UNIT III: INSTRUMENTAL

METHODS OF ANALYSIS

CHROMATOGRAPHY
Instructor
Dr. Najbul Hoque
 Chromatography
Chromatography
• Chromatography is taken to refer generally to the separation of components in a
sample by distribution of the components between two phases—one that is stationary
and one that moves, usually (but not necessarily) in a column.

• Russian botanist, Mikhail Tswett, invented adsorption chromatography during his

research on plant pigments.

• He separated different colored chlorophyll and carotenoid pigments of leaves by

passing an extract of the leaves through a column of calcium carbonate, alumina.

• He coined the term chromatography in a 1906 publication, from the Greek words
chroma meaning “color” and graphos meaning “to write.”
 Chromatography
Definition
IUPAC has drafted a recommended definition of chromatography: “Chromatography is a
physical method of separation in which the components to be separated are distributed
between two phases, one of which is stationary (stationary phase), while the other (the
mobile phase) moves in a definite direction”
 Chromatography
Apparatus in Chromatography
 Chromatography
Various
Terminologies
Stationary Phase

The stationary phase in chromatography is a phase that is fixed in place either in a column or on a
planar surface.

Mobile Phase

The mobile phase in chromatography is a phase that moves over or through the stationary phase
carrying with it the analyte mixture. The mobile phase may be a gas, a liquid, or a supercritical fluid.

Eluate

The mobile phase that exits the column is termed the eluate.
 Chromatography
Various
Terminologies
Eluent
An eluent is a solvent used to carry the components of a mixture through a stationary phase.

Adsorbate
A material that is capable of being adsorbed is known as adsorbate.

Example- Oxygen molecule adsorbed on cobalt. Here oxygen is adsorbate.

Retention time
it is specific time required for a particular analyte to pass through the system

Chromatogram
A chromatogram is a plot of some function of solute
concentration versus elution time or elution volume.
 Chromatography
Principle of Chromatography
• Chromatography is a separation method where the analyte is contained within a liquid or gaseous mobile phase, which is
pumped through a stationary phase.

• Usually, one phase is hydrophilic and the other lipophilic. The components of the analyte interact differently with these two
phases.

• Depending on the polarity they spend more or less time interacting with the stationary phase and are thus retarded to a
greater or lesser extent.

• This leads to the separation of the different components present in the sample.
 Chromatography
Advantages of chromatography

• Can separate complex mixtures with great precision. Even very similar components, such as proteins that may only vary by
a single amino acid.

• Can purify basically any soluble or volatile substance if the right adsorbent material, carrier fluid, and operating conditions
are employed.

• Exact quantitative analysis is done even from trace compounds.

• Small material consumption .

• Analyses of several compound can be done during one run.

• Chromatography is a fast analysis method.

 Chromatography
Classification of Chromatographic Techniques
Chromatographic processes can be classified according to the type of equilibration

process involved, which is governed by the type of stationary phase. Various bases

of equilibration are: (1) adsorption, (2) partition, (3) ion exchange, and (4) size

dependent pore penetration. More often than not, solute stationary-phase–mobile phase

interactions are governed by a combination of such processes.

 Chromatography
adsorption Chromatography
• Adsorption, or liquid-solid, chromatography is the classic form of liquid chromatography.

• In this chromatographic mode, the stationary phase is a finely divided solid to maximize the surface area. The stationary
phase (adsorbent) is chosen to permit differential interaction with the components of the sample to be resolved.

• The components distribute between the two phases through a combination of sorption and desorption processes.

• Thin-layer chromatography (TLC) is a special example of adsorption chromatography in which the stationary phase is
planar, in the form of a solid supported on an inert plate, and the mobile phase is a liquid.
 Chromatography
adsorption Chromatography: Column Chromatography
• The basic principle is selective adsorption.

• The long column is used to separate the mixture of substances.

• The solvent system that passes through the column is known as eluent.

• The process is known as elution.

• The substance having higher adsorbent capacity retains in upper part of column.

• The substance having less adsorbent capacity retains in lower part of column.

• As a result substances retain in different parts of column and separated partially.

• The solvent is passed again to have better separation.

 Chromatography
Column Chromatography: pictorial view
 Chromatography
Column Chromatography: requirements
 Chromatography
Column Chromatography: requirements
 Chromatography
Column Chromatography
 Chromatography
Column Chromatography
 Chromatography
adsorption Chromatography: Principle

• The mixture of gas or liquid gets separated when it passes over the adsorbent bed that adsorbs different compounds at
different rates.

• Adsorbent – A substance which is generally porous in nature with a high surface area to adsorb substances on its surface by
intermolecular forces is called adsorbent.

• Some commonly used adsorbents are Silica gel, hydrated silica gel, alumina etc.
 Chromatography
adsorption Chromatography: Principle

Caption
 Chromatography
adsorption Chromatography: Experiment

• Before starting with the adsorption chromatography experiment let us understand the two types of phases and the types of
forces involved during the mixture separation process.

• Stationary phase – Adsorbent is the stationary phase in adsorption chromatography. The forces involved help to remove
solutes from the adsorbent so that they can move with the mobile phase.

• Mobile phase – Either a liquid or a gas is used as a mobile phase in adsorption chromatography. Forces involved help to
remove solutes from the adsorbent so that they can move with the mobile phase. When a liquid is used as a mobile phase it
is called LSC (Liquid-Solid Chromatography). When a gas is used as a mobile phase it is called GSC (Gas-Solid
Chromatography).
 Chromatography
adsorption Chromatography: TLC
 Chromatography
adsorption Chromatography: TLC
 Chromatography
adsorption Chromatography: TLC
 Chromatography
adsorption Chromatography
 Chromatography
adsorption Chromatography: Application
• In monitoring the progress of reactions

• Identify compounds present in a given mixture

• Adsorption chromatography is used for separation of amino acids.

• It is used in the identification of carbohydrates, fats and fatty acids.

• Identification of medicinal plants and their constituents

 Chromatography
Classification of Chromatographic Techniques
Chromatography
Chromatography