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Chromatography

The document provides an overview of chromatography, a biophysical technique used for the separation, identification, and purification of mixture components. It details various chromatographic techniques including paper, thin layer, column, ion exchange, and electrophoresis, emphasizing their principles and applications in protein purification based on molecular characteristics. Key concepts such as partition coefficient, elution, and retention time are also explained, highlighting the importance of the interaction between mobile and stationary phases in the separation process.

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Misbah Ijaz
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1 views

Chromatography

The document provides an overview of chromatography, a biophysical technique used for the separation, identification, and purification of mixture components. It details various chromatographic techniques including paper, thin layer, column, ion exchange, and electrophoresis, emphasizing their principles and applications in protein purification based on molecular characteristics. Key concepts such as partition coefficient, elution, and retention time are also explained, highlighting the importance of the interaction between mobile and stationary phases in the separation process.

Uploaded by

Misbah Ijaz
Copyright
© © All Rights Reserved
We take content rights seriously. If you suspect this is your content, claim it here.
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Download as PDF, TXT or read online on Scribd
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Separation Methods

▪Principles of chromatography
▪classification of chromatographic
techniques
Chromatography ▪overview of paper chromatography
▪thin layer chromatography
▪Column chromatography
▪ion exchange chromatography
▪electrophoresis.
Chromatography is an important biophysical technique that
enables the separation, identification, and purification of the
components of a mixture for qualitative and quantitative
analysis.
Proteins can be purified based on characteristics such as
❑size and shape
❑total charge
❑hydrophobic groups present on the surface
❑binding capacity with the stationary phase.
Four separation techniques based on molecular characteristics
and interaction type are:
ion exchange
surface adsorption
size exclusion
Three separation techniques are based on stationery bed
Column
thin layer
paper chromatography
COLUMN CHROMATOGRAPHY IS ONE OF THE MOST COMMON
METHODS OF PROTEIN PURIFICATION.
Principle:
Chromatography is an extraction method in which the analyte and a liquid or gaseous mobile
phase are mixed and passed via a stationary phase.
The basic principle of chromatography is given below:
“Relative affinities of components of the mixture toward
mobile phase and stationary phase”
In general, it is the Distribution of analyte between the mobile phase and stationary
phase or the interaction of the sample with the mobile phase and stationary phase.
Greater the interaction of the analyte with the mobile phase faster would be
separation and greater the interaction of the analyte with the stationary phase
slower would be separation.
All chromatographic separations are based on the difference in the extent to which
solutes are partitioned between the mobile phase and the stationary phase.
The equilibria involved can be described quantitatively with the
temperature dependent constant
K=Cs /Cm
Partition/ Where

Distribution •K= partitioned coefficient


•Cs =solute concentration in stationary phase
coefficient •Cm =solute concentration in mobile phase
•K is Partition coefficient
K depends upon the following factors
•Temperature
•Type of compounds
•Stationary phase
•Mobile phase
Note that “K” with larger values has strong interaction between
solute and stationary phase and vice versa.
Chromatography can be
Types of
classified into different types on
chromatography the basis of the following
categories
1.Stationary phase
2.Mobile phase
3.Force / Method of separation
On the basis of the mobile phase
•Liquid chromatography
•Gas chromatography
On the basis of the stationary phase
❑Thin Layer chromatography
❑Paper chromatography
❑Column chromatography
On the basis of the method of
separation
❑Adsorption chromatography
❑Partition chromatography
❑Affinity chromatography
❑Ion exchange chromatography
❑Gel filtration chromatography
Working
Although, all types of chromatography are based on the same working principle
which is relative affinities of an analyte with mobile phase and stationary phase.
But the method of separation is different in
(i) Partitioned chromatography
In partitioned chromatography, an analyte is differently distributed between the
mobile phase and stationary phase. In partition chromatography, stationary
phases may be liquid or solid. Partitioned chromatography can be carried out
as
•Paper chromatography
•Column chromatography
•Gas chromatography
•Thin layer chromatography
(ii) Adsorption Chromatography
In adsorption chromatography, a suitable adsorbent is
used. The adsorbent acts as a stationary phase. The
working principle is the differential adsorption by the
adsorbent. In the adsorption chromatography, the
analyte is adsorbed on the stationary phase. It bonds
directly to the stationary phase. The adsorbent may also
produce a partitioned effect by retaining some of the
analytes. In adsorption chromatography, the stationary
phase is always solid.
(iii) Exclusion chromatography
In exclusion chromatography, sample components are separated according to their
molecular size. Exclusion chromatography can be carried out as
•Gel permeation
•Sieving separations
In gel permeation, there is three dimensional network of cross linked polymer
chains. In the presence of a suitable solvent, gel undergoes swelling and the space
between the polymer chains is increased. Molecules of smaller size are retarded
whereas large molecules pass unhinderedly.
In sieving separations, natural and synthetic zeolites are used as molecular sieves.
They have cavities and channels of different sizes in their structures. The compounds
of smaller size fall into the cavities while passing whereas larger particles pass
unhinderedly.
(iv) Ion exchange
chromatography
Ion exchange chromatography is the form of solid liquid chromatography. Bead
shaped ion-exchange resins act as stationary phase. These resins are highly
polymerized, crossed linked, organic materials containing a large number of acidic
and basic functional groups. These groups are known as active groups. These groups
are of hydrophilic nature. Cation exchangers contain cation whereas, anion
exchangers contain anions.
ion-exchange chromatography is mostly performed on columns with adsorbents and
cationic or anionic exchangers. The columns are designed in such a way that there
should be no hindrance in the flow of liquid. This form of chromatography relies on
the attraction between the oppositely charged ion exchanger, and the mobile phase
containing the analyte.
What is meant by the term
elution?
The term elution
means “wash out”. In
chromatography, Elution is What is Rf value in chromatography?
the process through which
analyte is passed through
stationary phase by the help Rf is known as the retardation/retention factor.
of mobile phase.
What is meant by retention It is the ratio of the distance moved by the solute
time?
The retention time is the time from the base to the distance moved by the solvent
taken by the solute (analyte) to from the base line.
pass through a column
(stationary phase) . It depends
upon relative affinities of solute Rf=distance moved by a compound form base
with mobile phase and
stationary phase. It is denoted line distance moved by a solvent form base line
by “RT”
Thin Layer Chromatography
TLC

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