This lab s topic was Gel Filtration Chromatography. Chromatography is a technique used to separate molecules on the basis of differences in size, shape, mass, charge, solubility and absorption properties. A specific kind of chromatography is gel filtration. This technique separates molecules on the basis of their size. This procedure is performed by passing molecules down a solution containing beads made of a polysaccharide or polyacrylamide gel with semi-uniform pores.
All of the beads have the same size pores with a specific fractionation range throughout the column. After the column is prepared a protein mixture is poured in the tube. The proteins with a molecular weight (MW) larger then the set fractionation range will flow straight through the tube without passing in any beads. Medium-size proteins will flow through some beads; proteins with a small MW will pass through all beads they encounter. A fraction collector gathers the proteins with the large molecules exiting the tube first and the smaller ones last. The molecules can then be detected by the use of a spectrophotometer.
In our lab, blue dextran, cytochrome c, DNP-glycine, bovine IgG, and ovalbumin were passed through the tubes. For our beads Sephacryl was used. Sephacryl is a polymer of allyl dextran covalently crossed g-linked with N, N -methylenebisacrylmide. S-200 was used because it has a fractionation range from 5 000 to 250 000. The process was run and the average fraction volume of our test was calculated to be 0. 63 mL.
Then to assist in the performance of the absorption spectra readings, 2. 5 mL of water was added to each tube. Then each tube s absorption spectrum was examined using a spectrophotometer. Each tube with a bluish tint was read at A 625 because of the known absorbance of blue dextran.
This was done also for red cytochrome c (A 412); yellow DNP-glycine (A 405); IgG, ovalbumin, and cytochrome c (A 280 for all tubes from firs blue dextran to last with cytochrome c). The data was then placed into the computer for evaluation. The significance of this procedure is the ability to purify or separate particles of a mixture. It is normally used along with other biochemical assays such as column chromatography to achieve the purest solution possible.