Chromatography is based on the principle where molecules in mixture applied onto the surface or into the solid, and fluid stationary phase (stable 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.

1. CHROMATOGRAPHY
AND
TYPES
by
LIKHITH K
BiSEP – 2021
Dept of Biotechnology
St Aloysius College
Mangaluru, Karnataka

2. CONTENTS
 Introduction
 History
 Purpose
 Principle
 Types
 Applications
 Conclusion
 Reference

3. INTRODUCTION
What is Chromatography?
 Derived from the Greek word Chroma meaning colour, chromatography provides a way to
identify unknown compounds and separate mixtures.
 The separation of a mixture by distribution of its components between a mobile and stationary
phase over time.
 A wide range of chromatographic procedures makes use of differences in size, binding
affinities, charge, and other properties to separate materials.

4.  Chromatography is a technique for,
 separating mixtures of compounds
 identifying unknown compounds
 establishing the purity or concentration of compounds
 monitoring product formation in the pharmaceutical
and biotechnology industries

5. HISTORY
 Chromatography (from Greek: chromatos -- color , "graphein" -- to write)
 1903 Tswett - plant pigments separated on chalk columns
 1931 Lederer & Kuhn – Liquid chromatography of carotenoids
 1938 Thin Layer Chromatography and ion exchange
 1950 Reverse phase Liquid chromatography
 1954 Martin & Synge (Nobel Prize)

6. Father of Chromatography Mikhail Tsvet

7. PURPOSE OF CHROMATOGRAPHY
 Analytical - determine chemical composition of a sample
 Preparative - purify and collect one or more components of a sample

8. PRINCIPLE OF CHROMATOGRAPHY
 Chromatography is based on the principle where molecules in mixture applied onto the surface
or into the solid, and fluid stationary phase (stable 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

9.  Three components thus form the basis of the chromatography technique.
1. Stationary phase: This phase is always composed of a “solid” phase or “a layer of a liquid
adsorbed on the surface solid support”.
2. Mobile phase: This phase is always composed of “liquid” or a “gaseous component.”
3. Separated molecules: The type of interaction between stationary phase, mobile phase, and
substances contained in the mixture is the basic component effective on separation of
molecules from each other

10.  Stationary phase in chromatography, is a solid phase or a liquid phase coated on the surface of
a solid phase.
 Mobile phase flowing over the stationary phase is a gaseous or liquid phase
 If mobile phase is liquid it is termed as liquid chromatography (LC) and if it is gas then it is
called gas chromatography

11. Retention factor (Rf):
• The retention factor is denoted by Rf and is a quantitative indication which tells about the
distance traveled by a particular compound in a particular solvent.
• This value helps to know that the known compound and unknown compound are similar or
not. If the value of Rf of a known compound is equal to the Rf value of unknown compound
then it means that the two compounds are same or identical.
• Retention factor is defined as a ratio of distance traveled by the solute to the distance traveled
by the solvent front.
• Rf = D1/D2
• Rf = distance traveled by solute/ distance traveled by solvent front

13. TYPES OF CHROMATOGRAPHY
The distinct types of chromatography are as explained below:
• Thin-layer chromatography
• Paper chromatography
• Size exclusion chromatography
• Ion exchange chromatography
• Affinity chromatography
• Reverse phase chromatography
• Hydrophobic interaction chromatography
• High-performance liquid chromatography

15. 1. 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
solid substances like alumina, silica gel and 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.
In cases where molecules of the sample are colourless, 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.
Formation of a visible colour can be observed under room light or UV light. The position of each
molecule in the mixture can be measured by calculating the ratio between the distances travelled
by the molecule and the solvent. This measurement value is called relative mobility, and
expressed with a symbol Rf. Rf. value is used for qualitative description of the molecules.

17. 2. 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 settle in its pores
make up the stationary “liquid phase.” Mobile phase consists of an appropriate fluid placed in a
developing tank. Paper chromatography is a “liquid-liquid” chromatography.

