Transcript Sep Tec Lecture Notes E

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:
·
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.