Size Exclusion chromatography is a useful method for separating molecules according their sizes and shapes. Stationary phase consists of beads with pores. Large molecules that has great volume than pore size can be excluded from solvent. Smaller molecules has to pass through those beads, this way smaller molecules obtained at last. Sample has to be dissolved in a liquid or gaseous fluid that called as mobile phase.Stationary phase consist of porous matrix to retard molecules by some properties.In the size exclusion method this matrix consist of porous structure with no binding affinity, and separate molecules by their size. Sample has to interact with these phases to purifying by their size through column. Eluent, has analyte, would be obtained this way. In this section stationary phase was Sephadex G75 that seperates molecules that has molecular weight between 3.000- 80.000 Da .(1*)
Materials and Methods:
200 mM NaCI 100mM Phospate buffer with pH 7 was prepared.Chromatography coloumn was prepared with bead powder G75. 80 μl sample was loaded with blue dextran and orange G.Loaded side were covered with piece of coton and 1 volume elution buffer added through experiment. When blue dextran reached to bottom of the column fractions were collected. 4 droplets for each tube was collected in this step. After orange G were obtained few more tube was collected, then collecting procedure was stopped.
Table 1. shows log MW and Ve/Vo values of standard samples and MBG 311’s Ve/Vo value.
Molecular weight calculation of MBG311 :
X=1.460722 when it placed in to equation
Y=1.765 that is logMW
101.765= 58.2 molecular weight.
Blue dextran was obtained first, because it has higher molecular weight than others.It can excluded from porous structure so it can reach faster than others.Oval albumin would be obtained before than lysosyzme because it has a greater molecular weight than lysosyzme. Oval albumin has 42.7 kDa and lysosyzme has 14.388 kDa molecular weight. Orange obatined last because it has smaller molecular and has to loss time when pass through bead’ porous structure of gel. Size exclusion one of the purifying methods for proteins. Other procedures can be applied for purification, like ion exchange,salting out or affinity chromatography . When applying ion exchange or salting out , generally obtained proteins not specific as size exclusion, because similarity between ionic forms and salt concentration not enough to seperate similar protein. For higher specific result size exclusion could be chosed in among them.However, affinity chromatography gives higher specific results and pure protein of interest. If specifity is important, affinity chromatography could be chosen rather than size exclusion. When higher amount of protein is needed an purity has second place than size exclusion could be chosen.2*