Page 145 - Academic Press Encyclopedia of Physical Science and Technology 3rd Analytical Chemistry

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Electrophoresis 373

lines such as Raman lines). If an external, polarized elec- III. ELECTROPHORESIS IN
tric field is applied to the solution, the molecules drift in A STATIONARY MATRIX
the fixed direction as well as diffuse. In the Fourier trans-
form this is equivalent to multiplying the transform by the Despite the decline in the application of free-solution elec-
transform of a uniform linear motion, which happens to trophoresis to physical chemistry, the results from this
be a single sine function. The result in QEL is to replace work showed that electrophoresis has a unique place in
the constantly decaying relaxation curve by a peak about studiesofbiologicalmacroions.Thus,attemptsweremade
which the relaxation occurs. The amount this peak is dis- to reduce the technical problems associated with elec-
placed from the zero time of the autocorrelation time scale trophoresing mixtures of macroions. Efforts were directed
is a measure of the constant velocity (or its period) of the at reducing the convective distrubances of the moving
scattering ions, while from the relaxation time the diffu- boundaries by introducing a neutral but physically inert
sion coefficient and hence the frictional coefficient can be matrix to support the solutions. Initially, paper saturated
estimated. Since the time required to collect the autocor- with suitable buffers was used, and a spot of the solution of
relation data following a single electrical pulse can be as macroion was placed on the paper before the electric field
low as 1 msec, this means that short repetitive pulses of was applied across the strip. The positions of the bands at
relatively high voltage can be applied across the solution the end of the experiment were found by selective staining.
and synchronized with the autocorrelation of the scattered This was reasonably successful for some applications, but
photons. Often, in this technique platinum electrodes are because particles moved in a solvent that was adsorbed
used to apply the voltage rather than reversible electrodes. to the surface of the cellulose fibers in the paper, there
Naturally, with these electrodes, electrolysis of the sol- were inconsistencies in the overall conduction of the ions.
vent occurs, and this generates a pH gradient between the Heating in regions of low electrical resistance dried the
electrodes. This effect can be reduced if the polarity of the paper unevenly even in a saturated atmosphere.
electrodes is reversed between pulses, but more reliable re- For these reasons paper was replaced by starch gels,
sults could be obtained if reversible silver/silver chloride and here the proportion of free solvent to inert matrix was
electrodes were used. The fact that reversing the polarity considerably higher than could be achieved with paper.
reverses the movement does not affect the analysis, pro- These gels are stabilized by weak intermolecular forces
vided that the voltages of the pulses remain constant and (hydrogen bonding and van der Waals dispersion forces).
movement is across the incident light beam. If the angle Thus, they require a backing plate for transferring the gel
between the incident light and scattered light is decreased, to a staining medium, and this disturbed the gels during
the transport vector decreases in magnitude until at full- handling. Furthermore, untreated starch has a number of
forward scattering only the diffusion is important. It is free phosphate groups covalently linked to the carbohy-
thus possible to discriminate between the two effects by drate chains, and these cause undesirable pumping of wa-
measuring the correlation function at a range of angles. ter(electroosmosis)whenanelectric fieldisappliedacross
Few results on the use of this procedure for macroions the starch strips. These difficulties prompted workers to
having relative masses of less than a million have been re- search for gels that were electrically neutral as well as
ported. This is because smaller biological macroions have physically strong enough to be handled without distro-
relatively small scattering cross, sections, and a lengthy tion. This led to the development of modern polyacry-
experiment is required to obtain a statistically significant lamide gels. These gels form the matrix for most present-
result. For small molecules the repetitive pulsing has to day electrophoretic analyses.
extend over many minutes, and in this time extraneous ef-
fects, such as the accumulation of electrolysis products (if
platinum electrodes must be used) or of gas bubbles, dis- A. Polyacrylamide Gel Electrophoresis
trub the result. The technique is ideal for simultaneously Acrylamide has the chemical structure given in Fig. 4a,
studying diffusion and charge on a macroion, because dif-
and it is the opening of the double (π) bond that leads
fusion can be converted directly to a frictional coefficient,
to the polymerization. It is necessary to activate this
which means that charge can be correctly calculated from
the mobility [see Eq. (6)]. Examples of suitable macroions
are viruses and particles from biological cells. Another ad-
vantage of the heterodyne method is that it is possible to
define a “frequency window” for the analysis of a given
size of molecule, so that specific ions can be followed even
when present in a mixture of smaller or very much larger FIGURE 4 Chemical structures of (a) acrylamide and (b) N,N -
ions. methylenebisacrylamide (bisacrylamide).