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Presentation Group No 2: Mehtab Nusrullah Waseem Abbas Dilshad Ahmad H Adeel Ahmad Muhammad Saeed Zeeshan Muzammil

Ion exchange chromatography is a powerful technique used to separate charged biomolecules like proteins. It works by exploiting the reversible interactions between charged sample molecules and oppositely charged ligands attached to the chromatography column. There are two main types: anion exchange chromatography where the stationary phase is positively charged, attracting negatively charged molecules; and cation exchange chromatography where the stationary phase is negatively charged, attracting positively charged molecules. The apparatus consists of solvent reservoirs, pumps, columns, detectors and other components to precisely control the mobile and stationary phases. The procedure involves preparing the protein sample, loading it onto the column, washing unbound proteins away with buffer washes, then eluting the bound proteins from the column using salt or pH gradients.

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30 views17 pages

Presentation Group No 2: Mehtab Nusrullah Waseem Abbas Dilshad Ahmad H Adeel Ahmad Muhammad Saeed Zeeshan Muzammil

Ion exchange chromatography is a powerful technique used to separate charged biomolecules like proteins. It works by exploiting the reversible interactions between charged sample molecules and oppositely charged ligands attached to the chromatography column. There are two main types: anion exchange chromatography where the stationary phase is positively charged, attracting negatively charged molecules; and cation exchange chromatography where the stationary phase is negatively charged, attracting positively charged molecules. The apparatus consists of solvent reservoirs, pumps, columns, detectors and other components to precisely control the mobile and stationary phases. The procedure involves preparing the protein sample, loading it onto the column, washing unbound proteins away with buffer washes, then eluting the bound proteins from the column using salt or pH gradients.

Uploaded by

muhammad saeed
Copyright
© © All Rights Reserved
We take content rights seriously. If you suspect this is your content, claim it here.
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Presentation Group No 2

Mehtab Nusrullah
Waseem Abbas
Dilshad Ahmad
H Adeel Ahmad
Muhammad Saeed
Zeeshan Muzammil
Presentation topic
Ion exchange Chromatograp
hy for protein sample
Ion exchange Chromatography

Theory:
Ion exchange chromatography is a
powerful liquid Chromatographic techniq
ue used for bioseparation.
The separationis done by a reversible inte
raction between charged molecules of sa
mple with charged ligands attached to a c
olumn.
IEC
3-It offers seazable sample handling capa
city, powerful resolving ability, broad app
licabilityb and moderate cast.
4-These are characteristics made IEC vers
atile and widely used technique.
5- There are two types of ion exchange C
hromatography.
Anion exchange:

1- Stationary phase is positively charged wh


ich attracts the negativly charges molecules
.
2-It is prominently used in protein purificati
on,water analysis and quality control.
Cation exchange chromatogra
phy
1-It is used for positively charged molecul
es.
2-Stationary phase is negatively charged
which attracts the positively charged mol
ecules.
Principle of IEC
1- It is based upon the separation of mol
ecules based on their respective charges.
2-The ion exchange chromatography con
sist of both mobile phase and stationary
phases.
Apparatus
1- IEC is consist of series of mobile phase res
ervoirs containing a different mobile phases.
2- Reservoirs are made of glass or plastic be
cause mobile phases can have extreme pH v
alues.
3- IEC system include pump,conduits,valves,
sampling devices,coloumns, and detector ce
lls.
Apparatus
4-Solvent reservoir are attached with solv
ent selection valve and programer.
5- Solvent passes through selector to hig
h pressure pump.
6- Mobile phase passes to the sampling d
evice from pump and relays onto the col
oumns.
Apparatus
7- The exit flow from coloumns passes th
e solvent to detector.
8- The detector could be electrical condu
ctivity detector or the UV detector..
Procedure
1- prepare the protein mixture by adding 0.2m
l to protein extract vial. Vortex the mixture un
til completely dissolved.
2-Centrifuge the tube for 2 mints at high spee
d.
3-Clamp chromatography coloumns on the sta
nd in upright position.
4-open the caps of the coloumns.
Procedure
5-Drain the buffer through coloumns,under gr
avity, in a waste container.
6-Ensure that coloumns coloumns resins settle
s down in coloumns.
7-Add 1ml equilibration buffer, and allow it to
drain out and then drain 2nd 1ml.
8-Carefully load the protein extract from step
2 to the coloumns.
Procedure
9-Wash the coloumns with buffer for 4 times t
o remove unbound protein. Collect 2ml wash f
raction as buffer drains into labeled 2ml collec
tion tube.
10- Elute the sample with salt gradient, buffer,
elution buffer, and a high salt buffer.
11-Apply 1ml gradient elution buffer to colou
mns.
Procedure
12- Collect 1ml fraction as the buffer drains into the c
ollection tubes. Collect all elutions in separate 2ml tu
bes.
13- Note the Colour Changes of fractions.
14- Determine the protein Concentration of the fracti
ons.
Precautions
1- Carefully handle the sample to eliminate co
ntamination by for high sensitivity analysis.
2- Carefully handle the coloumns as hitting the
coloumn or exposure to sudden pressure chan
ges.
3- Avoid precipitation in the coloumn
4- Adjust the pH of buffer at room temperatur
e.

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