EXPERIMENT

OBJECTIVE

To demonstrate the separation of biomolecules on the basis of their size by gel filtration
chromatography.

MATERIAL REQUIRED

1. Chromatographic kit that includes gel filtration column, gel filtration buffer (Phosphate
buffered saline; PBS) and test sample

2. Test tubes,

3. Distilled water,

4. Column stand

5. Micropipette.

PRINCIPLE

Chromatography is separation method that relies on difference in partitioning behavior between

mobile phase and stationary phase. The column hold the stationary phase and mobile phase carries the
sample. Gel filtration chromatography is also called size exclusion or gel permeation chromatography,
separate molecule on basis of size. The sample is applied on the top of the column containing the
porous beads as the molecule pass through the column of porous beads of cross field glycans they get
separated as follows.

 Large molecule cannot enter the porous beads and elute at the first peak in the chromatogram.
They elute first and this is called total exclusion.

 Intermediate molecule may enter the pores and may have an average residence time in the particles
depending on their size and shape. Different molecules have therefore different total transit time
through the column. This of a chromatogram is, called relative permeation region.

 Small molecules enter the pores and have the longest residence time on the column and elute
together as the last peak in the chromatogram which is called as permeation limit.
PROCEDURE

1. The column was fixed vertically at stand.

2. The column was equilibrated with 4 ml buffer and the buffer was drained out completely.
3. The column was loaded with 0.2 ml of sample on the column from its side.
4. When the sample was completely sunk, 1.0ml of buffer was loaded on it.
5. Buffer was allowed to flow out completely.
6. The column was kept filled with buffer until all colored biomolecules had eluted out.
7. The colored fractions were collected in different tubes.
8. The stop the flow, bottom stop cap was placed at their respective positions.

OBSERVATION

The order of elution of biomolecule was

1. Blue colored compound.

2. Brown colored compound.
3. Pink colored compound.

INTERPRETATION

 The blue colored component (dextron) were very large molecules, they exit fast from the
column.

 Brown colored component (heamoglobin) has intermediate size, they enter the porous bead but
their retention time was low.

 Pink colored component (cyanocobalmin) has small size, all of this enters the porous beads and
their retention time was high so they will late from the column.

PRECAUTIONS

1. The column should be placed vertically and should be steady

2. Column should not be dry

3. Column should be equilibrated properly with buffer (PBS)