Final year Bpharm 7th semester
Vikrant Ashok Shirke
Gsmcop, Wagholi, Pune
BP701T- INSTRUMENTAL METHODS OF ANALYSIS
(UNIT-V ) (03-08 MARKS)
Syallabus:-
Ion exchange chromatography:-
Introduction, classification, ion exchange resins, properties, mechanism of ion exchange
process, factors affecting ion exchange, methodology, and applications.
Gel chromatography:-
Introduction, theory, instrumentation and applications, Affinity chromatography
-Introduction.
Q.1. Attempt the following:- (3 Marks Each)
1) Discuss the principle and applications of ion exchange chromatography? (3/5Marks)
Write the principle involved in ion exchange chromatography?
★ Principle of Ion Exchange Chromatography:
1) Ion Exchange Chromatography (IEC) is a powerful separation technique
used for the purification and analysis of charged molecules like proteins,
amino acids, and nucleotides. It operates on the principle of electrostatic
interactions between charged analytes and oppositely charged stationary
phases.
2) It is based on the reversible exchange of ions between the analyte and a
charged resin (stationary phase). The resin contains fixed charged groups
and mobile counter ions.
3) When a solution containing sample ions passes through the column:-
A) Cation exchange chromatography: Uses negatively charged resins that
bind positively charged molecules (cations).
B) Anion exchange chromatography: Uses positively charged resins that bind
negatively charged molecules (anions).
i) Sample Loading: The sample is applied to the column containing the ion
exchange resin.
ii) Binding: Charged molecules bind to oppositely charged groups on the
resin.
iii) Elution: Bound molecules are eluted by changing pH or ionic strength
(e.g, using a salt gradient), which disrupts the ionic interaction.
★ Theory of Ion Exchange Chromatography
1) Ion exchange chromatography (IEC) is a separation technique based on
the electrostatic interactions between charged molecules in a solution and
, oppositely charged groups attached to an inert matrix (the stationary
phase).
2) It is widely used for separating and purifying proteins, amino acids,
nucleotides, and other charged biomolecules.
3) Ion exchange chromatography works on the principle of ion exchange
equilibrium.
4) The stationary phase consists of ion exchange resins that carry fixed
charged groups (either positive or negative).
5) These resins also have mobile counterions that are exchanged with ions
from the sample solution.
➢ Two Main Types:
1) Cation Exchange Chromatography:
The resin contains negatively charged groups (e.g., sulfonic or carboxylic acid).
Binds positively charged molecules (cations).
[ Example: Protein with net positive charge at a given pH. ]
2) Anion Exchange Chromatography:
The resin contains positively charged groups (e.g., quaternary ammonium).
Binds negatively charged molecules (anions).
[ Example: DNA, RNA, or proteins with net negative charge at a given pH. ]
➢ Key Factors Influencing Separation:
1) Charge of the Molecule:
Depends on the pH of the buffer and the molecule's isoelectric point (pI).
At pH < pI → molecule is positively charged → binds to cation exchanger.
At pH > pI → molecule is negatively charged → binds to anion exchanger.
2) Buffer Composition:
Buffer pH and ionic strength are crucial for controlling binding and elution.
Low salt = strong binding; high salt = weak binding/elution.
3) Ion Exchange Capacity:
Amount of charge the resin can carry and exchange.
➢ Mechanism of Separation:
1) Equilibration: Column is prepared with a buffer and resin in a specific
ionic form.
2) Sample Loading: Charged molecules bind to oppositely charged groups
on the resin.
3) Washing: Non-bound substances are washed away.
4) Elution: Bound molecules are displaced using a salt gradient or pH
change.
➢ Binding Strength Order:
The strength of binding depends on: a) Net charge magnitude.
b) Distribution of charge
Vikrant Ashok Shirke
Gsmcop, Wagholi, Pune
BP701T- INSTRUMENTAL METHODS OF ANALYSIS
(UNIT-V ) (03-08 MARKS)
Syallabus:-
Ion exchange chromatography:-
Introduction, classification, ion exchange resins, properties, mechanism of ion exchange
process, factors affecting ion exchange, methodology, and applications.
Gel chromatography:-
Introduction, theory, instrumentation and applications, Affinity chromatography
-Introduction.
Q.1. Attempt the following:- (3 Marks Each)
1) Discuss the principle and applications of ion exchange chromatography? (3/5Marks)
Write the principle involved in ion exchange chromatography?
★ Principle of Ion Exchange Chromatography:
1) Ion Exchange Chromatography (IEC) is a powerful separation technique
used for the purification and analysis of charged molecules like proteins,
amino acids, and nucleotides. It operates on the principle of electrostatic
interactions between charged analytes and oppositely charged stationary
phases.
2) It is based on the reversible exchange of ions between the analyte and a
charged resin (stationary phase). The resin contains fixed charged groups
and mobile counter ions.
3) When a solution containing sample ions passes through the column:-
A) Cation exchange chromatography: Uses negatively charged resins that
bind positively charged molecules (cations).
B) Anion exchange chromatography: Uses positively charged resins that bind
negatively charged molecules (anions).
i) Sample Loading: The sample is applied to the column containing the ion
exchange resin.
ii) Binding: Charged molecules bind to oppositely charged groups on the
resin.
iii) Elution: Bound molecules are eluted by changing pH or ionic strength
(e.g, using a salt gradient), which disrupts the ionic interaction.
★ Theory of Ion Exchange Chromatography
1) Ion exchange chromatography (IEC) is a separation technique based on
the electrostatic interactions between charged molecules in a solution and
, oppositely charged groups attached to an inert matrix (the stationary
phase).
2) It is widely used for separating and purifying proteins, amino acids,
nucleotides, and other charged biomolecules.
3) Ion exchange chromatography works on the principle of ion exchange
equilibrium.
4) The stationary phase consists of ion exchange resins that carry fixed
charged groups (either positive or negative).
5) These resins also have mobile counterions that are exchanged with ions
from the sample solution.
➢ Two Main Types:
1) Cation Exchange Chromatography:
The resin contains negatively charged groups (e.g., sulfonic or carboxylic acid).
Binds positively charged molecules (cations).
[ Example: Protein with net positive charge at a given pH. ]
2) Anion Exchange Chromatography:
The resin contains positively charged groups (e.g., quaternary ammonium).
Binds negatively charged molecules (anions).
[ Example: DNA, RNA, or proteins with net negative charge at a given pH. ]
➢ Key Factors Influencing Separation:
1) Charge of the Molecule:
Depends on the pH of the buffer and the molecule's isoelectric point (pI).
At pH < pI → molecule is positively charged → binds to cation exchanger.
At pH > pI → molecule is negatively charged → binds to anion exchanger.
2) Buffer Composition:
Buffer pH and ionic strength are crucial for controlling binding and elution.
Low salt = strong binding; high salt = weak binding/elution.
3) Ion Exchange Capacity:
Amount of charge the resin can carry and exchange.
➢ Mechanism of Separation:
1) Equilibration: Column is prepared with a buffer and resin in a specific
ionic form.
2) Sample Loading: Charged molecules bind to oppositely charged groups
on the resin.
3) Washing: Non-bound substances are washed away.
4) Elution: Bound molecules are displaced using a salt gradient or pH
change.
➢ Binding Strength Order:
The strength of binding depends on: a) Net charge magnitude.
b) Distribution of charge
