Separation 1

Separation of molecules according to their size through the use of Gel Filtration

and Dialysis

Tesnim Sairi

Dalhousie University

January 12, 2010

P. Briggs

BIOC 2610.03

Item Value
Flowchart /0.5
Presentation /1
Abstract /1
Introduction /1
Methods /1
Results /2
Discussion /1
Supplementary Questions /2
References /0.5
TOTAL /10

Abstract

Solutions have a variety of different molecules and to understand the type of

molecule located in these solutions one can separate them according to size. Two
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techniques that can be used to separate different molecules according to their size are gel

filtration and dialysis. The two work by either eluting molecules size by size or by

diffusing molecules through semi-permeable membranes. A solution was put into a

dialysis sac and the molecules inside the sac were allowed to diffuse through the sac’s

semi-permeable membrane into the diffusate according to the different in concentration.

Two compounds were placed into a column where they were eluted in respect to their

size. The different solutions were than further compared by measuring their absorbance at

two different wavelengths.

Introduction

Gel filtration and dialysis are two types of techniques that separate molecules

according to their size. The two techniques separate molecules in different ways. Gel
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Filtration works by separating molecules by the way they interact with the beads filling

up the column. The beads have pores that the molecules can penetrate. The small

molecules are able to diffuse the pores of the beads and continue going down the column

in that fashion. The large molecules cannot diffuse through the pores due to their size and

instead skip past the beads and flow down the column. Using that way the larger

molecules can be eluted out of the column faster than the smaller molecules that have to

diffuse in and out of the different beads. In Dialysis the separation of the molecules is

related to the concentration gradient inside and outside the dialysis sac. The molecules

inside the sac want to diffuse out of the sac to the area that has low concentration. To get

to the low concentration area the molecules must diffuse through the membrane of the

sac, which is semi-permeable and as a result will only let out small molecules. The

dialysis sac will therefore contain the large molecules while the outside solution will

contain the small molecules that were able to diffuse through the membrane. The gel

filtration is a better indicator of the actual size of the molecule because it is able to better

separate molecules according to the different sizes. The semi-permeable dialysis

categorizes the molecules as being either too large to pass or just small enough to diffuse.

The gel filtration is able to better separate because if the molecule was really large that it

would pass right through the beads but if the molecule was medium sized it could

penetrate the pores a little bit but not diffuse the entire way making that molecule slower

than the large ones yet faster than the smaller ones that can diffuse completely. The gel

filtration takes a longer time depending on how long the column is and how packed it is.

The dialysis depends on the concentration inside and out and would therefore take less

time to observe separation.

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Methods

Part 1: Column Chromatography

A sephadex column was filled with water and drained from the bottom. A test

tube was laid to collect the solution being drained. The blue solution was collected into

one test tube and the yellow solution was collected into a second test tube. The solutions

were than placed into cuvette and an absorbance reading was taken at 400 nm and 600

nm.

Part 2: Dialysis

In dialysis one 500 µ L of mixture was placed side a dialysis sac that was

previously washed with water. The air was removed from the sac and the top was tied up

just like the bottom. The sac was placed at the bottom of a beaker and water was poured

over the sac. The water over the sac was removed and replaced by new water ever 10

minutes. The water that was removed as placed into a designated test tube. The water was

replaced 6 times. The absorbance was measure for the water that was taken out of the test

tube every 10 minuets at a wavelength of 400 nm and 600 nm.

In dialysis two 250 µ L of mixture was placed into a closed off sac and tied u

from the top. The sac was placed in a beaker and 4 mL of water was added on top of the

sac. The water was taken out of the beaker after one minuet and the absorbance was

measure. The water was put back into the beaker and than taken out once again after

another minuet, this was done for a total of 5 minuets. After the 5 minuets the water was

left in the beaker for 5 minuets. Every 5 minuets an absorbance reading was taken for a

total of 30 minuets. Once the end point had been reached 250 µ L of the mixture was
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added straight into the water and the absorbance reading was taken. (Briggs, 2010)

Results

The absorption of the solution that came out of the dialysis sac was measured at

wavelength 400 and 600 for the two-dialysis procedures. The solutions being absorbed

are separated by different time intervals (See appendix figures 1-3). The gel filtration

eluted different amounts of two solutions Dextran Blue and Riboflavin phosphate. The

Dextran Blue came out first and the riboflavin came out second. The volume of the eluted

solutions was measured and the absorbance was taken at wavelength 400 and 600 (See

appendix table 1).

Percent recovery Calculation

Dextran Blue (DB):

Mg start = (6mg /mL) x (0.1 mL) = 0.6 mg

Mg end = (0.250 mg/mL) x (4.01 mL) x (0.078/0.206) = 0.379

Percent recovery = [(0.379) / (0.6)] x 100% = 63.3 %

Riboflavin phosphate:

Mg start = (2 mg/mL) x (0.1 mL) = 0.2 mg

Mg end = (0.02 mg/mL) x (6.21 mL) x (0.402/0.262) = 0.1905

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Percent recovery = [(0.1905)/(0.2)] x 100% = 95.3%

Discussion

Since dialysis relies on concentration gradient to separate the different molecules

the diffusate that the smaller molecules are being diffused into two must be replaced. In

the Dialysis sac the concentration is high and therefore the molecules want to leave the

sac and enter into the diffusate, which has a lower concentration. Eventually the

molecules that leave the sac and enter the diffusate will produce equilibrium between the

two solutions and that will result in no movement. The small molecules that are being

moved are usually salts of some kind and if they are no longer moving out of the sac that

means the proteins located in the sac are not fully purified. Replacing the diffusate

ensures that the concentration becomes lower than the concentration in the sac allowing

the small salt molecules to diffuse out of the sac and leave the large purified protein

molecules inside.

Reference

1. Briggs, P. (2010). Biochemistry, yours to Discover: Introductory Biochemistry

Lab. Halifax
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Appendix

Figure caption:

Figure 1: The absorbance of the solutions from dialysis one at 600 nm

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Figure 2: The absorbance of the solutions from dialysis one at 400 nm

Figure 3: The absorbance of the solutions from dialysis two at 400 nm. The dotted

line at the top represents the endpoint of the dialysis.

Table 1: Column chromatography raw data for compounds Dextran Blue and

Riboflavin phosphate including the percent recovery.

Table 1

Dextran Blue (DB) Riboflavin

phosphate (FMN)
Fraction Volume 4.01 6.21
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(mL)
A600 (fraction) 0.078 0.402
A600 (standard) 0.206 0.262
Conc. Standard 0.250 0.0200

(mg/mL)
Percent recoveries 63.3% 95.3%