A SEMINAR REPORT ON

CHROMATOGRAPHY
BY
FAITHFUL ONYENDI
19D/57MB/01323
INTRODUCTION
• Chromatography is based on the principle where molecules in mixture applied onto the surface or
into the solid, and fluid stationary phase is separating from each other while moving with the aid of a
mobile phase.
• The factors effective on this separation process include molecular characteristics related to adsorption
(liquid-solid), partition (liquid-solid), and affinity or differences among their molecular weights.
• Because of these differences, some components of the mixture stay longer in the stationary phase,
and they move slowly in the chromatography system, while others pass rapidly into mobile phase, and
leave the system faster (Harris, 2013).
• Based on this approach three components form the basis of the chromatography technique.
• Stationary phase: This phase is always composed of a “solid” phase or “a layer of a liquid adsorbed on
the surface a solid support”.
• Mobile phase: This phase is always composed of “liquid” or a “gaseous component.”
• Separated molecules
Background And Significance Of Study
• Chromatography means "to show with colors." It was the Russian botanist Mikhail Semyonovich
Tsvet (1872-1919) who invented the first chromatography technique in 1900, during his research
on chlorophyll.
• He used a liquid-adsorption column containing calcium carbonate to separate plant pigments
chromatography (McMurry, 2011).
• Although you might not be familiar with Chromatography, it has an impact on your everyday life.
• The process is used to find out what substances are composed of by separating compunds into
their various components and its use affects everything from what you eat  to how you fight
disease (McMurry, 2011). .
Types of chromatography
• Column chromatography
• Ion-exchange chromatography
• Gel-permeation (molecular sieve) chromatography
• Affinity chromatography
• Paper chromatography
• Thin-layer chromatography
• Gas chromatography
• Dye-ligand chromatography
• High-pressure liquid chromatography (HPLC) (Walls, 2011).
Column chromatography
• Since proteins have difference characteristic features as size, shape, net charge,
stationary phase used, and binding capacity, each one of these characteristic
components can be purified using chromatographic methods. Among these
methods, most frequently column chromatography is applied (Walls, 2011).
• Column Chromatography Applications
• Column Chromatography is used to isolate active ingredients.
• It is very helpful in separating compound mixtures.
• It is used to determine drug estimation from drug formulations.
• It is used to remove impurities.
• Used to isolate metabolites from biological fluids (Determann, 2012).
Paper chromatography
• In paper chromatography support material consists of a layer of cellulose highly saturated with
water.
• In this method a thick filter paper comprised the support, and water drops settled in its pores
made up the stationary “liquid phase.
• Mobile phase consists of an appropriate fluid placed in a developing tank.
• Paper chromatography is a “liquid-liquid” chromatography (Stoddard, 2007).
• Applications Of Paper Chromatography
• Separating Colored Pigments
• Reaction Monitoring
• Isolation And Purification
• Pathology and Forensic Science
• Food
Thin-layer chromatography

• Thin-layer chromatography is a “solid-liquid adsorption” chromatography.

