Practical 6

DESALTING OF A PROTEIN BY GEL FILTRATION

Introduction

Gel filtration chromatography is a separation based on size and shape. It is also called
molecular exclusion. Gel filtration chromatography separates proteins, peptides, and
oligonucleotides on the basis of size. Molecules move through a bed of porous beads,
diffusing into the beads to greater or lesser degrees. Smaller molecules diffuse further into the
pores of the beads and therefore move through the bed more slowly, while larger molecules
enter less or not at all and thus move through the bed more quickly.

In gel filtration chromatography, the stationary phase consists of porous beads with a
well-defined range of pore sizes. The stationary phase for gel filtration is said to have a
fractionation range, meaning that molecules within that molecular weight range can be
separated. Desalting and buffer exchange both entail recovering the components of a sample
in whatever buffer is used to pre-equilibrate the small, porous polymer beads (resin).
Desalting occurs when buffer salts and other small molecules are removed from a sample in
exchange for water with the resin being pre-equilibrated in water. Buffer exchange occurs
when the buffer salts in a sample are exchanged for those in another buffer.

Objective
Determining the presence of haemoglobin and ammonium sulphate with gel filtration method

Materials

Sephades G-25 Test tube

Haemoglobin Solution Pasture pipette
Distilled water Spectrophotometer
Nesslers reagent Micto-cuvettes
Glass tube
Procedures

1. A 10cm long column of Sephadex G-25 in the glass tubing was provided and series of test
tubes which was marked to show 1ml level was prepared.

2. A solution of haemoglobin was prepared by diluting with water a small portion of

crystalline suspension.

3. The solution was applied and allowed to soak in by using a Pasteur pipette. Water was then
added and eluted into the prepared test tubes.

4. The fractions were then collected.

5. Each fraction was read at 535m on the spectrophotometer, using the micro-cuvettes.

6. 0.5ml Nesslers reagent was added to each fraction and read without delay at 480m to
detect the presence of ammonium sulphate in the fractions.

7. The traces of turbidity developed into before the actual readings, it was filtered or
centrifuged.

Result

Fractions Fractions at 535m Fractions at 480m added 0.5ml Nesslers reagent

1 0.680 2.150
2 0.413 1.914
3 0.108 2.720
4 0.133 2.154
5 0.534 2.273
6 0.091 2.028
7 0.132 2.483
8 0.086 1.819
9 0.086 2.317
10 0.074 0.074
 Graphy of protein by gel filtration
3

2.72
2.5 2.483
2.273 2.317
2.15 2.154
2 2.028
1.914
1.819

1.5

0.68
0.5 0.534
0.413

0.108 0.133 0.091 0.132 0.086 0.086 0.074

0
1 2 3 4 5 6 7 8 9 10

535m 480m

Discussion

Sephadex is a bead-formed gel prepared by cross-linking dextran with epichlorohydrin. It is

supplied in its dry form. The gel swells in aqueous solutions. Different types of Sephadex
differ in their degree of cross-linking and hence in their degree of swelling and their
molecular fractionation range. Sephadex G-25 is suitable for removing lower molecular
weight from weight larger than 5000 due to porous resin inside the column. The lower
molecular weight will enter the pore of the resin and they are forced to follow the circuitous
path before exiting the beads while larger molecular weight molecules flow around the beads
taking direct to path down. Therefore, rate flow of lower molecular weight molecules is
slower than larger molecular weight.

From the graph ,we can see that first three fraction the absorbance reading is low as the
content of haemoglobin is less due to haemoglobin has not reached to the bottom of the
column. The reading of absorbance at fraction 4 and 5 increases as the haemoglobin has
reached to the bottom of the column. Therefore, the content of haemoglobin at fraction 5 is
the highest. The reading of absorbance decreases from fraction 6 to 10 because most of the
haemoglobin has been eluted into the previous fractions.
The presence of ammonium sulphate was detected using the fraction by adding 0.5ml of
Nesslers reagent to each fraction and reading the absorbance without delay at 480nm. .
Nesslers reagent is an alkaline aqueous solution of potassium tetraiodomercurate(II),

K2HgI4. The reagent is to test ammonium sulphate which contains ammonium ions in
haemoglobin fraction. K2HgI4 will react with ammonium ion and form a brown precipitate.

Conclusion

Gel filtration is a chromatography is a convenient method to perform separation of protein.

Based on the experiment, fraction 5 has the highest peak when tested under the absorbance of
535m which shows that it has the highest concentration of haemoglobin. However, there is
also a drastic drop from fraction 5 to 6.

References

L. Hagel, Gel Filtration in Protein Purification, Principles, High Resolution Methods

and Applications. J.-C. Janson and L. Rydn (editors), VCH Publishers Inc., New
York, 1989, p. 63106. [Accessed on 4th March 2017]
Man, P. (2017). Gel Filtration and Its Role in Desalting and Buffer Exchange. [online]
Info.gbiosciences.com. Available at: http://info.gbiosciences.com/blog/gel-filtration-
and-its-role-in-desalting-and-buffer-exchange [Accessed on 6th March 2017].