Gel chromatography uses cross-linked polysaccharide or polyacrylamide beads as a stationary phase to separate molecules based on size. The movement of solutes through the column depends on the flow rate of the mobile phase and diffusion of molecules through the pores of the stationary phase beads. Common stationary phases include Sephadex and Sepharose gels, with pore size determining the size of molecules that can diffuse into the beads. Gel chromatography is used for applications like group separation, molecular weight determination, and concentration of polymer solutions. Careful selection of the gel, column size, sample volume, eluent, and flow rate is required to plan a successful gel filtration experiment.

1. Gel chromatography

2. Molecular seving

3. Movement of the solutes depend on :
Flow rate of the mobile phase
Diffusion (( size ))

4. Classification
Gel
chromatography
Mobile
Filtration
Permeation
Stationary
Poly styrene
polymers
Sephadex
Pours glass

5. Sephadex chromatography
Se
Dextran
Epichlorohydrin

6. Cross linking

7. Types of sephadex according to cross
linking
G 10 G 25 G 100 G 200
Number refers to the pore size
Indicate the approximate molecular mass of exculded
molecules
Hydrophilic

8. Water regain
The weight of water taken up by
1g of dry gel is known as the
water regain
Wr
The approximate bed volume
= double the water regain value

9. Vi
Vo
Vt
Ve
(vr)
Vg

10. Technique of sephadex
chromatography
Swelling
Packing
Buffer))
Void volume
Column
development

11. Application of sephadex
Group separation &
fractionation &
purification
Molecular weight
determination

13. Vs = vi +(vg)
Large and small molecules

14. Application of
sephadex
Removal of small molecuels from polymer

15. Application of sephadex
Concentration of polymer
solution
Buffer change

16. Application of sephadex
Centrifugal
desalting
Highly
viscous
polymer
Polymer is
obtained the
void volume
poured into a basket
centrifugation
Slury of swelled sephadex
Sediment : Vg +Vi
Polymer
salt
Poured into the gel
Stirred & rotated

17. Application of sephadex
Adsorption
chromatography
Partition
chromatography
Aromatic amino
acid on sephadex
25
P- hydroxy phenyl
pyruvic acid
Hydroxy
nitrile
Innert suuport
Used to fractionate antibiotics ,
lipids and steroids , peptides ,
aminoacids and transfer RNA

18. Application of sephadex
Special sephadex gel
Sephadex LH
-20
Sepharose
gel
Sephadex ion exchanger

19. Planning a gel filtration experimet
Choosing the gel type
Choosing the particle size of the gel
Choosing the size of the column
Choosing the sample volume
Choosing the eluent
Choosing the flow rate of the eluent