PRACTICAL WRITEUP

ROLL-612
Demonstration of Separation of Molecules Based on Molecular Size by Gel
Chromatography
This laboratory exercise has been designed to visualize and analyze the chromatographic
separation of a mixture of blue dextran, α-chymotrypsinogen (protein) and potassium
ferricyanide on a Sephadex G-75 column (60 X 1.0 cm). Separation of the components i.e. blue
dextran (blue colour), α-chymotrypsinogen (colourless) and potassium ferricyanide (yellow
colour) of a green-coloured mixture can be visually seen in the form of blue- and yellow-
coloured bands distant by a colourless zone.

The approach is intended to be general but emphasis has been given to visualize the separation of
coloured molecules in the given mixture. The students prepare their own gel chromatographic
column, learn column packing and equilibration, load the sample, collect and monitor the
fractions, plot the elution profile and analyze their results.

This exercise can be completed in three days with three hours laboratory sessions. The whole
exercise is comprised of three parts: (i) packing and equilibration of the gel chromatographic
column, (ii) sample loading and elution of different components of the given mixture and (iii)
elution of individual components from the same column and analysis of results.

Materials and Methods

Materials
Sephadex G-75, α-chymotrypsinogen, blue dextran, potassium ferricyanide, PBS (phosphate
buffered saline) and distilled water. All materials used in this study were of analytical grade.
Absorbance measurements
The concentrations of blue dextran, protein and potassium ferricyanide were determined by
measuring the absorbance at 540, 280 and 420 nm, respectively, using spectrophotometer.
Preparation of a gel chromatographic column
Sephadex G-75 powder form (5 g) was allowed to swell in 200 ml of water at 90°C for 3 h. Fine
particles were removed by repeated decantation before packing of the gel into the column. A
glass burette was mounted onto a table in a vertical position with the help of an iron stand with
two clamps. The radius (r) of the glass burette was determined at three different places along the
height of the burette by collecting a known volume of water of 2 cm height (h) in the burette.
The volume (V) of the collected water was taken as equal to the volume of a cylinder and the
radius was obtained by substituting the values of V,  (3.14) and h (2 cm) into the formula, V=
r2h. The lower end of the burette received a disc of glass wool previously boiled in water and its
surface was covered by a few glass beads. The burette was filled with buffer [0.02M sodium
phosphate buffer, pH 7.0 containing
0.15M NaCl (PBS)] up to one fourth of its height and the gel slurry was poured slowly into the
column with the help of a glass rod in a single operation. The gel was left for 1 h to settle under
gravity and the outlet was opened slowly with a flow rate of 5 ml/h. The flow rate was increased
gradually after the gel settled down. Three bed volumes of the buffer (PBS) were passed through
the column at a flow rate of 40 ml/h to equilibrate and stabilize the gel bed. The column is
supposed to be stable if there is no change in the gel length during operation.
Sample application and elution
Before application of the sample, most of the buffer above the gel surface was removed and the
outlet was closed. The sample containing mixture of blue dextran (3 mg), α-chymotrypsinogen (6
mg) and potassium ferricyanide (2 mg) in 1 ml of PBS were layered gently on top of the gel bed
with the help of a micropipette and allowed to drain into the bed by slowly opening the outlet.
Once the sample had passed into the gel, 1 ml of PBS was applied in the same way at least two
times and finally connected to a reservoir containing PBS. The elution was performed with a
constant flow rate (30 ml/h) and fractions of 2 ml size were collected in tubes. Absorbance was
recorded at 540 nm for blue dextran (blue-coloured solution), 280 nm for protein (colourless
solution) and 420 nm for potassium ferricyanide (yellow-coloured solution). Absorbance values
were plotted against the elution volume to get the elution profile(s) of the given sample(s). The
volume required to elute the component at its maximum elution (peak position) was taken as the
elution volume of the component.

