Question

1.) Describe the goals of a Gel Filtration Chromotography Experiment???

2.) Explain each key theoretical principle of a Gel Filtration Chromotography, and how they help acheive the goal???.

4.) Explain the key equations used in the Gel Filtration Chromotography experiment and the terms involved in the equation????

HI im trying to prepare for a lab/report and i have some questions i could use help with please :) over all having trouble seeing how everything ties together etc :) thank you again for the help

volume inside the gel, Vi (the internal volume of the gel). Or: V = V. + V (1) These variables are illustrated in Figure 2. Large molecules that are completely excluded from the pores of the column will only have access to the V, Small molecules, that are completely included in the pores of the column, have access to both the void and internal volumes or V. + co WWW 0 Bu W Figure 2. Elution of proteins from a gel filtration column and the determination of molecular weight re to search o 2 goe W X 67%

Answer 1

1. Goals of Gel filtration chromatography

a. Gel filtration chromatography, also known as size exclusion chromatography is the method of separating differently sized molecules of proteins, polysaccharides or nucleic acids. We can even separate different components of DNA strands.

b. If at all, one has to do a specific study of a type of protein, values like molecular mass and the related parameters need to be known. This can be done only when the particular molecule is not in an unknown mixture.

c. Qualitative as well as quantitative analysis can be done. Identification and purity testing can be done, when a certain protein is separated out.

2.Theoretical principle

a. It is a partition type of chromatography where the sample will separate out on the basis of its size, i.e. the size of its molecules. A mixture of proteins would be run through a stationary phase, which consist of a powder, which swells up and gives a beaded slurry.

b. The gaps between the bead are the ones which will capture the similar sized molecules and hence bring about the separation.

c. An eluting agent will bring out the large sized molecules first followed by the small sized ones

3. Important expressions

There are two expressions and only 5 parameters to be known.

V_t = Total liquid volume of the column

V₀ = void volume (liquid contained in the voids)

V_i = liquid volume inside the gel

_Ve = eluted volume

K_d = distribution or partition coeffiecient

V_t = V₀ + V_i

K_d = $\frac{V_{e}-V_{0}}{V_{t}-V_{0}}$