Chromatography is an analytical technique used to separate mixtures based on differences in physical properties of constituents. It involves a stationary phase and mobile phase. In chromatography, mixtures are separated as they travel through the stationary phase at different rates depending on interactions with the phases. Common types include paper chromatography, thin layer chromatography, column chromatography, and high performance liquid chromatography which uses high pressure to enhance separation.

1. CHROMATOGRAPHY

2. Chromatography
The term “chromatography” is derived from Greek, chroma meaning, “colour,” and
graphein meaning “to write.” The discovery of chromatography is generally attributed
to the Russian botanist Mikhail S. Tsvet (also spelled Tswett) in 1906.
Chromatography is an analytical technique which is used to separate the individual
constituents within a sample on the basis of differences in their physical characteristics,
e.g. molecular size, shape, charge, volatility, solubility and/or adsorptivity.

3. MObile PHAse
sTATiOnARY
PHAse
Ø Liquid or gas that flows through a chromatography system, moving
the materials to be separated.
Ø Mixture of substances to be separated dissolved in a liquid or gas
Ø A porous solid matrix through which the sample contained in
the mobile phase penetrates

4. Principle of Chromatography
Chromatography separate the components from a mixture based on the interaction of component
with the mobile / stationary phase and the properties of the compound.
Example: Proteins separation will be dependent upon size, shape, net charge, binding capacity,
stationary phase and mobile phase used.

5. Types of Chromatography
Ø Paper Chromatography
Ø Thin Layer Chromatography
Ø Column Chromatography
Ø High Performance Liquid Chromatography
Components
1. Stationary phase: This phase is always composed of a “solid” phase or “a layer of a liquid
adsorbed on the surface, a solid support”.
2. Mobile phase: This phase is always composed of “liquid” or a “gaseous component.”

6. Paper Chromatography
ü Stationary phase in paper chromatography consists of a layer of cellulose, highly saturated
with water.
ü Thick filter paper with water drops settled in its pores make up the stationary “liquid
phase”.
ü Mobile phase consists of an appropriate fluid (immiscible) placed in a developing tank
ü Used for separation of amino acids, sugars, sugar derivatives and peptides.

7. Paper Chromatography Procedure
1. Spot of sample applied to a strip of chromatography paper (stationary phase)
2. Paper bottom is dipped in appropriate solvent (DO NOT dip the spot in solvent).
3. Solvent moves up by capillary action & dissolves / may dissolve sample mixture.
4. Different compounds in mixture move at different rates due to differences in their solubility &
attraction to the mobile & stationary phases.
5. May take several minutes to several hours.

8. Paper Chromatography Analysis
Ø After separation,the chromatogram either shows compounds by their color, or may be developed by
UV light, Ninhydrin or treating with Iodine vapours.
Ø Chromatogram is the paper remaining after the experiment
Ø Calculate the retention factor ( Rf) = distance traveled from the spot or origin by the solute / distance
traveled from the spot or origin by the solvent
i. If Rf = 0 then solute remains in stationary phase and is immobile
ii. If Rf =1 then solute has no affinity with the stationary phase

9. Thin Layer Chromatography (TLC)
ü It involves a stationary phase consisting of thin layer of adsorbent material, usually silica
gel, aluminum oxide or cellulose immobilized onto a flat, inert carrier sheet.
ü Compound to be separated is dissolved in an
appropriate solvent and is pulled up via capillary
action, separating the solution based on the
polarity of the components of the compound
Sample applied

10. Advantages ofTLC over Paper Chromatography
1. Paper chromatography takes longer time (14-16hours) for separation of
compounds however TLC takes few hours (3-4hours) to complete.
2. Corrosive reagents like H2SO4 can be used in TLC unlike paper chromatography.
3. Convenient to visualize the separated compounds.
4. Multiple samples can be run on single stationary phase.
5. Relatively low cost.

11. Column Chromatography
Ø The stationary phase used in column chromatography is a solid (silica gel is most commonly
used, 2nd most is alumina) and is housed in a cylindrical column of 5mm to 50mm in
diameter and 5cm to 1m in height.
Ø Glass columns used to house stationary phase may range in size depending upon scale of
separation.
Ø Stationary phases are generally ground to powder or gel to increase the surface area
Ø Mobile phase (eluent) may be a pure solvent or mixture of different solvents. Eluent is
chosen selectively for optimum flow rate for each particular separation.

13. High Performance Liquid Chromatography
(HPLC)
HPLC is an abbreviation for High Performance Liquid Chromatography. "Chromatography" is a technique
for separation, "chromatogram" means a plot obtained via chromatography, and "chromatograph" is the
instrument used to conduct chromatography.
Principle:
The high-performance liquid chromatography works on the principle that some components of the sample
take longer than others to pass through the chromatography column.
The time taken by the molecules depends on the affinity of the molecule with the mobile phase (liquid or
gas) and the stationary phase (solid or liquid).
The ones with greater affinity with the stationary phase take longer to pass through the column and vice
versa.

14. High Performance Liquid Chromatography (HPLC)
Ø The separation of compounds is enhanced by application of high pressure (5000-10000
pounds per square inch) hence this technique was also known as high pressure liquid
chromatography.
Ø It requires non-compressible resin and strong metal columns, elutes containing separated
compounds are detected by methods such as UV-absorption & fluorescence.
Ø Stationary phase consists of an immobilized thin layer of a liquid on the micro glass, resin or
plastic beads, tightly packed into a narrow column.
Ø Mobile phase comprises of a buffered solvent which is passed under high pressure through the
column for eluting the solutes of the sample.

15. HPLC Workflow

16. HPLC Working:
There are two phases for HPLC: the mobile phase and the stationary phase.
i. The mobile phase is the liquid that dissolves the target compound.
ii. The stationary phase is the part of a column that interacts with the target compound.
In the column, the stronger the affinity (e.g.; van der waals force) between the component and the mobile
phase, the faster the component moves through the column along with the mobile phase. On the other
hand, the stronger the affinity with the stationary phase, the slower it moves through the column.

17. The solvent used to separate components in a liquid sample for HPLC analysis is called the mobile phase.
The mobile phase is delivered to a separation column, otherwise known as the stationary phase, and then
to the detector at a stable flow rate controlled by the solvent delivery pump.
A certain amount of sample is injected into the column and the compounds contained in the sample are
separated.
The compounds separated in the column are detected by a detector downstream of the column and each
compound is identified and quantified.

18. Advantages of HPLC:
1. HPLC offers a quick, automated and highly accurate method to identify certain chemical components
in a sample.
2. Compared to other chromatographic techniques, such as TLC, HPLC is extremely quick and efficient
3. The process can be completed in roughly 10 to 30 minutes, and it delivers high resolution. It is accurate
and highly reproducible.
4. Because it is largely automated, basic HPLC runs can be performed with minimal training.
Limitations:
1. Despite its advantages, HPLC can be costly, requiring large quantities of expensive organics.
2. HPLC does have low sensitivity for certain compounds, and some cannot be detected as they are
irreversibly adsorbed.