3. Results and Discussion

Figure 1. Separation of various components of a given coloured mixture on a Sephadex G-

75 column (60 X 1.0 cm). The green-coloured mixture (3 mg blue dextran + 6 mg α-
chymotrypsinogen + 2 mg potassium ferricyanide) in 1 ml of 0.02M sodium phosphate buffer,
pH 7.0 containing 0.15M NaCl was applied onto the column and the elution was performed at a
flow rate of 30 ml/h. Lane 1 shows photograph taken soon after loading the green coloured
sample. Lanes 2 and 3 show photographs taken 5 min and 20 min, respectively after application
of the sample.

Figure 1 shows visual chromatographic separation of different components of the mixture (green
in colour) containing blue dextran (blue in colour), α-chymotrypsinogen (colourless) and
potassium ferricyanide (yellow in colour) on a Sephadex G-75 column. The first lane shows the
sample (green-coloured mixture), when loaded onto the column. Lane 2 shows separation of
different components of the mixture into distinct blue- and yellow-coloured bands. The blue-
coloured band represented blue dextran while potassium ferricyanide band was of yellow colour.
Being bigger in size with very high molecular weight (2 X 106), blue dextran was completely
excluded by Sephadex G-75 gel particles and moved faster through interstitial spaces available in
the column. On the other hand, potassium ferricyanide, being a low molecular weight (329)
compound had both the inner spaces and interstitial spaces of the gel column available to it and
therefore, took longer time to pass through the gel column. Lane 3 shows a very clear visual
demonstration of the separation of two coloured components of the mixture, as they were seen to
be well separated and far apart from each other on the column. The middle colourless zone
between the blue- and the yellow-coloured bands was the protein, α-chymotrypsinogen with a
molecular weight of 25000. Elution of these components from the column in different fractions
also reflected their separation from each other as fractions collected very early were of blue
colour representing elution of blue dextran, followed by colourless fractions of the protein,α-
chymotrypsinogen and finally yellow-coloured fractions of potassium ferricyanide. Therefore,
visual monitoring of coloured bands on a column helps to understand the principle of gel
chromatography involving separation of the molecules based on their size.
Figure 2. Elution profile of the mixture containing blue dextran, α-chymotrypsinogen and
potassium
ferricyanide on Sephadex G-75 column (60 X 1.0 cm). The column was monitored for blue
dextran (fraction number 8-15) at 540 nm (A), α- chymotrypsinogen (fraction number 13-21) at
280 nm (B) and potassium ferricyanide (fraction number 22-30) at 420 nm (C).

Figure 2 shows elution profiles (absorbance versus elution volume) of three components of the
mixture, when monitored at 540, 280 and 420 nm for blue-coloured, colourless and yellow-
coloured fractions, respectively. Peak A represents elution profile of blue dextran, monitored at
540 nm with an elution volume of 22 ml. Elution volume of the blue dextran represented void
volume (Vo) of the column, as it was completely excluded by all the gel particles, resulting its
elution with the interstitial volume (void volume) of the column. The protein, α-
chymotrypsinogen eluted right after the blue dextran peak with an elution volume of 30 ml, when
monitored at 280 nm (Peak B). This is understandable as its molecular weight (25, 000) lies
between blue dextran and potassium ferricyanide. Therefore, some of the inner spaces and all
interstitial spaces of the gel column would have been available to it. The last peak (Peak C)
eluted from the column showed elution of potassium ferricyanide, when monitored for
absorbance at 420 nm. Its elution volume (50 ml) was equal to the sum of the void volume (Vo)
and the inner volume (Vi) of the column, as both the interstitial spaces and inner spaces of the
gel column were available to it. Therefore, subtracting the void volume (22 ml) from the elution
volume of potassium ferricyanide (50 ml) yielded the value of the inner volume (28 ml) of the
column. Thus, all the three components of the mixture, differing in their molecular weights, were
successfully separated by this column.

In conclusion, the exercise described here is a simple visual method for the introduction of gel
chromatographic technique. The technique is useful in being simple, economical, independent of
the use of any advanced instrumentation and interesting to visualize the separation of molecules.
Similar kind of exercises can be developed to teach other chromatographic techniques to these
students using coloured mixtures.