v is proportional to the electric field strength E if the field is not too strong. Using this assumption makes possible the introduction of electrophoretic mobility μe as coefficient of proportionality between particle speed and electric field strength:
Multiple theories were developed during 20th century for calculating this parameter.
The most known and widely used theory of electrophoresis was developed by Smoluchowski in 1903 
, where ε is the dielectric constant of the dispersion medium, ε0 is the permittivity of free space (C² N-1 m-2), η is dynamic viscosity of the dispersion medium (Pa s), and ζ is zeta potential (i.e., the electrokinetic potential of the slipping plane in the double layer). Smoluchowski theory is very powerful because it works for dispersed particles of any shape and any concentration, when it is valid. Unfortunately, it has limitations of its validity. It follows, for instance, from the fact that it does not include Debye length κ-1. However, Debye length must be important for electrophoresis, as follows immediately from the Figure on the right. Increasing thickness of the DL leads to removing point of retardation force further from the particle surface. The thicker DL, the smaller retardation force must be.
Gel electrophoresis is a technique used for the separation of deoxyribonucleic acid, ribonucleic acid, or protein molecules using an electric current applied to a gel matrix. It is usually performed for analytical purposes, but may be used as a preparative technique prior to use of other methods such as mass spectrometry, RFLP, PCR, cloning, DNA sequencing, or Southern blotting for further characterization.
The term "gel" in this instance refers to the matrix used to contain, then separate the target molecules. In most cases the gel is a crosslinked polymer whose composition and porosity is chosen based on the specific weight and composition of the target to be analyzed. When separating proteins or small nucleic acids (DNA, RNA, or oligonucleotides) the gel is usually composed of different concentrations of acrylamide and a crosslinker, producing different sized mesh networks of polyacrylamide. When separating larger nucleic acids (greater than a few hundred bases), the preferred matrix is purified agarose. In both cases, the gel forms a solid, yet porous matrix. Acrylamide, in contrast to polyacrylamide, is a neurotoxin and must be handled using appropriate safety precautions to avoid poisoning.
Gel electrophoresis apparatus - An agarose gel is placed in this buffer-filled box and electrical current is applied via the power supply to the rear. The negative terminal is at the far end (black wire), so DNA migrates toward the camera. Classification Electrophoresis
"Electrophoresis" refers to the electromotive force (EMF) that is used to move the molecules through the gel matrix. By placing the molecules in wells in the gel and applying an electric current, the molecules will move through the matrix at different rates, usually determined by mass, toward the cathode if negatively charged or toward the anode if positively charged .
I Agarose gel prepared for DNA analysis - The first lane contains a DNA ladder for sizing, and the other four lanes show variously-sized DNA fragments that are present in some but not all of the samples.
After the electrophoresis is complete, the molecules in the gel can be stained to make them visible. Ethidium bromide, silver, or coomassie blue dye may be used for this process. Other methods may also be used to visualize the separation of the mixture's components on the gel. If the analyte molecules fluoresce under ultraviolet light, a photograph can be taken of the gel under ultraviolet lighting conditions. If the molecules to be separated contain radioactivity added for visibility, an autoradiogram can be recorded of the gel. If several mixtures have initially been injected next to each other, they will run parallel in individual lanes. Depending on the number of different molecules, each lane shows separation of the components from the original mixture as one or more distinct bands, one band per component. Incomplete separation of the components can lead to overlapping bands, or to indistinguishable smears representing multiple unresolved components. Bands in different lanes that end up at the same distance from the top contain molecules that passed through the gel with the same speed, which usually means they are approximately the same size. There are molecular weight size markers available that contain a mixture of molecules of known sizes. If such a marker was run on one lane in the gel parallel to the unknown samples, the bands observed can be compared to those of the unknown in order to determine their size. The distance a band travels is approximately inversely proportional to the logarithm of the size of the molecule.
