Size Elusion Chromotography

Topics: Vitamin B12, High performance liquid chromatography, Folic acid Pages: 4 (698 words) Published: October 5, 2010

Size elusion chromatography is used to filter macromolecules according to their size, where large molecules pass around a gel matrix and is eluded first, while smaller molecules are eluded more slowly as they pass through a maze of pores created by the gel matrix. We used size elusion chromatography to separate a solution containing three components: blue dextran, methyl orange and vitamin B12. The elusion profile was constructed and the Kb value was calculated.


Chromatography is a method of macromolecule separation. Macromolecules can be separated according to a variety of properties: size, charge, mass, density and affinity. The method used is usually based upon the macromolecule target for separation. Proteins for example can be isolated according to their charge or polarity, depending on their R-group. Ion exchange chromatography is able to take advantage of these properties, and can be used to isolate a charged protein from solution. Another example would be the use of affinity chromatography to isolate receptors or enzymes that have affinities to specific ligands.

Chromatography is usually an easy process and is able to provide accurate and reliable results. We used size exclusion chromatography for the following experiment. A solution of methyl orange, blue dextrin and vitamin B 12 was passed through a gel (Sephadex G75) in attempt to isolate each comonent.

The sephedex G75 gel is used to separate molecules of large size (greater than 6x10⁴ Da), which is ideal for our solution: blue dextran greater than 2000000 Da, methyl orange 327.3g/mol and vitamin B 1350Da.


Column Preparation

No preparation was done. The columns were prepared and provided by the lab technicians

Sample Loading

The column was washed with elusion buffer by slowly dripping a total of 10 ml elusion buffer into the column, while the preparation solution was eluded, taking care not to disturb the...

References: Voet, D., Voet, J.G. (2004) Biological Chemistry 3 rd Edition pp. 459, USA, Wiley
Reed, R et al (2003) Practical Skills in Biomolecular Sciences 2e, Pearson Education Ltd, Edinburgh Gate.
Boyer, R (2006) Biochemistry Laboratory - Modern theory and Techniques, Benjamin Cummings, San Francisco.
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