Size exclusion column chromatography of Hemoglobin and Vitamin B12 mixture.

SIZE EXCLUSION COLUMN CHROMATOGRAPHY OF HEMOGLOBIN AND VITAMIN

B12 MIXTURE.

By

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Size exclusion column chromatography of Hemoglobin and Vitamin B12 mixture.
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Introduction

Chromatography is a technique that is used to separate substances of a sample mixture in form of

a solution on the basis of difference in electrical charge, size, ability to bind to other molecules

or individual molecules size. Gel filtration or Size exclusion chromatography separates

components of a mixture on basis of difference in size of molecules. This is accomplished by

using a column filled with beads with pores of a polymeric gel. The gel allows smaller molecules

of a mixture to pass through its pores and within the beads while excluding bigger ones.

Substance with larger molecules go through a shorter distance since they don’t pass through the

interior part of the bed hence move quicker than smaller molecules.

Based on this principle, we set up the SEC column to separate vitamin B12 from hemoglobin.

Bio-gel P 60 was used for our case due its ability to exclude molecules having molecular mass

greater than 60,000 Daltons. Therefore hemoglobin and vitamin B12 can be separated as

hemoglobin molecular weight is 68,500 Daltons while else that of Vitamin B12 is 1355 Daltons.

Aims.

1. To show an understanding of the principles and operations of size exclusion

chromatography

2. To demonstrate the separation of hemoglobin and vitamin B12 mixture on the basis of

their size by gel filtration.

3. To learn how to set up a size exclusion chromatography experiment.

Hypothesis.

Ho: Hemoglobin and Vitamin B12 will separate on the basis of their size.

Ha: Hemoglobin and Vitamin B12 will not separate on the basis of their size

Method.
Size exclusion column chromatography of Hemoglobin and Vitamin B12 mixture.
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 The column was fixed vertically at stand.

 10 collection tubes were labelled sequentially (1-10).

 Last 2 tubes were labelled “waste” and “column buffer”

 Aliquot 4ml of Column buffer was put into the tube labeled column buffer

 The column was then equilibrated with 4 ml buffer and the buffer allowed to drain out completely.

 250ml of column buffer was carefully added to the top of the column (2x) and the process

of collecting drops into tube started (Size separation will work best when the column is

left undisturbed)

 3ml of the buffer was added to ensure the column does not run dry.

 5 drops were collected into tube 2 and then the column transferred to tube 3

 Same procedure was repeated in collecting fractions for tube 4, 5, 6, 7 and 8

 10 drops were collected for tube 10.

 To stop the flow, bottom stop cap was placed at their respective positions.

To stop the flow, bottom stop cap was placed at their respective positions.

Risk assessment.

Some of the reagents provided and process involving this experiment can be risky to student’s

health, therefore good laboratory management and procedures should adhered. They include

putting on safety clothing like gloves, goggles and laboratory coats. The following equipment’s

and process need to be handled with particular high level of caution.

Size exclusion column chromatography of Hemoglobin and Vitamin B12 mixture.
4

Activity Hazards Control Seriousness Likelihood Risk Rating

identified measures/pre of hazard of potential (S*L)

procedures\s cautions harm

ubstances occurring

Glass wear Shattering Be carful 40% 10% 4%

Hydrate Cause skin Put on 30% 15% 4.5%

copper irritation laboratory

sulphate coat and

gloves.

0.2 M Toxic fumes Avoiding 58% 36% 20%

Sulphuric and irritation direct

acid inhalation

and using it

in fume

hood.

Accidents Causing Long hair 39% 16% 6.24%

due spills, tied back,

movements Physical minimal

in the lab injuries, movements

breaking up (all

equipment. equipment

and reagents
Size exclusion column chromatography of Hemoglobin and Vitamin B12 mixture.
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should be

provided

within the

work bench).

Potassium Corrosive Put on 32% 23% 7.36%

hydroxide laboratory

0.4 M coat and

solution gloves
Size exclusion column chromatography of Hemoglobin and Vitamin B12 mixture.
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Results and analysis of results

Analysis

The order of elution of components was

 Brown colored compound then (tube 2 hemoglobin). Eluted the first because of having a greater

molecular weight of 68, 500 thus was excluded.

 Pink colored compound (tube 6 Vitamin B12). Eluted last because of smaller molecular weight

of 1355

More hemoglobin was collected in tube 2 than Vitamin B12 in tube 6

Size exclusion column chromatography of Hemoglobin and Vitamin B12 mixture.
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Hemoglobin and vitamin B12 are the two molecules which are being separated in this lab

activity. Hemoglobin, which is brown, has a molecular weight of 68,500 Daltons and is thus

excluded from the column. Hemoglobin will pass more quickly through the column and appear

in the early collection tubes, or fractions. Vitamin B12, which is pink, has a molecular weight of

1,355 Daltons and is thus fractionated by the column. The vitamin B12 molecules penetrate the

pores of the beads, becoming temporarily trapped. As a result, they pass much more slowly

through the column and appear in the later fractions.

