SEPARATION TECHNIQUES
B.Pharm PCG 301
Dr. Ibrahim Malami
Department of Pharmacognosy & Ethnopharmacy, Faculty of
Pharmaceutical Sciences, Usmanu Danfodiyo University, Sokoto
Centre For Advanced Medical Research and Training (CAMRET),
Usmanu Danfodiyo University, Sokoto
GEL FILTRATION CHROMATOGRAPHY
Introduction
Gel filtration chromatography (GFC) – size-exclusion
chromatography.
Techniques – employs a cross-linked dextran which, when added to
a suitable solvent (e.g. chloroform, ethyl acetate or water), swells to
form a gel matrix.
Solid support (gel) – contains a controlled pore size that allow small
molecules (<500 Da) to be retained in the matrix, larger molecules
(>500 Da) are excluded and move quickly through the gel.
Principle – based on the separation of solute molecules according
to their size in solution.
Gel is loaded into a column and the extract is added to the top of
the column. Larger molecules are the first to elute, followed by
molecules of smaller size.
Function – is to provide a continuous
decrease in accessibility for molecules
of increasing size.
As a result, molecules elute in order of decreasing size; the largest
molecules are eluted from the column first and the smallest are
eluted last.
Materials for GFC
Stationary phases
Polyacrylamide: are porous gel matrix beads – particle size 45
to180 μm.
Used as a stationary phase to carry out purification of
macromolecular natural products, e.g., carbohydrates, peptides,
and tannins.
They swell in water and are almost exclusively used with water
as the mobile phase, although up to 20% alcohol can be used to
improve the solubility of the sample.
E.g., Bio-gel gels – in various sizes (e.g., P-
10 gels allows separation of molecules
within range of 1,500 – 20,000 MW)
Carbohydrates: A cross-linking of polysaccharides leads to the
formation of porous polymer of carbohydrates beads.
E.g., Sephadex
They are gel filtration resins, with varying degrees of porosity
(bead sizes – ranges between 20 and 300 μm).
Column preparation using Sephadex
Choose a glass column (size depends on the required bed volume)
– 1 mL of swollen gel is required for the separation of 1 mg of
extract.
Suspend the gel in a mobile phase (CHCl3, EtOAc or MeOH) and
allow to swell overnight to ensure the gel is completely swollen.
Partially open the outlet valve on the column and slowly fill the
column with the gel with gentle tapping during the packing to ensure
a good column.
Adjust the flow rate to between 1 and 5 mL/min to allow the packing
bed to settle.
After the bed is settled, allow the solvent level to drop to a level just
above the packing material.
Sample application
Dissolve the sample in the mobile phase and carefully apply the
sample solution onto the column taking care not to disturb the bed.
Open the outlet valve and allow the sample to flow into the bed.
Add another appropriate volume of the mobile phase to the bed and
allow to flow through so that the sample solution will be completely
washed into the bed.
Sample elution and collection
Open the column outlet and adjust the flow rate to 2–4 mL/min. The
flow rate should be set to give a contact time of approximately 50–
100 min.
Collect the fractions, normally about 5 mL/ fraction.
