Column Chromatography

Question 1

Column Chromatography

Answer 1

is a process used to separate molecules based on their molecular mass, charge, or solubility.

Question 2

How do molecules separate in column chromatography?

Answer 2

Molecules that are strongly attracted to the stationary phase will be retained and molecules that are not attracted will be eluted faster, resulting in a separation.

Question 3

What is the stationary phase and mobile phase in column chromatography?

Answer 3

Sample is passed over a stationary phase (fine granular or bead like matrix) by the flow of a mobile phase (cutting solvent or eluent).

Question 4

Gel Filtration Chromatography

Answer 4

in this type of column chromatography molecules are separated by molecular mass, i.e. size.

Question 5

What is special about the stationary phase in gel filtration chromatography?

Answer 5

Stationary phase consist of fine beads containing pores of controlled size. Large molecules can not enter the beads and they are eluted first and small molecules enter pores and are eluted later.

Question 6

At what volume do large molecules elute?

Answer 6

Vo

Question 7

What is Ve?

Answer 7

Molecules that are small enough to pass through some of the pores of the beads, elute at various volumes Ve.

Question 8

At what volume do small molecules elute?

Answer 8

Vt

Question 9

Partition Coefficient Formula

Answer 9

Kav = (Ve-Vo)/(Vt-Vo)

Question 10

How does the gel filtration chromatography diagram look like?

Question 11

Limitations of gel filtration chromatography?

Answer 11

the most remarkable limitation is sample dilution.

Question 12

What does the semilogarithmic plot of the relationship between Kav and MW suggest?

Answer 12

Lower molecular weight, greater Kav.

Question 13

Semilogarithmic plot of the relationship between Kav and MW

Question 14

What are the four main steps of column chromatography?

Answer 14

1. Equilibration: a buffer that is compatible with the portion of interest and the resin of choice is passed over the column.
2. Binding: proteins bind to the column via several non-covalent interaction. Proteins that interact only weakly or non-specifically with the resin are removed by washing the column.
3. Washing: ensures proteins that are bound by non-specific interaction are removed and only specific binding is favored.
4. Elution: proteins that interact strongly with the resin are eluted from the column.