Factors affecting electrophoresis The electric field ·       Voltage. If the separation of the electrodes is d (cm) and the potential difference between them is V volts, the potential gradient is V/d. The force on an ion bearing a charge q coulombs is then Vq/d newtons. The force causes migration and the rate of migration is proportional to Vq/d. The rate of migration under unit potential gradient is called the mobility of the ion. An increase in the potential gradient will therefore increase the rate of migration proportionally. ·       Current. When a potential difference is applied between the electrodes, a current is generated, measured in coulombs sec or amperes. The size of this current is determined by the resistance of the medium and is proportional to the voltage. The current in the solution between the electrodes is conducted mainly by the buffer ions with a small proportion being conducted by the sample ions. An increase in voltage will increase the total charge per second conveyed towards the electrode. The distance migrated by the ions will be proportional to both current and time. ·       Resistance. Ohm’s law expresses the relationship between current I (measured in amperes, A), voltage V (measured in volts, V) and resistance R (measured in ohms, W) in which: V/I=R ·       The current, and hence the rate of migration are thus inversely proportional to the resistance, which in turn is a function of the medium, the buffer and its concentration. Resistance will increase with the length of the supporting medium but will decrease with its cross-sectional area and with increasing buffer ion concentration. During electrophoresis the power dissipated in the supporting medium (W, measured in watts) is such that: W=I2R ·       An increase in temperature will cause the resistance to fall. Part of this effect is due to an increase in the mobility of the ions as a result of a decrease in the viscous resistance offered by the liquid to the motion of the ions through it as the temperature rises. The heating will produce evaporation of the solvent from the supporting medium causing a decrease in resistance. Although the rate of migration and the total charge per second conveyed towards the electrode will increase, the increase in buffer ion concentration will result in slower migration of the sample.

·       When constant voltage is applied, the current will increase during electrophoresis due to a decrease in resistance of the medium with the rise in temperature. Consequently, more heat will be produced resulting in more evaporation of solvent and a decrease in resistance. A constant current avoids these problems but may lead to a drop in voltage due to decreased resistance, resulting in reduced rate of migration. ·       If a number of gels are run in parallel with one power supply, then: 1/R= 1/r1 +1/r2+…. 1/rn where R is the total resistance and r1,r2, etc. are the resistances of each gel. The total current supplied must be increased in proportion to the number of gels used, assuming that they all have the same resistance.   The resistance does decrease as the temperature increase for semiconductors. Since the gel is a semiconductor so it make sense for the decrease in resistance. This is  because increasing temperature means increasing the velocity of charged particles so conductivity will increase.  However, the resistance in metals does increase as the temperature increase.  When the temperature of a metal is increased its resistance also increases this is because the increased vibration of the matel lattice makes it harder for the free electrons to flow easily through the material. When a semiconductor is heated its resistance goes down as more electrons have the energy to become free from the lattice.

  The supporting medium ·       Adsorption. This is the retention of sample molecules by the supporting medium. Adsorption causes tailing of the sample and reduces the rate and resolution of the separation. ·       Electro-osmosis. This results from a relative charge being produced between water molecules in the buffer and the surface of the supporting medium. The charge may be caused by surface adsorption of ions from the buffer and the presence of stationary carboxyl groups on paper or sulphonate groups on agar. The effects of electro-osmosis can normally be ignored. ·       Molecular Sieving. Molecular sieving occurring in agar, starch and polyacrylamide gels is that the movement of large molecules is hindered increasingly by decreasing the pore size since all molecules have to traverse through the pores.

The sample ·       Charge. The rate of migration increases with an increase in the net charge. The magnitude of the charge is generally pH dependent. ·       Size. The rate of migration decreases for larger molecules, due to the increased frictional and electrostatic forces exerted by the surrounding medium. ·       Shape. Molecules of similar size but different shapes such as fibrous and globular proteins exhibit different migration characteristics because of the differential effect of frictional and electrostatic forces.

The buffer ·       Composition. The buffers in common use are formate, acetate, citrate, barbitone, phosphate, Tris, EDTA and pyridine. The buffer should not bind to the sample. In some cases, however, binding can be advantageous, for example borate buffers are used to separate carbohydrates since they produce charged complexes with carbohydrates. Since the buffer acts as a solvent for the sample, some diffusion of the sample is inevitable, being particularly noticeable for small molecules such as amino acids and sugars. The extent of diffusion can be minimized by not overloading the sample. ·       Concentrations As the ionic strength of the buffer increase, the proportion of current carried by the buffer will increase and the share of the current carried by the sample will decrease, thus slowing the sample migration. High ionic strength of the buffer will also increase the overall current and heat will be produced. At low ionic strengths the proportion of current carried by the buffer will decrease and the share of the current carried by the sample will increase, thus increasing its rate of migration. A low ionic buffer strength reduces the overall current and results in less heat production, but diffusion and the resulting loss of resolution are higher. The compromise is to use between 0.05 to 0.1 M. ·       PH determines extent of ionisation.

Gel Filtration Chromatography (also called "Molecular Sieve" chromatography, or "Size Exclusion" chromatography) stationary phase (column matrix) = "beads" of a polysaccharide material that separates proteins based on size and shape.  Different column packing materials (hydrated, porous beads of carbohydrate polymer (e.g. dextran or agarose) or polyacrylamide) available, with wide range of molecular exclusion limits, for separating proteins of all sizes. Solution of mixture of proteins, small molecules, etc. "filters" through the beads: Large molecules can’t get into the smaller pores in the beads and move more rapidly through the column, emerging (eluting) sooner. Smaller molecules and ions can enter all the pores in the beads with the buffer, and thus have more space to "explore" on their way down the column, and elute later. For any particular column dimensions and material, volume of buffer required to elute a specific protein depends mostly on molecular weight of the protein (but shape plays an important role also -- separation is really based on differences in hydrodynamic volume). Thus, one can separate proteins by size. Fig. 5-18a (Nelson & Cox, Lehninger Principles of Biochemistry, 3rd ed.): Size exclusion chromatography

                                           This animation illustrates how size exclusion chromatography works.  Note how the small red spheres pass into the channels in the beads, whereas the large blue spheres do not.  Thus, the small spheres have a longer "distance" to transverse than the large spheres to get to bottom of column, which means that a larger volume of solvent must pass through the column before the red spheres are eluted. The following plot of relative amount of the large solute (blue) and of the smaller solute (red) goes with the animation. Larger solutes elute EARLIER, smaller solutes LATER, from a size exclusion column.                                                                                                                    calibrate the column: determine elution volumes of proteins with known molecular weights construct a calibration curve relating(known) molecular weight to (measured) elution volume specifically for that column.   Such a calibration curve can then be used to estimate the molecular weight of an unknown protein.