Gel Filtration Chromatography: protein fractionation

Well known as GFC, this is usually used to fractionate protein. It is based on molecular size, when larger particles will eluete first, followed by smaller molecule. In my experiment, I will employ this to fractionate humic substance. There are several points from which the method in using this GFC. GFC is included in Size Exclusion Chromatography. Size exclusion chromatography (SEC) is a chromatographic method in which particles are separated based on their size. When an aqueous solution is used to transport the sample through the column the technique is known as gel filtration chromatography.

GFC needs an apparatus such as column with small pores polymer beads inside that have pores of different sizes. More information can be seen in here

So, what you need are:
1. Glass column (such as C 16/70 Pharmacia):
be careful, make sure you really know what you need. There are two types of column, one with gel prepacked column such as superdex 200 column or empty column for which you have to pack it by your self, this column such as C16/70, XK groups column.

2. Gel filtration media (such as Sephadex 15, Sephadex 20 etc.)
Choosing best gel medium is really crucial to get high resolution of your chromatography process. Make sure you pick the correct one.
3. Blue dextran 2,000,000 MW, I use this to measure void volumn, but there are plenty of course out there, chemicals you can pick to measure void volumn.
4. Buffer solution:
0.1 M Sodium Phosphate or Tris-buffer, at pH 7

Good guidance about how to work on this GFC can be found at mol wt chromatography, I found this really helpful for me to get some insight, especially for my experiment. I just found a good site where you can get very basic and complete information about gel filtration chromatography, you can get the handbook and products catalog in GE Healthcare Sciences.

My Own Procedure

Like what I mentioned above, GFC is a form of column chromatography. To use GFC, you will deal with a solid stationary phase (column or glass tube) and a liquid mobile phase (buffer or solvent). Mobile phase is allowed to flow through the solid phase in the column. Oke, let’s get thru with step by step procedure, well MY procedure.

1. Prepare GFC components, in this case the column. I would probably use C 16/70 Pharmacia column, this is equilibrated with mobile phase PBS (phosphate buffer saline) of sodium phosphate 0.1 M, pH 7.

2. Buffer should flow pass column with flowrate between 0.8-1 mL/min. Fill the reservoir of column with buffer by slow pipetting PBS onto the top of the gel matrix, until buffer is 5 cm above gel surface.

Next procedure can be seen in mol weight determination. The difference between my procedure with this site, is that, I will look for 10 mL sample instead of 0.4 mL. This to allow good measurement due to TOC sample requirement. Samples will be taken from humic acid solution 9 ppm, instead of protein.

What to record are: fraction for each 10 mL samples taken (DOC and UV purposes), elution volumn (Ve) and MW (of known standard). Note and plot graph for log (MW) and volumn samples for standard. Determine the formula, and measure UV254 absorbance. Fill the column with humic acid solution (without TiO2-UV), get the fraction in terms of sample volumn (elute volumn, Ve), plug in with the formula, obtain the MW, and determine the UV254 absorbance. The analogue with samples after UV irradiation only and TiO2-UV irradiation. Thus, we can know the molecular weight distribution.

Feed water
I will use commercial humic acid (Aldrich) and its MW (molecular weight) fractions will be used as the feed water. A stock solution is perapred by dissolving 1 gr humic acid in 1 L deionized water (Milli-Q) and filtering through 0.45 micro CA (cellulose acetate, Whatman) membrane filter. The filtrate is stored at 4 degree C for subsequent uses.

Fractionation of humic acid
The humic acid solution is subject to GFC for fractionating humic substances with respect to its apparent molecular weight (AMW). The packing material used probably Sephadex G-75 gel (Pharmacia). The chemical PEG, polyethylene glycol (Merck) with its respective MW of 400, 1500, 6000, 10,000 and 20,000, will be used to calibrate the column AMW. The eluent will be collected in a fraction collector and four fractions aer thus will be obtained for subsequent UF experiments. Each fraction will be analyzed for dissolved organic carbon (DOC), UV absorbance and FTIR.