Chromatography is a technique used to separate mixtures by distributing components between two phases, one stationary and one mobile. It was first developed in 1903 by Russian scientist Mikhail Tswett to separate plant pigments like chlorophyll. There are several types including liquid chromatography, gas chromatography, paper chromatography, thin-layer chromatography, gel filtration, ion exchange, affinity chromatography, HPLC, and HPTLC. It has many applications in fields like pharmaceuticals, manufacturing, forensics, and environmental testing to analyze, identify, purify, and quantify components in a mixture.

1. BASICS OF CHROMATOGRAPHYBASICS OF CHROMATOGRAPHYBASICS OF CHROMATOGRAPHYBASICS OF CHROMATOGRAPHY
Presented by- Pankaj KushwahaPresented by- Pankaj Kushwaha
Dept. of Biomedical ScienceDept. of Biomedical Science
Bundelkhand University JhansiBundelkhand University Jhansi

2. INTRODUCTORY PRINCIPLESINTRODUCTORY PRINCIPLES
Chromatography is a combination of two words;
* Chromo – Meaning color
* Graphy – representation of something on
paper

3. HISTORY OF CHROMATOGRAPHYHISTORY OF CHROMATOGRAPHY
Chromatography, literally "color writing", was first employed byChromatography, literally "color writing", was first employed by
Russian scientist Mikhail Tswett in 1903/1906. He continued toRussian scientist Mikhail Tswett in 1903/1906. He continued to
work with chromatography in the first decade of the 20th century,work with chromatography in the first decade of the 20th century,
primarily for the separation of plant pigments such as primarily for the separation of plant pigments such as chlorophyllchlorophyll, ,
carotenescarotenes, and , and xanthophyllsxanthophylls. Since these components have different. Since these components have different
colors (green, orange, and yellow,respectively) they gave thecolors (green, orange, and yellow,respectively) they gave the
technique its name.technique its name.

4. It is a physical separation method of separation in which
the components of a mixture are separated by differences in
their distribution between two phases, one of which is
stationary (stationary phase) while the other (mobile phase)
moves through it in a definite direction. The substances
must interact with the stationary phase to be retained and
separated by it.

5. DEFINITION OF CHROMATOGRAPHY
IUPAC definition (International Union of pure and applied
Chemistry) (1993):
Chromatography is a physical method of separation in which the
components to be separated are distributed between two phases, one
of which is stationary while the other moves in a definite direction.
The stationary phase may be a solid, or a liquid supported on a
solid or gel, the mobile phase may be either a gas or a liquid.

6. Mixture
Separat
e
 Analyze
• Identify
• Purify
• QuantifyComponent
s
CHROMATOGRAPHYCHROMATOGRAPHY

7.  Chromatograph:Chromatograph: Instrument employed for a chromatography.Instrument employed for a chromatography.
 Eluent:Eluent: Fluid entering a column.Fluid entering a column.
 Eluate:Eluate: Fluid exiting the column.Fluid exiting the column.
 Elution:Elution: The process of passing the mobile phase through theThe process of passing the mobile phase through the
column.column.
 Flow rate:Flow rate: How much mobile phase passed / minute (ml/min).How much mobile phase passed / minute (ml/min).
 Linear velocity:Linear velocity: Distance passed by mobile phase per 1 min in theDistance passed by mobile phase per 1 min in the
column (cm/min).column (cm/min).

8. Mobile Phase – gas or liquid that carries the mixture
of components through the stationary phase.
Stationary Phase – the part of the apparatus that
holds the components as they move through it,
separating them.

9. Uses for ChromatographyUses for Chromatography
Chromatography is used by scientists to:
•Analyze – examine a mixture, its components, and their
relations to one another
•Identify – determine the identity of a mixture or
components based on known components
•Purify – separate components in order to isolate one of
interest for further study
•Quantify – determine the amount of the a mixture
and/or the components present in the sample

10. Real-life examples of uses for chromatography:
Pharmaceutical CompanyPharmaceutical Company
HospitalHospital
Law EnforcementLaw Enforcement
Environmental AgencyEnvironmental Agency
Manufacturing PlantManufacturing Plant

11. Chromatogram:
It is the visual output of the chromatograph.
Chromatograph:
It is equipment that enables a sophisticated Separation.
Stationary phase (bounded phase):
It is a phase that is covalently bonded to the support particles or to the
inside wall of the column tubing.

12. Mobile phase:
It is the phase which moves in a definite direction.
Analyte (Sample):
It is the substance to be separated during chromatography.
Eluate:
It is the mobile phase leaving the column.

13. Retention time:
It is the characteristic time it takes for a particular analyte to pass
through the system (from the column inlet to the detector) under set
conditions.
Eluent:
It is the solvent that will carry the analyte.

14. Retardation factor (R):
Fraction of an analyte in the mobile phase of a chromatographic
system.

15. •Liquid Chromatography – separates liquid samples with a liquid
solvent (mobile phase) and a column composed of solid beads (stationary
phase)
• Gas Chromatography – separates vaporized samples with a carrier
gas (mobile phase) and a column composed of a liquid or of solid beads
(stationary phase)
•Paper Chromatography – separates dried liquid samples with a
liquid solvent (mobile phase) and a paper strip (stationary phase)
•Thin-Layer Chromatography – separates dried liquid samples with
a liquid solvent (mobile phase) and a glass plate covered with a thin layer
of alumina or silica gel (stationary phase)

17. GEL FILTRATION
 Gel filtration separates molecules according to the differences
in size as they pass through the filtration medium packed in
the column.
 It is well suited for biomolecules that are sensitive to pH
,concentration and harsh environment.
 Parameters that affects gel filtration are, particle size, flow rate,
packaging density, porosity of the particle and viscosity of the
mobile phase.

19. MATERIALS REQUIRED
 Cross linked dextrans (sephadex)
 Agarose (sepharose)
 Polyacrylamide
 Porous glass gel.
APPLICATIONS
 Fractionation (purification of the desired protein using suitable gel)
 Molecular weight determination

20. ION EXCHANGE

21. ION EXCHANGERS
 Cation exchangers (negative ions – stationary)
 Anion exchangers (positive ions - stationary)
Four types of polymers are commonly used.They are,
 Synthetic hydrophobic polymer resins crosslinked with
divinylbenzene.
 Naturally occuring as well as synthetic polymers(cellulose)
 Synthetic hydrophilic polymers
 Silica gel

22. AFFINITY CHROMATOGRAPHYAFFINITY CHROMATOGRAPHY

23. HPLCHPLC
 HPLC is a physical separation technique in which a sample
dissolved in a liquid is injected into a column packed with
small particles and it is separated into its constituent
components
 HPLC is probably the most important and widely used
analytical technique for quantitative analysis of organics and
biomolecules
 HPLC is applicable to many kind of samples:
 Most useful for pharmaceuticals, biomolecules, and labile
organics

24. HPLC INSTRUMENTATION OVERVIEWHPLC INSTRUMENTATION OVERVIEW
24
Principle Pattern An Example
Detector
Thermostatted
Column Compartment
Autosampler
Binary Pump
Vacuum DegasserSolvent Cabinet
Solvent Reservoirs
Controller

26. HPTLCHPTLC
 HPTLC is a sophisticated form of TLC.
 Fastest of all chromatographic techniques.
 Any combinations of stationary and mobile phases can be
used.
 Analytical HPTLC is used for micro preparative analysis
(ie., separation of milligram scale for analysis of fraction )
 Gives more sharper and compact bands with minimum
distance of migration.
 Used for both qualitative and quantitative analysis.

27. HPLC VS. HPTLCHPLC VS. HPTLC