Discussion

The aim of this report is to run a mixture of hemoglobin and vitamin B12 through a size exclusion

chromatography column in order to separate the two biomolecules making up our sample. This method

is also known as gel permeation chromatography. This chromatography technique allows biomolecules

to separate by size. It relies on portioning behavior between the liquid phase and solid phase. The column

hold the solid or stationary phase which is made up of beads and liquid or mobile phase carries the

sample that is the buffer. The sample mixture to be analyzed is put at the top of the column containing

the porous beads, then individual molecules are allowed pass through the column of cross field glycan’s

as they get to separate as follows: Larger molecule cannot permeate into the porous beads and elute as

early fractions. They are collected first and this volume is known as the total exclusion. Relatively

smaller molecules will enter the spaces between beads and will be exposed to resistant in movement.

They will move at slow rate and could have an average residence time in the particles depending on

their size and shape. Different biomolecules contained in the sample to be tested will show different
Size exclusion column chromatography of Hemoglobin and Vitamin B12 mixture.
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total movement period through the SEC column. This part of the column is known as relative permeation

region. Smaller biomolecules will enter the pores and have the longest movement period along the

column and will be collected as last fractions of the experiment and this volume is known as permeation

limit. The liquid that is used to dissolve our mixture components, hemoglobin and vitamin B12, to

develop the mobile or liquid phase is known as a buffer solution. Hemoglobin and Vitamin B12 mixture

is the test sample and is put on the size exclusion chromatography column set up then the individual

components within the buffer enter the top of the bed and permeate into and around the porous beads

and finally move through a small opening at the bottom end of the column. More buffer is added to the

buffer in order to complete the process after the sample mixture has entered the column bed. This is

followed by collecting the mobile phase in a number of test tubes. Into each tubes a given number of

drops is obtained depending on the rate of flow. Individual mixture component with larger molecules

will be collected in first tubes as it will pass quickly through the column. Sample component with

relatively smaller molecules will pass slow and hence collected in later tubes.

In this experiment Vitamin B12 and Hemoglobin are the components making up our mixture sample to

be separated by size exclusion chromatography. Bio gel P-60 was used, a polyacrylamide gel, it has the

ability to exclude biomolecules with a molecular weight more than 60,000 Daltons. After 10 fractions

were collected and the color of the individual content in the tubes observed it was found and proved

Hemoglobin was the larger while Vitamin B12 is the smaller of the two biomolecules. Test tube 2

contained brown color contents, which is the hemoglobin color and tube 6 color was found to be pink

the color of vitamin B12. The void volume (Vo) which is the volume found outside the beads was found

to be 29% of the (Vt) the total volume of the column. This meant that the hemoglobin and Vitamin B12

in our sample mixture co eluted therefore, the two individual biomolecules did not get to completely

separate in the case our experiment. Hemoglobin is larger in size with a molecular weight of 68,500
Size exclusion column chromatography of Hemoglobin and Vitamin B12 mixture.
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Daltons while else that of Vitamin B12 is 1355 Daltons, hence hemoglobin eluted in early fractions and

Vitamin B12 was the second to elute. During this experiment care should be taken not allow the column

to dry off and this is prevented by periodically adding a small amount of the buffer while collection

process is taking place. It should be ensured, the column is positioned vertically and very steady to

improve accuracy and reliability of the results. Additionally the column at the starts of the experiment

should be properly equilibrated.

Further suggestions on the experiment.

The success of a size exclusion column chromatography relies primarily on conditions that give high

selectivity and reduce internal pressure within the column when molecules are separating. They are pre

packed columns that are preferred with conditions which give reliable results. However there is need to

optimize the column in use to obtain better resolution. When a similar experiment is done in future using

size exclusion chromatography method, to separate a mixture that involves a protein, a reducing agent

should be incorporated in the column at low concentrations in order to maintain the native structure of

the protein. Avoid artificial formation of oligomers of the proteins to separate. The method can also be

scaled up by, after a separation has been done using a smaller column, larger columns can be prepared

to separate the same sample as smaller columns are prone to generating internal pressure that can limit

accuracy of separation. When doing the second separation using a column with larger cross section the

initial velocity used in smaller columns should be maintained. Additionally buffer that will give the best

protein stability should be chosen, volatile buffers like ammonium bicarbonate increase are
Size exclusion column chromatography of Hemoglobin and Vitamin B12 mixture.
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recommended. Number of additives should be reduced as they reduce the flow rate during separation

process and also may provide extra contamination to the sample.