Gel electrophoresis is used in forensics, molecular biology, genetics, microbiology and biochemistry. The results can be analyzed quantitatively by visualizing the gel with UV light and a gel imaging device. The image is recorded with a computer operated camera, and the intensity of the band or spot of interest is measured and compared against standard or markers loaded on the same gel. The measurement and analysis are mostly done with specialized software. Depending on the type of analysis being performed, other techniques are often implemented in conjunction with the results of gel electrophoresis, providing a wide range of field-specific applications.
In the case of nucleic acids, the direction of migration, from negative to positive electrodes, is due to the naturally-occurring negative charge carried by their sugar-phosphate backbone. Double-stranded DNA fragments naturally behave as long rods, so their migration through the gel is relative to their radius of gyration, or, for non-cyclic fragments, size. Singlestranded DNA or RNA tend to fold up into molecules with complex shapes and migrate through the gel in a complicated manner based on their tertiary structure. Therefore, agents that disrupt the hydrogen bonds, such as sodium hydroxide or formamide, are used to denature the nucleic acids and cause them to behave as long rods again. Gel electrophoresis of large DNA or RNA is usually done by agarose gel electrophoresis. See the "Chain termination method" page for an example of a polyacrylamide DNA sequencing gel.
11 SDS-PAGE autoradiography - The indicated proteins are present in different concentrations in the two samples.
Proteins, unlike nucleic acids, can have varying charges and complex shapes, therefore they may not migrate into the gel at similar rates, or at all, when placing a negative to positive EMF on the sample. Proteins therefore, are usually denatured in the presence of a detergent such as sodium dodecyl sulfate/sodium dodecyl phosphate (SDS/SDP) that coats the proteins with a negative charge. Generally, the amount of SDS bound is relative to the size of the protein (usually 1.4g SDS per gram of protein), so that the resulting denatured proteins have an overall negative charge, and all the proteins have a similar charge to mass ratio. Since denatured proteins act like long rods instead of having a complex tertiary shape, the rate at which the resulting SDS coated proteins migrate in the gel is relative only to its size and not its charge or shape. Proteins are usually analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), by native gel electrophoresis, by quantitative preparative native continuous polyacrylamide gel electrophoresis (QPNC-PAGE), or by 2-D electrophoresis.
• • • • • • • • • • •
1930s - first reports of the use of sucrose for gel electrophoresis 1955 - introduction of starch gels, mediocre separation 1959 - introduction of acrylamide gels (Raymond and Weintraub); accurate control of parameters such as pore size and stability 1964 - disc gel electrophoresis (Ornstein and Davis) 1969 - introduction of denaturing agents especially SDS separation of protein subunit (Weber and Osborn) 1970 - Laemmli separated 28 components of T4 phage using a stacking gel and SDS 1975 - 2-dimensional gels (O’Farrell); isoelectric focusing then SDS gel electrophoresis 1977 - sequencing gels late 1970s - agarose gels 1983 - pulsed field gel electrophoresis enables separation of large DNA molecules 1983 - introduction of capillary electrophoresis
A 1959 book on electrophoresis by Milan Bier cites references from the 1800s. However, Oliver Smithies made significant contributions. Bier states: "The method of Smithies ... is finding wide application because of its unique separatory power." Taken in context, Bier clearly implies that Smithies' method is an improvement.
Capillary electrophoresis (CE), also known as capillary zone electrophoresis (CZE), can be used to separate ionic species by their charge and frictional forces. In traditional electrophoresis, electrically charged analytes move in a conductive liquid medium under the influence of an electric field. Introduced in the 1960s, the technique of capillary electrophoresis (CE) was designed to separate species based on their size to charge ratio in the interior of a small capillary filled with an electrolyte. While its use has been sporadic, CE offers unparalleled resolution and selectivity allowing for separation of analytes with very little physical difference. Efficiencies of millions of plates are routinely reported. Once thought impossible, separation of large proteins differing in only one amino acid (ie. DLysine substituted for L-Lysine) and even an isotopic separation of 14N and 15N ammonium hydroxide have been reported. No other technique has shown such powerful selectivity with the ability for extremely high sensitivity. As few as 6 molecules of a substance have been separated and detected with the help of laser-induced fluorescence (LIF).
