The prestation describes ion exchange chromatography

1. Ion Exchange Chromatography

2. 1850: Thompson studies the adsorption of ammonium ions to soils
1947: Spedding and Powell demonstrated first practical preparative
separation of the rare earths.
1950s: Kraus and Nelson separation of metal ions such as fluoride,
chloride, nitrate or sulfate complexes by anion exchange chromatography.
1956: Peterson and Sober separated proteins
197: Small, Stevens and Bauman gave present form

3. Principle
• the separation of charged analytes takes
place on the basis of ionic (or electrostatic)
interactions between analytes and the
stationary phase consisting of ionic
functional groups (ionic exchangers).
• The driving force for the separation of ions is
competitive ionic binding and repulsion
between the similarly charged analyte
ions/stationary phase ions.

4. Theory of ion exchange
• The basic steps and components of ion exchange
chromatography are same as the liquid column
chromatography i.e. mobile phase, stationary phase
and eluent.
• The stationary phases used in ion exchange
chromatography consist of insoluble surfaces with
exchangeable ions and are called as ion exchangers.
• The ionic functional groups fixed on the surface of the
stationary phase are referred as fixed ions while the
exchangeable ions with opposite charge are referred
as counter ions (tendency to move under the influence
of electric field, diffusion or via exchange with similarly
charged external ions. )
• The counter ions :protons (H+ ), hydroxide groups (OH-
), unipositive/negative ions (Na+ , K+ , Cl- ), double
charged ions (Ca2+, Mg2+), and polyatomic ions (SO4
2- , PO4 3- ), organic bases (NR2H + ) and acids (COO-
), etc.

5. Ion exchangers
Cation
exchang
ers
fixed ion is
negatively
charged
Anion
exchang
ers.
fixed charge is
positively
charged
The charged functional groups may be cross-linked to
polystyrene, sephadex, sepharose, cellulose or polyacrylic
beads.

6. a) Crude extracts
are introduced to the
columns.
b) Through the washing
step, the unwanted
contaminants are eluted from
the column. In this step, the
fine-tuning of the pH of the
buffers is essential for
the efficient removal of
the contaminant and the
minimal loss of the target
protein.
c) After complete elution of
the contaminants, the ion
exchanger-tag interactions
are weakened via an
optimized pH-assisted
elution strategy the
pure fusion protein
is recovered from the column.
Schematic illustration of ion-exchange chromatography.

7. STEPS OF ION EXCHANGE CHROMATOGRAPHY
Column preparation (Swelling the gel/addition of
buffer/ degassing/ packaging column/ Equilibration of
the column
Sample preparation & loading (20 mg/ml protein,IF
SALTS, PURIFICATION)
Choosing Buffer selection AND Flow rate (elution and
require high ionic strength for elution cause
precipitation of proteins) ((CEC)buffers citrate and
phosphate buffers etc. N-methyl piperazine, bis-tris,
tris, phosphate and piperidine (AEC)Elutions

8. STEPS OF ION EXCHANGE CHROMATOGRAPHY
Flow rate (Slow in loading/ High in washing and
elution)
Elution:
The bound high salt concentration in the equilibration
buffer, either by gradient (e.g. 0.1 – 0.5 M NaCl) or step
wise (such as 0.1M, 0.3M, 0.5M NaCl) elution.
Alternatively, buffer with varying pH can be used to
elute bound proteins. After completion of each run, the
column washed thoroughly with high salt concentration

9. media composition of buffer
sample preparation -
volume and
concentration, sample
loading, flow rate
composition of buffer,
length of tubing and
fraction size.
Factors affecting ion-exchange chromatography

10. Applications of Ion Exchange Chromatography
concurrent analysis of inorganic anions/ carbonates
in consumed water and waste water
Determination of petrochemicals viz.
amines, mineral acids, cyanides etc
Evaluation of heavy metals and cyanide
polyphosphates in electrochemical and tannery
waste
Chemical purity and heavy metal analysis in fertilizers Analysis of alkaline earth metals, sulfur, and chlorine in paper
industry.

11. heavy
metal/galactose/lactose/glucose in
dairy products
Evaluation of sulphates, chlorides nitrates
and phosphates in sweeteners and sugar
syrups
Fluoride content in teas.
segregation of anions in decongestant and analgesic drugs.
Heavy metals in cosmetics