Separation of Colored Molecules Based on their Molecular Lab Report

Separation of Colored Molecules Based on their Molecular - Lab Report Example Chromatography is the method used in separating and analyzing complex mixtures. The separation process consists of a moving phase and a stationary phase. The mixture that is to be isolated is in the moving phase and it is made to pass through the immobile phase. Gel filtration is a procedure by which molecules are removed from a mixture based on their molecular shape and size. In gel filtration chromatography, the stationary phase consists of beads that have pores which span a narrow range that determines the size of the macromolecules to be fractioned. Large molecules that are large to enter the pores of the beads are separated thus comes out of the column first. Examples of gels used in gel filtration are Dextran, Polyacrylamide, and Agarose. When the column of Sephadex gel particles are packed: the volume of the solvent in the beads makes the internal volume (Vi) and that outside makes the external volume (Ve). The volume of the polymer forms the gel matrix volume (Vg). Bed volume is given by: The elution volume of a molecular substance is the volume of solvent that is needed to move out of that substance. Its least amount is the void volume. Small molecules have higher volume available them than the void volume. Where Kd is the coefficient of distribution and molecules have Kd between 0 and 1.The gel filtration column is divided into proteins of known molecular weight and then the Kd of each protein is calculated. The graph of Kd against log10 molecular weight is plotted and this graph allows the molecular weight of the unknown protein to be read from it. The buffer on top of the column was allowed to drain up to the level of the gel until the meniscus just disappeared.