The instrumentation needed to perform capillary electrophoresis is relatively simple. A basic schematic of a capillary electrophoresis system is shown in figure 1. The system's main components are a sample vial, source and destination vials, a capillary, electrodes, a highvoltage power supply, a detector, and a data output and handling device. The source vial, destination vial and capillary are filled with an electrolyte such as an aqueous buffer solution. To introduce the sample, the capillary inlet is placed into a vial containing the sample and then returned to the source vial (sample is introduced into the capillary via capillary action, pressure, or siphoning). The migration of the analytes is then initiated by an electric field that is applied between the source and destination vials and is supplied to the electrodes by the high-voltage power supply. It is important to note that all ions, positive or negative, are pulled through the capillary in the same direction by electroosmotic flow, as will be explained. The analytes separate as they migrate due to their electrophoretic mobility, as will be explained, and are detected near the outlet end of the capillary. The output of the detector is sent to a data output and handling device such as an integrator or computer. The data is then displayed as an electropherogram, which reports detector response as a function of time. Separated chemical compounds appear as peaks with different retention times in an electropherogram. (PRAVEEN KUMAR,BBI.PATNA,D.Y.PATIL,PUNE)
Figure 1: Diagram of capillary electrophoresis system Detection
Separation by capillary electrophoresis can be detected by several detection devices. The majority of commercial systems use UV or UV-Vis absorbance as their primary mode of detection. In these systems, a section of the capillary itself is used as the detection cell. The use of on-tube detection enables detection of separated analytes with no loss of resolution. In general, capillaries used in capillary electrophoresis are coated with a polymer for increased stability. The portion of the capillary used for UV detection, however, must be optically transparent. Bare capillaries can break relatively easily and, as a result, capillaries with transparent coatings are available to increase the stability of the cell window. The path length of the detection cell in capillary electrophoresis (~ 50 micrometers) is far less than that of a traditional UV cell (~ 1 cm). According to the Beer-Lambert law, the sensitivity of the detector is proportional to the path length of the cell. To improve the sensitivity, the path length can be increased, though this results in a loss of resolution. The capillary tube itself can be expanded at the detection point, creating a "bubble cell" with a longer path length or additional tubing can be added at the detection point as shown in figure 2. Both of these methods, however, will decrease the resolution of the separation.
14 Figure 2: Techniques for increasing the pathlength of the capillary: a.) a bubble cell and b.) a z-cell (additional tubing).
Fluorescence detection can also be used in capillary electrophoresis for samples that naturally fluoresce or are chemically modified to contain fluorescent tags. This mode of detection offers high sensitivity and improved selectivity for these samples, but cannot be utilized for samples that do not fluoresce. The set-up for fluorescence detection in a capillary electrophoresis system can be complicated. The method requires that the light beam be focused on the capillary, which can be difficult for many light sources. Laser-induced fluorescence has been used in CE systems with detection limits as low as 10-18 to 10-21 mol. The sensitivity of the technique is attributed to the high intensity of the incident light and the ability to accurately focus the light on the capillary. In order to obtain the identity of sample components, capillary electrophoresis can be directly coupled with mass spectrometers or Surface Enhanced Raman Spectroscopy (SERS). In most systems, the capillary outlet is introduced into an ion source that utilizes electrospray ionization (ESI). The resulting ions are then analyzed by the mass spectrometer. This set-up requires volatile buffer solutions, which will affect the range of separation modes that can be employed and the degree of resolution that can be achieved. The measurement and analysis are mostly done with a specialized gel analysis software. For CE-SERS, capillary electrophoresis eluants can be deposited onto a SERS-active substrate. Analyte retention times can be translated into spatial distance by moving the SERSactive substrate at a constant rate during capillary electrophoresis. This allows the subsequent spectroscopic technique to be applied to specific eluants for identification with high sensitivity. SERS-active substrates can be chosen that do not interfere with the spectrum of the analytes.
