Gel Chromatography Technique

Study Notes

Sample should be spotted at the top of Sepxadex gel or Bio-Rad gel and soaked in an appropriate solvent, then eluted with the same solvent.
Separation depends on the molecular size of the separated species, with larger ones eluted faster.
Sequential fractions of the eluate are collected, and the radioactivity is measured in each fraction.
The radioactivity is plotted versus fraction number, giving the relative concentration of different molecular size components in a given sample.
The amount of component is expressed as the ratio of its radioactivity to the total radioactivity placed on the column.
Gel chromatography is useful in separating proteins of different molecular weights and detecting impurities in 99mTc radiopharmaceuticals.

Sample is applied on a paper or polyacrylamide gel soaked in a suitable buffer, and then an appropriate voltage is applied across the paper or gel for a specified time.
Components move across the paper or gel according to their charge or ionic mobility.
After electrophoresis, the distribution of activity along the strip or gel column can be determined by a counter or a radiochromatographic scanner.
Since protein molecules become charged in buffer solutions above or below their isoelectric pH, most proteins can be separated by this method with the use of appropriate buffers.
For example, a good separation of free iodide and radioiodinated proteins can be achieved by electrophoresis in buffer.

Resins are polymerized, high molecular weight, insoluble electrolytes composed of a large, heavy polymeric ion and an oppositely charged small ion that is exchangeable.
Different components of the sample distribute themselves between the adsorbent (stationary layer) and the solvent (mobile phase) depending on their distribution coefficients.
Electrostatic forces of the stationary phase tend to retard various components, while the mobile phase carries them along at different speeds.
This effect and varying solubilities of different components in a solvent cause the individual components to move at different speeds and to appear at different distances along the paper or ITLC strip.
Polarity of the solvent also affects the chromatographic separation of different components in a sample.
Paper or ITLC can be ascending or descending.
Each component has a Rf value, which is the ratio of the distance traveled by the component to the distance the solvent front has moved from the original point.
Rf values are established with known components and used preliminary for identification of different components from a given sample.

A solution containing one or more chemical compounds is shaken with an immiscible liquid.
Separation is affected by the preferential solubility of individual components in one solvent.
Partition coefficient is the ratio of solubility's of a component in two immiscible liquids.
Solvent extraction of 99mTcO4- with MEK (methyl ethyl ketone) from 99MoO4-2 has been a successful method of avoiding various radiocontaminants in the 99mTc-eluate.

Separation of components with high resolution is achieved.
Advantages include high resolution, speed, and high recovery of solutes.
Columns are heavy-walled tubes of glass or stainless steel, packed with appropriate packing material.
Sample size is very small (5-250 µl), and the sample is injected into the column.