MSM5003-ANALYTICAL TECHNIQUES LAB

Date: 20 October 2022

EXPT 4: GEL FILTRATION CHROMATOGRAPHY (GPC)

Structure
1.1 AIM
OBJECTIVES
1.2 INTRODUCTION
1.3 PRINCIPLE
1.4 REQUIREMENTS
1.5 PROCEDURE
1.6 OBSERVATION
1.7 CALCULATIONS
1.8 RESULTS
1.9 CONCLUSION
1.1 AIM
To introduce the principles of Gel Filtration Chromatography as a method that separates molecules
according to their size and shape.
OBJECTIVES:
To learn the chromatographic separation technique of a mixture of biomolecules by Gel Filtration
Chromatography using Sephadex G-50 column.
1.2 INTRODUCTION
Gel-filtration chromatography is also called as gel permeation chromatography (GPC) or size
exclusion chromatography (SEC). It is a form of column chromatography in which molecules are
separated based on their molecular mass or, more precisely, their Stokes radius. The Stokes radius is
the effective radius a molecule has as it tumbles rapidly in solution. A long, extended molecule has
a larger Stokes radius than a compact molecule of the same molecular mass. It is generally used to
separate biological molecules and to determine molecular weights and molecular weight distributions
of polymers, such as proteins.
1.3 PRINCIPLE
Gel filtration separates molecules based solely on their size.

➢ Larger molecules elute from (i.e., flow off) the column first because they have a shorter path
through the gel filtration beads (They’re too large to enter the beads).

➢ Smaller molecules elute from the column later than large molecules because they have a longer
path through the gel filtration beads (They enter the beads, which makes their path through the
column longer than the path for molecules that cannot enter the beads).
 ➢ Gel filtration chromatography is used primarily to purify proteins or other molecules of interest.

➢ Gel filtration chromatography can be used to estimate the size of unknown proteins.
The stationary phase in gel-filtration chromatography consists of fine beads that contain pores of
controlled size. The space between the beads is referred to as the void space (V0). The space within
the beads is referred to as Vi. The inert volume of the stationary phase is referred to as Vg. The
mobile phase fills the space between the beads and within the beads. The chemical and physical
properties of both the stationary and mobile phases in gel filtration are chosen to prevent, as nearly
as possible, interactions of proteins with the beads that are due to properties other than molecular
size. This is in direct contrast to other forms of chromatography in which interactions with the
stationary phase provide the basis of separation.
The sample is applied in a narrow band at the top of the column and then it is washed through the
column by the mobile phase. Large molecules in the sample that cannot pass through the pores of the
beads are excluded from the beads and are restricted to the outer voids. They elute from the column
after an amount of the mobile phase equal to V0 has passed through the column. Molecules that are
much smaller than the pores equilibrate with the entire liquid volume and elute at a volume equal to
Vt, which is the sum of V0 and Vi. Molecules that are small enough to pass through some of the
pores of the beads, however, elute at various volumes (Ve), depending on how small they are and
what fraction of the pores of the beads are accessible to them. Such molecules are referred to as
partially included. Molecules in the sample can be separated in order of their size by collecting
fractions as the mobile phase is eluted through the column, with the largest molecules eluting first
and the smallest last.
The figure below represents the principle of gel-filtration chromatography. A partition coefficient
can be calculated from the values above using the equation
Kav = (Ve-V0)/(Vt-V0)
A semi logarithmic plot of the relationship of Kav to molecular weight can elucidate the efficiency
of separation of molecules. Assuming that all molecules are of similar shape, the separation of
molecules on the basis of molecular weight will be greatest in the linear range of the curve.

Figure 1. Diagrammatic representation of Vt and Vo.

Figure 2. Gel filtration chromatography principle. Figure 3. Elution profile from

filtration Chromatography
 Figure 4. Molecular weight vs. elution volume. Figure 5. Partial structure of Sephadex

In this experiment, Sephadex G-50 column is prepared and three substances based on their molecular
weight are separated. A mixture containing three colored molecules: blue dextran (MW 2000 KDa),
cytochrome c (a red protein, MW = 12.4 KDa), and an unknown (Methylene blue MW:14.3KDa) is
given, and the separation occurs across the Sephadex column.

1.4 REQUIREMENTS
Apparatus: Sephadex G-50 (Sigma), Size exclusion column, ring stand with clamp for each column,
plastic transfer pipettes, Eppendorf tubes
Chemicals: Gel filtration buffer [Phosphate buffer (0.05M, pH: 6.5)], Gel filtration sample [Blue
dextran (2000 KDa), Methylene blue (14.3 KDa) and Cytochrome c (12.4 KDa).