Modes of separation
The separation of compounds by capillary electrophoresis is dependent on the differential migration of analytes in an applied electric field. The electrophoretic migration velocity (up) of an analyte toward the electrode of opposite charge is:
where μp is the electrophoretic mobility and E is the electric field strength. The electrophoretic mobility is proportional to the ionic charge of a sample and inversely proportional to any frictional forces present in the buffer. When two species in a sample have different charges or experience different frictional forces, they will separate from one another as they migrate through a buffer solution. The frictional forces experienced by an analyte ion depend on the viscosity (η) of the medium and the size and shape of the ion. Accordingly, the electrophoretic mobility of an analyte at a given pH is given by:
where z is the net charge of the analyte and r is the Stokes radius of the analyte. The Stokes radius is given by:
where kB is the Boltzmann constant, and T is the temperature, D is the diffusion coefficient. These equations indicate that the electrophoretic mobility of the analyte is proportional to the charge of the analyte and inversely proportional to its radius. The electrophoretic mobility can be determined experimentally from the migration time and the field strength:
where L is the distance from the inlet to the detection point, tr is the time required for the analyte to reach the detection point (migration time), V is the applied voltage (field strength), and Lt is the total length of the capillary. Since only charged ions are affected by the electric field, neutral analytes are poorly separated by capillary electrophoresis. The velocity of migration of an analyte in capillary electrophoresis will also depend upon the rate of electroosmotic flow (EOF) of the buffer solution. In a typical system, the electroosmotic flow is directed toward the negatively charged cathode so that the buffer flows through the capillary from the source vial to the destination vial. Separated by differing electrophoretic mobilities, analytes migrate toward the electrode of opposite charge. As a result, negatively charged analytes are attracted to the positively charged anode, counter to the EOF, while positively charged analytes are attracted to the cathode, in agreement with the EOF as depicted in figure 3.
Figure 3: Diagram of the separation of charged and neutral analytes (A) according to their respective electrophoretic and electroosmotic flow mobilities
The velocity of the electroosmotic flow, uo can be written as: uo = μoE where μo is the electroosmotic mobility, which is defined as:
where ζ is the zeta potential of the capillary wall, and ε is the relative permittivity of the buffer solution. Experimentally, the electroosmotic mobility can be determined by measuring the retention time of a neutral analyte The velocity (u) of an analyte in an electric field can then be defined as: up + uo = (μp + μo)E
Since the electroosmotic flow of the buffer solution is generally greater than that of the electrophoretic flow of the analytes, all analytes are carried along with the buffer solution toward the cathode. Even small, triply charged anions can be redirected to the cathode by the relatively powerful EOF of the buffer solution. Negatively charged analytes are retained longer in the capilliary due to their conflicting electrophoretic mobilities. The order of migration seen by the detector is shown in figure 3: small multiply charged cations migrate quickly and small multiply charged anions are retained strongly. Electroosmotic flow is observed when an electric field is applied to a solution in a capillary that has fixed charges on its interior wall. Charge is accumulated on the inner surface of a capillary when a buffer solution is placed inside the capillary. In a fused-silica capillary, silanol (Si-OH) groups attached to the interior wall of the capillary are ionized to negatively charged silanoate (Si-O-) groups at pH values greater than three. The ionization of the capillary wall can be enhanced by first running a basic solution, such as NaOH or KOH through the capillary prior to introducing the buffer solution. Attracted to the negatively charged silanoate groups, the positively charged cations of the buffer solution will form two inner layers of cations (called the diffuse double layer or the electrical double layer) on the capillary wall as shown in figure 4. The first layer is referred to as the fixed layer because it is held tightly to the silanoate groups. The outer layer, called the mobile layer, is farther from the silanoate groups. The mobile cation layer is pulled in the direction of the negatively charged cathode when an electric field is applied. Since these cations are solvated, the bulk buffer solution migrates with the mobile layer, causing the electroosmotic flow of the buffer solution. Other capillaries including Teflon capillaries also exhibit electroosmotic flow. The EOF of these capillaries is probably the result of adsorption of the electrically charged ions of the buffer onto the capillary walls. The rate of EOF is dependent on the field strength and the charge density of the capillary wall. The wall's charge density is proportional to the pH of the buffer solution. The electroosmotic flow will increase with pH until all of the available silanols lining the wall of the capillary are fully ionized.
