Gel chromatography by Mr. Vinayak Bodhankar

Introduction, theory
Instrumentation
Applications
TYPES OF GEL USED
SEPARATION PROCEDURE

2. ntent = **
> Introduction, theory => *

> Instrumentation

> Applications

6. + TYPES OF GEL ge

> They are uncharged and ini
analyzed. a

> Three types of gels are used:

1) Dextran (Sephadex

2. Agarose gel .

3. Acrylamide gels (synthetic gel)
Nature and properties of Gel:

= Chemically inert

= Mechanically stable

= Ideal porous structure

= Wide pore size give low resolution

= Uniform particle size

8. Characters of Sephadex: ge .
able gels.
Stable at pH 2-12. = u
= Their particles are free from i
= Insoluble in water and organic coal
= Swell in water and other hydrophilic si

= Requires bactericidal such as Hg acetate.

2. AGAROSE GEL f
> Obtained from agar and composed of alternating units of 1, Beta-D-galactose and
1,4 linked 3,6-anhydro alpha, L-galactose.

> This was subjected to epichlorohydrin to give agarose.

5

» 4
Characters: en À = d
= It dissolves in H,O at 50°C and on cooling form gel. ¿

= Insoluble below 40°C.

= Freezing destroys the gel.

10. 2 BG GB vpo
O Mobile Phase q 5
Q Columns

O Pump à. LA

U Detectors añ

1. Stationary Phase:

Composed of semi-permeable, porous
pore sizes.

Properties of gel beads:

Y” Chemically inert.

ll Sn u
Y” Mechanically stable.

v Has ideal and homogeneous porous structure

Y” Uniform particle and pore size.

—u
v The pore size of the gel must be carefully controlled.
—

11. GPC Column

Mobile phase

12. Synthetic elastomers (polybutadiene,
polyisoprene)

PS, PVC, Styrene-Butadiene Rubber, Epoxy resins

Polyolefins
Polyurethane
Proteins, polysaccharides

Toluene

Tetrahydrofuran (THF)

Tri-chloro-benzene
Di-methylformamide (DMF)
Water / Buffers

13. ercially available columns include:
= Analytical column: Be
" Preparative columns: 22-25 iar eter.
= Usual column lengths: 25,.30,,50)
4. PUMP:
= These are either syringe pumps or

flow rate.

15. à +
-
+ SEPARATION PR@CEDURE
1. Preparation of column for gel filtration:

L

2. Loading the sample onto the column: 4
The sample must enter the resin in the form of soluti ion using a syringe.
3. Eluting the sample and detection of compo: = E

Fractions are collected as the sample elutes from the tote TE

Swelling of the gel: Some esin come in ap

first in the eluent or ie desired buffer to swe.
Packing the column: Make a slurry of gel +
which is one third filled with the buf
Washing the resin: After packing, ar volt f

column to remove any air bubbles and to test the cole een

17. 3. FRACTIONATION: se >
ethod of separation, the similar substar

Thus, the separation of substangesfich haine

separated.

4. PROTEIN-BINDING Sur =

5. DETERMINATION OF MOLECULAI

It is assumed that the size of moleculei