High pressure liquid chromatography (HPLC) uses very fine silica gel particles in columns, greatly increasing separation power but severely decreasing mobile phase flow rate. High pressure pumps are needed to push the mobile phase through stainless steel columns. HPLC allows for improved separation compared to regular column chromatography due to the small particle size increasing surface area, but requires specialized equipment like high pressure pumps and stainless steel columns to accommodate the low flow rates. HPLC finds applications in forensic analysis of substances in the body and pharmaceutical analysis to test drug purity.

1. 5) HIGH PRESSURE COLUMNS (HPLC) :In this columns we use very fine silica gel so
great increaser in separation power. However, the flow rate of the mobile phase is severely
decreased. High pressure pumps are used to push the solvent through the column which in
this case must be made of stainless steel.
ADVANTAGES
 Column chromatography is inexpensive
 Any type of mixture can be separated
 Any quantity of mixture can be separated
 Wider choice of Mobile Phase
DISADVANTAGES
 The process is Time consuming
 It requires ample amount of mobile phase
DO’S
 Increase the length of the column improves separation.
 Reducing the particle size increases the surface area and improve separation.
 The choice of the suitable adsorbent is very important
 Uniformity in packing of the column
 The properratio between sample to be separated and the amount ofstationary phase should
be maintained
DONT’S
 Higher flow rate affects the rate of separation
 Inconsistency of flow of mobile phase affects separation so there should be consistency of
flow
 Reduction of the particle size will decrease the flow rate of the mobile phase.
 Handling of equipment should be proper
 Do not increase the temperature of column as it affects separation
APPLICATION
 Forensic testing to determine levels of alcohol, drugs or poisonous substances in the body.
 Chromatography can identify substances in the bloodstream make it valuable in testing for
doping or performance enhancing drugs in athletes, too.
 Pharmaceuticalindustry: In this field, (Includes Cosmetics and Herbal products too)it is
mainly used to assertion purity of drugs. Identify impurities and develop chromatographic
methods to quantify impurities.
COLUMN CHROMATOGRAPHY
LABORATORY GUIDE
SUBMITTED TO : SUBMITTED BY:
Mrs . Varsha srivastava Simran Sood
Shuchita Anand
Temsusangla Aier
DEPARTMENT OF NUTRITION AND DIETETICS
FACULTY OF APPLIED SCIENCE
MANAV RACHNA INTERNATIONAL INSTITUTE OF
RESEARCH AND STUDIES
(2018)

2. COLUMN CHROMATOGRAPHY
Introduction
 The basic definition of column chromatography is “method used to purify
individual chemical compounds from mixtures of compounds.”
 Chromatography is able to separate substances based on differential absorption of
compounds to the absorbent; compounds move through the column at different
rates, allowing them to be separated into fractions.
 It consists mainly of two phases which are stationery phase and mobile phase.
Stationery phase is easily disposable in it.
Principle
 An impure sample is loaded onto a column of adsorbant (Adsorbents are also used
for extraction purposes removing traces of organic materials from large volumes of
water very efficiently) suchas silica gel or alumina. An organic solvent or a mixture
of solvents (the eluent) flows down through the column. Components of the sample
separate from each other by partitioning between the stationary packing material
(silica oralumina) and the mobile eluent. Molecules with different polarity partition
to different extents, and therefore move through the column at different rates. The
eluent is collected in fractions. Fractions care typically analyzed by “THIN
LAYER CHROMATOGRAPHY” to see if separation of the components was
successful.
Figure 1 : Showing the working of column chromatography in diagrammatic
form
 There are four types of column chromatography
 PARTITION CHROMATOGRAPHY.
 ADSORPTION CHROMATOGRAPHY.
 ION EXCHANGE CHROMATOGRAPHY.
 GEL CHROMATOGRAPHY.
 HIGH PERFORMANCE LIQUID CHROMATOGRAPHY.
 GAS CHROMATOGRAPHY.
STATIONARY PHASE
 The stationary phase or adsorbent in column chromatography is a solid.
 the most common stationary phase for column chromatography is silica gel, the
next most common being alumina. Cellulose powder has often been used in the past.
 The stationery phase are usually finely ground powders orgels and/or are microporous
for an increased surface.
MOBILE PHASE
 It is a mixture oforganic solvents (unusually onesolvent only) the choiceof the column
mobile phase is achieved after TLC study in different solvent systems.
 The mobile phase, a liquid, is added to the top and flows down through the column by
either gravity or external pressure.
 It helps in carrying the component of the mixture being used while stationery phase
remains same in it.
TYPES OF COLUMNS:
1) GRAVITY COLUMNS: The mobile phase move through the stationary phase by
gravity
force.
2) FLASH COLUMNS: (Air or nitrogen pressure): The mobile phase is pushed by
stream of air or nitrogen using special using special values (Adaptors).
3) LOW AND MEDIUM PRESSURE COLUMNS: (pumped):The movement of
mobile phase is accelerated by using pump that generate low or medium pressure. The
increase in the flow
rate shorten the time of separation.
4) VACCUM COLUMNS [Vacuum liquid chromatography (VLC)]: The adsorbent
is applied dry into a sintered glass funnel. The sample is applied by dry method or as
solution. Then the mobile phaseis added portionby portionand vacuum is applied after
each portion to collect each fraction