Introduction to

Chromatography
Dr. Ali Talha Khalil
General Info.
• Chromatography is the combination of two Greek words
• Chroma - Color
• Graphy-Graphein- writing

• Chromatography is a laboratory technique for the separation of a

mixture

• The term was coined in 20th Century, and the technique initially
included separating plant pigments
• It is defined as the process of separation of the individual
components of a mixture based on their relative affinities towards
stationary and mobile phases
• Principle: The samples are subjected to flow by mobile liquid onto or
through the stable stationary phase. The sample components are
separated into fractions based on their relative affinity towards the
two phases during their travel
• The fraction with greater affinity to stationary layer travels slower and
shorter distance while that with less affinity travels faster and longer
• The mixture is dissolved in a fluid called the mobile
phase, which carries it through a structure holding another
material called the stationary phase
• The various constituents of the mixture travel at different
speeds, causing them to separate
• The separation is based on differential partitioning between
the mobile and stationary phases
History

• Chromatography was first employed in Russia by the Italian-born

scientist Mikhail Tsvet in 1900 AD
• Chromatography technique developed substantially as a result of the
work of Archer John Porter Martin and Richard Laurence Millington
Synge during the 1940s and 1950s, for which they won the
1952 Nobel Prize in Chemistry
Chromatography terms
• The stationary phase is the substance fixed in place for the
chromatography procedure. Its also called immobilized phase
• The mobile phase is the phase that moves in a definite direction. The
mobile phase consists of the sample being separated/analyzed and
the solvent that moves the sample through the column
• The analyte is the substance to be separated during chromatography.
It is also normally what is needed from the mixture
• The eluent is the solvent that carries the analyte
• The eluate is the mobile phase leaving the column. This is also called
effluent
• The eluite is the analyte, the eluted solute
• A chromatograph is equipment that enables a sophisticated
separation
• The retention time is the characteristic time it takes for a particular
analyte to pass through the system
Broad types

• Preparative chromatography is used to purify sufficient quantities of

a substance for further use, rather than analysis.
• Analytical chromatography is done normally with smaller amounts of
material and is for establishing the presence or measuring the relative
proportions of analytes in a mixture
Paper chromatography

• Paper chromatography is an analytical method used to separate

colored chemicals or substances
• The mobile phase is generally mixture of polar organic solvent with
water, while the stationary phase is water
• Paper is used to support stationary phase (water)
• The retention factor (Rƒ) may be defined as the ratio of the distance
traveled by the solute to the distance traveled by the solvent
• It is used in chromatography to quantify the amount of retardation of
a sample in a stationary phase relative to a mobile phase
• If Rƒ value of a solution is zero, the solute remains in the stationary
phase and thus it is immobile
• If Rƒ value = 1 then the solute has no affinity for the stationary phase
and travels with the solvent front
Types of Chromatography:
• There are three ways to classify chromatography.
• They are-
• (a)Classification of chromatography according to mobile phase:
• 1. Liquid chromatography: mobile phase is a liquid.
• 2.Gas chromatography: mobile phase is a gas.
• (b)Classification according to the packing of the stationary phase
• 1.Thin layer chromatography (TLC): the stationary phase is a thin layer supported on
glass, plastic or aluminum plates
• 2.Paper chromatography (PC): the stationary phase is a thin film of liquidsupported on
an inert support
• 3.Column chromatography (CC): stationary phase is packed in a glass column
• c)Classification according to the force of separation:
• 1.Adsorption chromatography.
• 2.Partition chromatography.
• 3.Ion exchange chromatography.
• 4.Gel filtration chromatography.
• 5.Affinity chromatography
Applications
• General uses of chromatography in our real life are
• Pharmaceutical Company – determine amount of each chemical found in new
product
• Hospital – detect blood or alcohol levels in a patient’s blood stream
• Law Enforcement – to compare a sample found at a crime scene to samples
from suspects
• Environmental Agency – determine the level of pollutants in the water supply
• Environmental Agency – determine the level of pollutants in the water supply
• Biotechnology industry – establishing the purity or concentration of
compounds in biotechnological research
Etc.