Page 104 - Carrahers_Polymer_Chemistry,_Eighth_Edition

Molecular Weight of Polymers 67

Buffer
Buffer
Negatively charged
protein

Electrophoresis strip

Positively charged
protein
Cathode (−) Anode (+)

Power supply

FIGURE 3.10 Basic components of an electrophoresis apparatus.

This migration is called electrophoresis. The velocity at which molecules move is mainly dependant

upon the electric field and charge on the polymer driving the molecule toward one of the electrodes,
and a frictional force dependant on the size and structure of the macromolecule that opposes the
movement. In general, the larger and more bulky the macromolecule the greater the resistance to
movement and the greater the applied field and charge on the molecule the more rapid the move-

ment. While electrophoresis can be conducted on solutions, it is customary to use a supporting

medium of a paper or gel. For a given system, it is possible to calibrate the rate of flow with the
molecular weight and/or size of the molecule. Here, the flow characteristics of the calibration mate-

rial must be similar to those of the unknown.
Generally though, electrophoresis is often employed in the separation of complex molecules such
as proteins where the primary factor in the separation is the charge on the species. Some amino
acids such as aspartic acid and glutamic acid contain an “additional” acid functional group, while
amino acids such as lysine, arginine, and histidine contain “additional” basic groups. The presence
of these units will confer to the protein tendencies to move toward the anode or cathode. The rate of
movement is dependent on a number of factors, including the relative abundance and accessibility
of these acid and base functional groups.
Figure 3.10 contains an illustration of the basic components of a typical electrophoresis appara-
tus. The troughs at either end contain an electrolyte buffer solution. The sample to be separated is
placed in the approximate center of the electrophoresis strip.
Gel permeation chromatography (GPC) is a form of chromatography that is based on separation
by molecular size rather than chemical properties. GPC or Size exclusion chromatography (SEC) is
widely used for molecular weight and MWD determination. In itself, SEC does not give an absolute
molecular weight and must be calibrated against polymer samples whose molecular weight has been
determined by a technique that does give an absolute molecular weight.
Size exclusion chromatography is an HPLC technique whereby the polymer chains are separated
according to differences in hydrodynamic volume. This separation is made possible by the use of
special packing material in the column. The packing material is usually polymeric porous spheres
often composed of polystyrene cross-linked by addition of varying amounts of divinylbenzene.
Retention in the column is mainly governed by the partitioning (or exchanging) of polymer chains

between the mobile (or eluent) phase flowing through the column and the stagnate liquid phase that
is present in the interior of the packing material. The column-packing approaches being spherical.
In reality, the packing material has various clefts that ensnare the passing polymer chains such that
the progress down the column of smaller chains is preferentially slowed (Figure 3.11).

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