1.5 PROCEDURE
➢ Swelling of sephadex:
✓ Sephadex is supplied as a dry powder and must be allowed to swell in excess buffer before use.
✓ Filter all buffers through a 0.22-μm filter to help prevent microbial growth or add 0.02% sodium
azide in the buffer. During swelling excessive stirring should be avoided as it may break the beads.
Do not use magnetic stirrers.
1. For five experiments, weigh out 8 g of dry sephadex G-50 in 150 mL of phosphate buffer, (0.05M,
pH 6.5) to obtain volume of your ~72-88 ml bed volume of your column.
2. Swelling times for the different types of Sephadex are different. The process of swelling is
accelerated by using a boiling water bath. At room temperature, you need at least 3-4 hours for
swelling to complete. Note: Prepare the slurry in advance for the practical.
3. After swelling is complete, allow to cool, decant the supernatant and floating fine particles by
aspiration.
4. Add buffer to make a 75% suspension (slurry) that is suitable for packing.
5. Degas the suspension before packing.
➢ Packing the column:
1. Choose a column of appropriate size for the application. The volume of the column should be 20 to
50-fold that of the sample to be applied. Usually tall thin columns with lengths 20-40 times their
diameter is used.
2. Now set up the column in a burette stand, making sure that the column is vertical and confirm this
by looking at it from two directions.
3. Tighten the screw clip at the bottom of the glass column provided and fill the column with water to
ensure that the clip is capable of stopping the solvent flow. Empty out the water. Note: Bed volume
of sephadex is 9-11ml (bed volume is volume of sephadex (ml) swelled per gram of dry sephadex
powder). Dry bead diameter is ~50- 150μm.
4. When packing the column, fill the column about one third full of elution buffer. Open the valve at
the bottom of the column so the liquid starts to drip out. Then, using a pipette, add dilute slurry
(swollen Sephadex G-50) of the column matrix material to the top of the column while stirring gently.
Keep adding slurry to the column while gently stirring intermittently. The goal is to produce a
uniform bed of desired height.
5. Using a minimal amount of Sephadex G-50. Please pour any unused slurry back into the bottle. Note:
Sephadex is very expensive and can be re-used.

➢ Equilibrate the column:

1. Once the gel is thoroughly packed into the column to the desired height, adjust the flow adaptor to
the surface of the gel bed.
2. Pass a further 2–3 column volumes of the buffer to be used in the separation. This stabilizes and
equilibrates the bed.
3. Readjust the flow adaptor to the surface of the gel bed.
4. The bed material should never be allowed to go dry. If the column has bubbles or does go dry it
should be repacked.
5. Checking the packed bed by running a freshly prepared and filtered solution of a colored substance.
Blue Dextran 2000 at a concentration of 2 mg/ml can be used for this purpose. The quality of the
packing can be checked by watching the progress of a zone of this substance through the bed. Visual
inspection of the bed in transmitted light may also reveal heterogeneities and air bubbles.

➢ Void volume (Vo) determination:

1. Dissolve the blue dextran in an equilibration buffer containing 5% glycerol at a concentration of 2
mg/ml. This concentration of blue dextran will give an A280 of ~1.0 in the peak fraction. Glycerol
is added to increase the density of the solution, but its use is optional.
2. The recommended sample volume is less than 2% of the total gel bed volume. Carefully apply the
blue dextran sample to the column (avoid disturbing the gel bed surface) to determine Vo and to
check column packing.
3. The flow rate should be ~7% of the column volume per hour. The skewing of the blue dextran band
represents a fault in the column, although some tailing is normal. The leading peak indicates the void
volume. Determine spectrophotometrically the elution volume for blue dextran (Vo for the column)
at 280 nm or 610 nm by measuring the volume of effluent collected from the point of sample
application to the center of the effluent peak.
4. The void volume (Vo), total volume (Vt) and the volume of mobile phase present inside the gel (Vi)
can also be calculated in the following ways:
a) Vt = Πr2 h, where Π = 3.14, r is the radius of the column and h is the height of the gel column.
b) Vo =1/3 Vt (void volume is about one third of the total bed volume)
c) Vi = Vt-Vo
➢ Sample application:
✓ Considerable care must be taken to avoid disturbing the bed surface. An uneven bed surface leads to
uneven separated bands and poor resolution.
1. Dissolve 40 mg of Cytochrome c (A) and 10 mg of methylene blue (B) in 1 ml of phosphate buffer
individually.
2. Mix 150µl of A and 150µl of B and Load the 300µl of the sample into the column.
3. Open the column outlet (clip) slightly and allow the sample to drain into the bed and then close the
column outlet immediately.
4. Slowly wash down the walls of the column with 0.3 ml buffer. Allow this to run in until the meniscus
just sink into the gel bed.