• In this method stationary phase is a solid adsorbent substance coated on glass plates.
• As adsorbent material all solid substances used. in column chromatography (alumina, silica gel,
cellulose) can be utilized.
• In this method, the mobile phase travels upward through the stationary phase The solvent
travels up the thin plate soaked with the solvent by means of capillary action.
• During this procedure, it also drives the mixture priorly dropped on the lower parts of the plate
with a pipette upwards with different flow rates. Thus the separation of analytes is achieved.
• This upward travelling rate depends on the polarity of the material, solid phase, and of the
solvent (Donald, 2006).
• In cases where molecules of the sample are colorless, florescence, radioactivity or a specific
chemical substance can be used to produce a visible coloured reactive product so as to identify
their positions on the chromatogram (Donald, 2006).
Applications of Thin layer
Chromatography
• Thin-layer chromatography can be used to monitor the progress of a
reaction, identify compounds present in a given mixture, and
determine the purity of a substance.
• Specific examples of these applications include:
analyzing ceramides and fatty acids, detection
of pesticides or insecticides in food and water, analyzing the dye
composition of fibers in forensics, assaying the radiochemical
purity of radiopharmaceuticals, or identification of medicinal
plants and their constituents (Reich, 2007).
Gas chromatography
• In this method stationary phase is a column which is placed in the device, and contains
a liquid stationary phase which is adsorbed onto the surface of an inert solid (Harris,
2016).
• Gas chromatography is a “gas-liquid” chromatography. Its carrier phase consists of gases
as He or N2.
• Mobile phase which is an inert gas is passed through a column under high pressure. The
sample to be analyzed is vaporized, and enters into a gaseous mobile phase phase.
• The components contained in the sample are dispersed between mobile phase, and
stationary phase on the solid support.
• Gas chromatography is a simple, multifaceted, highly sensitive, and rapidly applied
technique for the extremely excellent separation of very minute molecules.
• It is used in the separation of very little amounts of analytes (Harris, 2016).
GENERAL APPLICATION OF
CHROMATOGRAPHY
• 1. Creating vaccinations
• Chromatography is useful in determining which antibodies fight various diseases and viruses. Scientists used Chromatography in the fight against
the Ebola virus, responsible for over 11,000 deaths, to develop the experimental immunisation Zmapp. The process was used to find out which
antibodies are the most effective at neutralising the deadly virus (Harris, 2016).
• 2. Food testing
• The 2013 horsemeat scandal, in which horsemeat passed off by vendors as beef came to light, highlighted the ineffectiveness of traditional food
analysis methods and positioned chromatography as the frontrunner in determining the contents of processed meat. The traditional methods of
analysis were effective at determining the composition of raw samples but inconclusive when analysing processed meats so a more precise
method was called for. High performance liquid chromatography, combined with mass spectrometry (HPLC-MS) was then successfully used to
find out if meat that had been labelled as beef was beef, horse or a mix of these and various other ingredient (Harris, 2016).
• 3. Beverage testing
• Many drinks manufacturers use this technique to ensure each bottle of their product is exactly the same, so you can rely on a consistent taste.
One such brand is Jägermeister which uses chromatography to monitor the levels of sugar in their final product (Harris, 2016).
• 4. Drug testing
• As chromatography can accurately identify substances within the bloodstream, it is widely used in sport to test athletes for doping or
performance enhancing drugs, something to think about the next time you're watching your favourite sport.
• 5. Forensic testing
• Chromatography is also used to help catch criminals. In line with programmes like CSI, gas chromatography is used to analyse blood and cloth
samples, helping to identify criminals and bring them to justice. 
 CONCLUSION AND
RECOMMENDATION
• Initially chromatographic techniques were used to separate substances based on their
color as was the case with herbal pigments. With time its application area was
extended considerably.
• Nowadays, chromatography is accepted as an extremely sensitive, and effective
separation method.
• Column chromatography is one of the useful separation, and determination methods.
• Column chromatography is a protein purification method realized especially based on
one of the characteristic features of proteins. Besides, these methods are used to
control purity of a protein.
• High Performance liquid chromatography (HPLC) technique which has many superior
features including especially its higher sensitivity, rapid turnover rate, its use as a
quantitative method, can purify amino acids, proteins, nucleic acids, hydrocarbons,
carbohydrates, drugs, antibiotics, and steroids.
REFERENCES
• Donald PL, Lampman GM, Kritz GS, Randall G. Engel introduction to organic
• laboratory techniques. 4th ed. Thomson Brooks/Cole; (2006). [Google Scholar].
• Harris, Daniel C. (2016). Quantitative chemical analysis. Charles A. Lucy (Ninth ed.). New
York. Kumar, Pranav (2018). Biophysics and Molecular Biology. New Delhi: Pathfinder
Publication.
• McMurry J (2011). Organic chemistry: with biological applications (2nd ed.). Belmont, CA:
Brooks/Cole.
• Walls D, Loughran ST. Protein chromatography:Methods and protocols, methods in
molecular biology. (2011) [Google Scholar}.