Figure 4: Depiction of the interior of a fused-silica gel capillary in the presence of a buffer solution.  Efficiency and resolution
The number of theoretical plates, or separation efficiency, in capillary electrophoresis is given by:
where N is the number of theoretical plates, μ is the apparent mobility in the separation medium and Dm is the diffusion coefficient of the analyte. According to this equation, the efficiency of separation is only limited by diffusion and is proportional to the strength of the electric field. The efficiency of capillary electrophoresis separations is typically much higher than the efficiency of other separation techniques like HPLC. Unlike HPLC, in capillary electrophoresis there is no mass transfer between phases. In addition, the flow profile in EOFdriven systems is flat, rather than the rounded laminar flow profile characteristic of the pressure-driven flow in chromatography columns as shown in figure 5. As a result, EOF does not significantly contribute to band broadening as in pressure-driven chromatography. Capillary electrophoresis separations can have several hundred thousand theoretical plates.
Figure 5: Flow profiles of laminar and electroosmotic flow.
The resolution (Rs) of capillary electrophoresis separations can be written as:
According to this equation, maximum resolution is reached when the electrophoretic and electroosmotic mobilities are similar in magnitude and opposite in sign. In addition, it can be seen that high resolution requires lower velocity and, correspondingly, increased analysis time.
As discussed above, separations in a capillary electrophoresis system are typically dependent on the analytes having different electrophoretic mobilities. However, some classes of analyte cannot be separated by this effect because they are neutral (uncharged) or because they may not differ significantly in electrophoretic mobility. However, there are several techniques that can help separate such analytes with a capillary electrophoresis system. Adding a surfactant to the electrolyte can facilitate the separation of neutral compounds by micellar electrokinetic chromatography. Charged polymers such as DNA can be separated by filling the capillary with a gel matrix that retards longer strands more than shorter strands. This is called capillary gel electrophoresis. This is a high-resolution alternative to slab gel electrophoresis. Some capillary electrophoresis systems can also be used for microscale liquid chromatography or capillary electrochromatography. A capillary electrophoresis system can also be used for isotachophoresis and isoelectric focusing.
1. ^ http://www.ncbi.nlm.nih.gov/pubmed/5806584 2. ^ Milan Bier (ed.) (1959). Electrophoresis. Theory, Methods and Applications, 3rd printing, Academic Press, 225. LCC 59-7676. OCLC 1175404. 3. ^ a b c Berg JM, Tymoczko JL Stryer L (2002). Molecular Cell Biology, 5th ed., WH Freeman. ISBN 0-7167-4955-6. 4. ^ Robyt, John F. (1990). Biochemical Techniques Theory and Practice. Waveland Press. ISBN 0-88133-556-8. 5. ^ Lodish H, Berk A, Matsudaira P, et al (2004). Molecular Cell Biology, 5th ed., WH Freeman: New York, NY. ISBN 978-0716743668. 6. ^ Troubleshooting DNA agarose gel electrophoresis. Focus 19:3 p.66 (1997). 7. ^ http://www.ncbi.nlm.nih.gov/pubmed/5806584 8. ^ Milan Bier (ed.) (1959). Electrophoresis. Theory, Methods and Applications, 3rd printing, Academic Press, 225. LCC 59-7676. OCLC 1175404. 9.