➢ Elution of the column:

1. Fill the column with buffer carefully.
2. As the molecules (mixture) gradually separate in the column, periodically add fresh buffer to the
reservoir to keep it full.
3. Continue collecting 1 ml fractions in each of the 2-8 tubes from the outlet of column.
4. Use different tubes for collection of the different coloured fractions.

1.6 OBSERVATION
The following are the observation in which coloured molecules are separated within the column
and finally eluted out.

Fig 6: Mixture of Cytochrome C & Methylene Blue added to column

chromatography as Sample 2
Fig 7: Orange color (high intensity) Cytochrome c was eluted in the 6th fraction, and dark
blue color (high intensity) Methylene Blue was eluted in the 11th fraction, and the color
intensity decreased with the increasing fraction

1.7 CALCULATION
Height of the column = 7.5 cm
Column diameter = 1.4 cm
Column radius = 1.4 cm/2 = 0.7 cm
Total volume/ Bed volume (Vt) = ᴨ r2 h
= 3.14 × (0.7cm × 0.7cm) × 7.5 cm
= 11.5395 cm3
= 11.5395 ml
1
Hence, Void volume (Vo)= Vt
3

= 1 × 11.5395ml = 3.8465ml
3

Flow rate calculation:

Flow rate is calculated by passing the buffer solution through the column and recording the time by
when the 1ml of buffer solution is obtained in the flow through.
Buffer solution Volume Time taken to elute
1. 1ml 40.67 seconds
2. 1ml 39.13 seconds
3. 1ml 39.38 seconds

119.18
The average time taken to elute 1ml of buffer solution = = 39.726 seconds = 0.66 minutes
3
1 1
Flow rate = = = 1.5152 ml / min
mean time 0.66 minutes
▪ The column length was 7.5 cm. After washing the column with buffer, the sampling was started.
▪ Phosphate buffer was added drop by drop, so that column won’t get dry.
▪ Orange colour (high intensity) Cytochrome c was eluted in 6th fraction.
▪ Dark blue colour (high intensity) Methylene Blue was eluted in 11th fraction and the color intensity
decreases with the increasing fraction.
▪ The absolute time of elution of 6th and 11th fraction was calculated:
For Cytochrome c:
Time taken to elute 1.5152ml of buffer solution = 1min
6ml ×1min
Time taken to elute 6ml of Cytochrome c solution = = 3.96 min
1.5152ml
Flow rate 1.5152 ml/min
Dead time (tD) Cyt c = = = 0.26 min
Vv 6ml

For Methylene blue:

Time taken to elute 1.5152ml of buffer solution = 1min

Time taken to elute 11ml of Methylene blue solution = 11ml ×1min = 7.26min
1.5152ml
Flow rate 1.5152 ml/min
Dead time (tD) Methylene blue = = = 0.1378 min
Vv 11ml

1.8 RESULT
▪ In the first sample blue color elute was obtained in the 6th fraction. The absolute time of elution was
3.96 minutes.
▪ In the second sample, orange-colored elute was observed at the 6th fraction, and the color intensity
of the 5th and 6th fractions was high.
▪ In the second sample, dark blue colored elute was obtained at the 11th fraction after which the color
intensity decreased with increasing fractions.
▪ The absolute time for elution of Cytochrome c and Methylene blue is 3.96 minutes and 7.26
minutes, respectively.
▪ This validates the principle of GFC that the high molecular weight analyte (Cytochrome c) is eluted
first and followed by the low molecular weight analyte (Methylene Blue),
▪ The separation of the biomolecules was observed concerning the above result.

1.9 CONCLUSION
Therefore, gel filtration chromatography is a technique that depends upon the molecular weight of
the biomolecule, size distribution of gel beads and elution limit. The movement of small molecules
is slower than the large molecules due to rapid diffusion into the pores. Oppositely, the larger
particles cannot move into the pores, move more quickly and leave the column rapidly.