Genei: Gel Filtration Chromatography Teaching Kit Manual

Gel Filtration Chromatography GeNeiTM Gel Filtration Chromatography GeNeiTM
GeNeiTM Gel Filtration

Chromatography
Teaching Kit
Manual
Cat No. 6103900011730

Revision No.: 00050516
 Genei Laboratories Private Limited, 2016  Genei Laboratories Private Limited, 2016
CONTENTS
Page No.
v Objective 1
v Principle 1
v Kit Description 4
v Materials Provided 5
v Procedure 7
v Observation & Interpretation 8
v Ordering Information 9
 Genei Laboratories Private Limited, 2016  Genei Laboratories Private Limited, 2016
Objective:
To learn the technique of separation of biomolecules on
the basis of their size by Gel filtration chromatography.
Principle:
Chromatography is a method of separation of
biomolecules that is based on the differences in partitioning
behavior between a flowing mobile phase and a stationary
phase to separate the components in a mixture. A column
holds the stationary phase and the mobile phase carries the
sample.
Gel-filtration chromatography, also called size-exclusion

or gel-permeation chromatography, separates molecules
based on the differences in their size. The sample is applied
on top of a column containing porous beads. As the molecules
pass through the column of porous beads of crosslinked
agarose, they get separated as follows:
• Large molecules cannot enter the pores and elute as

the first peak in the chromatogram. They elute fast and
this is called total exclusion.
• Intermediate molecules may enter the pores and may
have an average residence time in the particles
depending on their size and shape. Different molecules
therefore have different total transit times through the
column. This portion of a chromatogram is called the
selective permeation region.
• Small molecules enter the pores and have the longest
residence time in the column and elute together as the
last peak in the chromatogram. This last peak in the
chromatogram is the total permeation limit
(Refer figure 1).
 Genei Laboratories Private Limited, 2016  Genei Laboratories Private Limited, 2016 1
Column contains stationary phase
Porous gel bead
Smaller molecules retarded by

entering pores in beads
Molecules too large to enter

pores pass rapidly through
column
Fig 1: Schematic Diagram of a Gel Filtration Chromatography Components collected in different tubes
Column
Molecule Size
Fig 2: Schematic representation of sample separation by

gel filtration chromatography
 Genei Laboratories Private Limited, 2016 2  Genei Laboratories Private Limited, 2016 3
Kit Description: Materials Provided:

In this kit, a mixture of three different biomolecules The list below provides information about the materials
ranging in molecular size from 376 Da to 2000 kDa are supplied in the kit. The components should be stored as
supplied. These are separated on the basis of their size suggested.
through a gel filtration column. The movement of the samples
through the column is easily monitored as the biomolecules Quantity
are coloured, which also aids in collection of the resolved
biomolecules. Materials 6103900011730 Store
(5 Expts.)
6103900011730 : The kit is designed to carry out Gel Filtration Column (2 ml) 2 Nos. 4°C
5 experiments.
Gel Filtration Buffer 125 ml 4°C
Duration of experiment: Approximately 1 hour. Sample 5 x 0.2 ml 4°C
Materials Required:
Glassware : Test tubes.
Reagent : Distilled water.
Other Requirements : Column stand, Micropipette, Tips.
Note: Procedure:
• Read the entire procedure carefully prior to starting 1. Fix the column vertically to a stand.
the experiment 2. Equilibrate the column with 4 ml Gel Filtration Buffer.
• Allow the components to attain room temperature, Drain out the Gel Filtration Buffer completely.
1 hour before use. 3. Load 0.2 ml of the Sample onto the column, along the
• Reconstitute one sample vial per experiment, with sides of the column.
0.2 ml of Gel Filtration Buffer to get a homogenous 4. Allow the Sample to sink completely and then add
solution. Store the sample at 4°C and use within 0.2 ml of Gel Filtration Buffer.
3 months. 5. Allow the Gel Filtration Buffer to flow out completely.
• Do not let the column to dry, at any time. 6. Keep toping the column with Gel Filtration Buffer, till all
• Always open the upper cap first and then the lower cap the coloured biomolecules have eluted out.
to start the flow of Gel Filtration Buffer through column. (Approximately 20 ml of Gel Filtration Buffer is required/
• Similarly to stop the flow of Gel Filtration Buffer or to experiment).
store the column, fix the lower cap first and then the 7. Collect the colored fractions in different tubes.
upper cap. (Refer fig 2).
• Column can be used 2-3 times only. 8. Fix the lower cap and then the upper cap to stop the
• Store the column at 4°C after each use. flow of buffer. Store at 4°C for next use.
Observation: Ordering Information

Note down the order in which various molecules are
collected and interpret the results. Product Size Cat #
Interpretation: GeNei™ Gel Filtration 1 EA 6103900011730

• The Blue coloured component is blue dextran having a Chromatography Teaching Kit
molecular weight of 2,000,000 Da. These are very large (Consumables for 5 experiments)
molecules that exit fast from the column and are
collected as first fraction.
Email:
• The Brownish red coloured component collected as Sales:
second fraction is haemoglobin having a molecular sales@geneilabs.com
weight of 64500 Da. The molecules are of intermediate
size and they enter the pores but their retention time is Customer Support:
low, and hence take less time to exit the column. techsupport@geneilabs.com
• The Pink coloured component is vitamin B12 having
molecular weight of 376 Da. These are very small
molecules that enter or permeate the pores of the beads
and are retained in the bead for a longer time. They
take a long time to exit the column and are collected
as the third fraction.

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Gel Filtration Chromatography GeNeiTM Gel Filtration Chromatography GeNeiTM

GeNeiTM Gel Filtration

Cat No. 6103900011730

v Observation & Interpretation 8

Gel-filtration chromatography, also called size-exclusion

• Large molecules cannot enter the pores and elute as

Column contains stationary phase

Porous gel bead

Smaller molecules retarded by

Molecules too large to enter

Fig 2: Schematic representation of sample separation by

Kit Description: Materials Provided:

Observation: Ordering Information

Interpretation: GeNei™ Gel Filtration 1 EA 6103900011730

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