HBC 1019 – Biochemistry I
Practical 4 : Protein Separation; Size
Exclusion Chromatography (SEC)
Tan Ka Yun
1. To gain basic gel filtration chromatography techniques.
2. Compare and contrast the use of different types of column chromatography in the
purification of protein.
3. Explain how naturally occurring or recombinant proteins are separated and purified using
4. Discuss how the structure and biochemical properties of proteins relate to purification
using column chromatography.
5. Apply the scientific method to solve problems.
Chromatography is a technique used to separate the component of a chemical mixture
which is mobile phase by moving the mixture along a stationary phase, such as gelatin. Different
components of mixture are caught by the material at different rates and form isolated bands that
can be analyzed. Chromatography often used in biotechnology for separation or purifying
biological molecules, like protein.
There are some of the common types of chromatography are gel filtration chromatography,
affinity chromatography and ion exchange chromatography.
Gel filtration chromatography is a technique that separates proteins, peptides, and
oligonucleotides on the basis of size. Gel filtration chromatography is also may be used for
analysis of molecular size, for separation of component in a mixture, or for salt removal or buffer
exchange from a preparation of macromolecules.
Size Exclusion Chromatography (SEC) is a widely used technique for the purification and
analysis of synthetic and biological polymers, such as proteins, polysaccharides and nucleic
In gel filtration chromatography, size exclusion chromatography (SEC) commonly referred to
microscopic beads which the column is packed with a matrix of fine porous beads. When the
protein solution is poured on the column, small molecules enter the pores in the beads. Larger
molecules are excluded from the holes, and pass quickly between the beads. These larger
molecules are eluted first. The smaller molecules have a longer path to travel, as they get stuck
over and over again in the maze of pores running from bead to bead. These smaller molecules,
therefore, take longer to make their way through the column and are eluted last. Depending on
the molecules, proteins may be separated, based on their size alone, and fractions containing the
isolated proteins can be collected.
The column bed is often referred as the mass of beads within the columns. The matrix of fine
porous beads works somewhat like a sieve, but in reverse. The beads have in them very small
holes. The beads act as “trap” or “sieves” and function to filter small molecules which become
temporarily trapped within the pores. Larger molecules pass around are “excluded” form, the
beads. In this practical, each column is provided and prefilled with beads that effectively separate
or “fractionate” molecules that are below 60,000 Daltons. Molecules below 60,000 Daltons
entered the beads and passed through the column more slowly as the liquid flows through the
column. The smaller the molecule, the slower they move through the column. Molecules greater
than 60,000 Daltons passed around the beads and are excluded from the column – also referred
to as the exclusion limit.
Materials, reagents and equipments:
Protein Mix that contains BSA (Bovine Serum Albumin) and Vitamin B1
Size exclusion chromatography columns
12 Collection tubes
Test Tube rack for holding 12 tubes
The 8 collection tubes are labelled sequentially from 1 to 8. The last two collection tubes are
labelled “Waste” and “Tube 1”. 4ml of Column Buffer is pipette into the chromatography column and
the buffer is drained into the “Waste” collection tube. The column is then placed onto “Tube 1”. A drop
of protein mix is carefully loaded onto the top of the column bed. The protein mix is allowed to enter
the column bed. After that, 250µl of column buffer is carefully added to the top of the column. The
buffer is allowed to run down the side of the tube and onto the top of the bed. The drops are collected
in “Tube 1”. When all the liquid has drained from the column, another 250µl of column buffer is added
to the top of the column. The drops are still collected in “Tube 1”. After all the liquid has drained from
the column, 3ml of column buffer is added to the top of the column by adding 1ml from the pipette 3
times. The column is transferred to tube 1 and 5 drops of the buffer is collected. After tube 1, 5 drops of
the buffer are then collected into tube 2 and this process is repeated until tube 8. The observation are
recorded in a table as below.
2 Slightly pink
3 Darker pink
10 Darker Yellow
1. Two different types of column chromatography techniques are gel filtration and affinity.
2. Gel Filtration Chromatography is used in this lab activity.
3. The column is packed with a matrix of fine porous beads. When the protein solution is
poured on the column, the larger molecules are excluded from the holes, and pass quickly
between the beads while the smaller molecules have a longer path to travel, as they get
stuck over and over again in the maze of pores running from bead to bead. This is
because the smaller molecules will enter the pores and move out from the pores again
while the larger molecules need only move straightly from the top to the bottom.
4. If a size exclusion chromatography is said to have an exclusion limit of 40,000 Daltons,
BSA (67,000 Daltons) would be excluded from the column because the size is bigger
than the exclusion limit while the vitamin B12 (1,350 Daltons) would be fractionated
from the column because the size is smaller than the exclusion limit.
5. We need to add more buffer after the protein mixture was loaded onto the column (at step
7 and 8 of the protocol) to prevent the column from drying because if the column dry, the
molecules cannot move through it.
6. BSA would exit the column first. The BSA has higher Daltons than vitamin B12 because
it is a larger molecule compare to vitamin B12.
7. If the following mix of molecules were purified using size exclusion chromatography
with exclusion limit of 80,000, the order in the molecules pass through the opening in the
bottom of the column would be:
First molecule to appear: Myosin
Second molecule to appear: Hemoglobin
Third molecule to appear: Myoglobin
As the protein solution is poured on the column, the molecules enter the pores in the beads. The
larger molecules will travel faster compare to small molecules because of the size exclusion
limit. The larger molecules can move straightly from top to bottom while the small molecules
will get stuck over and over again in the maze of pores running from bead to bead.