Dept. of Pharmacy
Chromatography (from Greek chroma "color and graphein "to write") is
the collective term for a set of laboratory techniques for
the separation of mixtures. 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.
mobile phase = solvent or gas
stationary phase = column packing material
Examples of Chromatography
Used to determine the chemical
composition of unknown
substances, such as the different
compounds in gasoline shown by
each separate peak in the graph
Can be used to separate the
components of inks, dyes, plant
compounds (chlorophyll), make-
up, and many other substances
Used to identify unknown plant
pigments & other compounds.
Uses thin plastic or glass trays to
identify the composition of pigments,
chemicals, and other unknown
Application of chromatography
• The chromatographic technique is used for the
separation of amino acids, proteins & carbohydrates.
• It is also used for the analysis of drugs, hormones,
vitamins & brain amines.
• Helpful for the qualitative & quantitative analysis of
• The technique is also useful for the determination of
molecular weight of proteins.
• 1903: Michael Tswett (or Tsvet) a Russian botanist separated the pigments in green
plant leaves using a glass column packed with 2 μm inulin and ligroin as mobile phase.
• 1938: Izmailov & Schreiber worked out a procedure where the solid stationary
chromatographic phase is distributed as a thin film on a glass plate.
• 1941: Archer John Porter Martin & Richard Laurence Millington Synge published a
paper on liquid partition chromatography that set the stage for gas liquid
• 1952: Martin and Synge were awarded the Nobel prize in chemistry for the invention
of column partition chromatography, and particularly paper chromatography.
• 1956: Martin & James applied gas chromatography to the separation of acids and
Michael Tswett Archer John Porter Martin Richard Laurence Millington Synge
Methods of chromatography:
Two phases chromatography are:
1. Mobile phase
2. Stationary phase
Classification of chromatographic methods
Mobile Phase Gas Liquid
phase Liquid Solid Bonded Liq. Liq Solid Bonded Liq. Ion Ex Polymer
Process (part’n) (adsorp’n) (adsorp’n- (part’n) (adsorp’n) (adsorp’n- (ion (part’n
part’n) HPLC part’n) exc’ng) sieving)
paper HPLC exclusion
Techniques of chromatography
• Techniques by Chromatographic bed shape
-The stationary phase is held in to a tube made of glass or metal (gel-ion exchange-
- A specific type of papers is used as stationary phase.
Thin layer chromatography
- The stationary phase is spread on glass or plastic or aluminum sheets.
• Techniques by Physical state of mobile phase
-The mobile phase is an inert gas nitrogen or helium. Again if the stationary phase is
solid it is called: Gas-Solid Chromatography(GSC). When stationary phase is liquid it is
called: Gas-Liquid Chromatography(GLC).
-The mobile phase is liquid. In case of separation by adsorption the stationary phase is
solid so it is called: Liquid-Solid Chromatography(LSC). If separation occurs through
partition, the stationary phase is liquid so it is called: Liquid-Liquid
• Affinity chromatography
Supercritical fluid chromatography
Mechanisms of separation in chromatography are:
1. ADSORPTION (LIQUID-SOLID) CHROMATOGRAPHY
2. PARTITION (LIQUID-LIQUID) CHROMATOGRAPHY
3. ION-EXCHANGE CHROMATOGRAPHY
4. Molecular Exclusion Chromatography
5. Affinity chromatography
6. HIGH PERFORMANCE LIQUID CHROMATOGRAPHY
1. Adsorption chromatography: It utilizes a mobile liquid or gaseous phase that is adsorbed
onto the surface of a stationary solid phase. The equilibration between the mobile and
stationary phase accounts for the separation of different solutes.
2. Partition chromatography: This form of chromatography is based on a thin film formed
on the surface of a solid support by a liquid stationary phase. Solute equilibrates between the
mobile phase and the stationary liquid.
Ion Exchange chromatography : In this type of chromatography the use of a resin (the
stationary solid phase) is used to covalently attach anions or cations onto it. Solute ions of the
opposite charge in the mobile liquid phase are attracted to the resin by electrostatic forces. It
has 2 prinicipal types of ion- exchanger is cationic and anionic.
Molecular Exclusion Chromatography (gel permeation/gel filtration): This type of
chromatography lacks an attractive interaction between the stationary phase and solute. The
liquid or gaseous phase passes through a porous gel which separates the molecules according
to its size. The pores are normally small and exclude the larger solute molecules, but allows
smaller molecules to enter the gel, causing them to flow through a larger volume.
Affinity chromatography :
This technique separates proteins on the basis of a reversible interaction between a protein or
group of proteins and a specific ligand coupled to a chromatography matrix. The technique
can be used to separate active bio molecules from denatured or functionally different forms,
to isolate pure substances present at low concentration in large volumes of crude sample and
also to remove specific contaminants.
High-performance liquid chromatography (HPLC)
Liquid chromatography (LC) is a separation technique in which the mobile phase is
a liquid. Liquid chromatography can be carried out either in a column or a plane.
Present day liquid chromatography that generally utilizes very small packing
particles and a relatively high pressure is referred to as high performance liquid
• It has the ability to separate, identify, and quantitate the compounds that are
present in any sample that can be dissolved in a liquid.
• HPLC can be, and has been, applied to just about any sample, such as
pharmaceuticals, food, nutraceuticals, cosmetics, environmental matrices, forensic
A chromatography detector is a device used in gas chromatography (GC) or liquid
chromatography (LC) to detect components of the mixture being eluted off the
chromatography column. There are two general types of detectors: destructive and
non-destructive. The destructive detectors perform continuous transformation of
the column effluent (burning, evaporation or mixing with reagents) with
subsequent measurement of some physical property of the resulting material
(plasma, aerosol or reaction mixture). The non-destructive detectors are directly
measuring some property of the column effluent (for example UV absorption) and
thus affords for the further analyte recovery.
• Flame ionization detector (FID)
• Aerosol-based detector (ABD)
• Flame photometric detector (FPD)
• Atomic-emission detector (AED)
• MS detector
• Nitrogen Phosphorus Detector (NPD)
• Evaporative light scattering detector (ELSD)
• UV detectors : Fixed or variable wavelength, which includes diode array detector (DAD or
PDA). The UV absorption of the effluent is continuously measured at single or multiple
wavelengths. These are by far most popular detectors for LC.
• Thermal conductivity detector (TCD): Measures the thermal conductivity of the effluent.
Only used in GC.
• Fluorescence detector : Irradiates the effluent with a light of set wavelength and measure
the fluorescence of the effluent at a single or multiple wavelength. Used only in LC
• Electron capture detector (ECD): The most sensitive detector known. Allows for the
detection of organic molecules containing halogen, nitro groups etc.
• Conductivity monitor: Continuously measures the conductivity of the effluent. Used only in
LC when conductive eluents (water or alcohols) are used.
• Photoionization detector (PID): Measures the increase in conductivity achieved by ionizing
the effluent gas with UV radiation.
• Refractive index detector (RI or RID): Continuously measures the refractive index of the
effluent. Used only in LC. The lowest sensitivity of all detectors. Useful when nothing else
works and at high analyte concentrations.
• Radio flow detector: Measures radioactivity of the effluent. This detector can be destructive
if a scintillation cocktail is continuously added to the effluent.
• Chiral detector: Continuously measures the optical angle of rotation of the elutant. Used
only in LC when chiral compounds are being analyzed.
• It is a number that represents how far a compound
travels in a particular solvent.
• It is measured by measuring the ratio of the
distance traveled by the compound and the
distance traveled by the solvent.