18. 3. Size exclusion, Gel- permeation (molecular sieve) chromatography
The basic principle of this method is to use dextran containing materials to separate
macromolecules based on their differences in molecular sizes. This procedure is basically used to
determine molecular weights of proteins, and to decrease salt concentrations of protein solutions.
In a gel- permeation column stationary phase consists of inert molecules with small pores. The
solution containing molecules of different dimensions (analyte) are passed continuously with a
constant flow rate through the column. Molecules larger than pores can not permeate into gel
particles, and they are retained between particles within a restricted area.
Larger molecules pass through spaces between porous particles, and move rapidly through inside
the column. Molecules smaller than the pores are diffused into pores, and as molecules get
smaller, they leave the column with proportionally longer retention times. Sephadeks G type is
the most frequently used column material. Besides, dextran, agorose, polyacrylamide are also
used as column materials

20. 4. Ion- exchange chromatography
Ion- exchange chromatography is based on electrostatic interactions between charged protein
groups, and solid support material (matrix). Matrix has an ion load opposite to that of the protein
to be separated, and the affinity of the protein to the column is achieved with ionic ties.
Proteins are separated from the column either by changing pH, concentration of ion salts or ionic
strength of the buffer solution. Positively charged ion- exchange matrices are called anion-
exchange matrices, and adsorb negatively charged proteins. While matrices bound with negatively
charged groups are known as cation-exchange matrices, and adsorb positively charged proteins

22. 5. Affinity chromatography
This chromatography technique is used for the purification of enzymes, hormones, antibodies,
nucleic acids, and specific proteins. A ligand which can make a complex with specific protein
binds the filling material of the column.(dextran, polyacrylamide, cellulose etc)
The specific protein which makes a complex with the ligand is attached to the solid support
(matrix), and retained in the column, while free proteins leave the column. Then the bound protein
leaves the column by means of changing its ionic strength through alteration of pH or addition of
a salt solution.

24. 6. Reversed-phase HPLC (RP-HPLC)
This is one of most important techniques for protein separations and the method of choice for
peptide separation. RP-HPLC has been applied on the nano, micro, and analytical scale, and has
also been scaled up for preparative purifications, to large industrial scale. Because of its
compatibility with mass spectrometry, RP-HPLC is an indispensable tool in proteomic research.
With modern instrumentation and columns, complex mixtures of peptides and proteins can be
separated at the molar levels for further analysis. In addition, preparative RP-HPLC is often used
for large-scale purification of proteins. This unit provides protocols for packing and testing a
column, protein separation by use of gradient or step elution, desalting of protein solutions, and
separation of enzymatic digests before mass spectrometric analyses.

26. 7. Hydrophobic interaction chromatography (HIC)
In this method the adsorbents prepared as column material for the ligand binding in affinity
chromatography are used. HIC technique is based on hydrophobic interactions between side
chains bound to chromatography matrix.

27. 8. High-pressure liquid chromatography (HPLC)
Using this chromatography technique it is possible to perform structural, and functional analysis,
and purification of many molecules within a short time, This technique yields perfect results in the
separation, and identification of amino acids, carbohydrates, lipids, nucleic acids, proteins,
steroids, and other bio chemical active molecules, In HPLC, mobile phase passes through
columns under 10–400 atmospheric pressure, and with a high (0.1–5 cm//sec) flow rate.
In this technique, use of small particles, and application of high pressure on the rate of solvent
flow increases separation power, of HPLC and the analysis is completed within a short time.
Essential components of a HPLC device are solvent depot, high- pressure pump, commercially
prepared column, detector, and recorder. Duration of separation is controlled with the aid of a
computerized system, and material is accrued.

29. Applications of Chromatography in the Pharmaceutical Industry
The technique of chromatography is extensively employed in the pharmaceutical industry in order
to analyze and identify the presence of any trace amounts of chemicals and elements in a given
sample.
Another vital application of chromatography in this sector is in the separation of certain chemical
compounds based on their molecular masses (and sometimes on the basis of the elements that
constitute them).
The technique of chromatography also plays a crucial role in the development of new drugs. For
example, the presence of impurities and other unknown compounds can be detected in the drug
sample with the help of chromatography. Furthermore, the purity of the drug sample can also be
analyzed with the help of this technique .
Applications of Chromatography in the Food Industry
In the food industry, the technique of chromatography plays a vital role in the determination of the
shelf life of food substances by helping in the analysis of the point at which food spoils.
Furthermore, the presence of chemical additives in food can also be determined with the help of
this technique.
The nutritional value of the food sample can also be determined by employing chromatographic
techniques.

30. Applications of Chromatography in the Chemical Industry
 Chromatography plays a vital role in the chemical industry for the testing of water samples for
purity.
 The testing of air samples for their purity is also accomplished by chromatographic techniques
in the chemical industry.
 The presence of toxic contaminants in oils and pesticides (the most notable of which being
polychlorinated biphenyls, often abbreviated to PCBs) can be determined with the help of
specialized chromatographic techniques such as GC and HPLC.
 It can also be noted that chromatography also has many applications in the life sciences.
Applications of Chromatography in the Field of Molecular Biology
 In the field of molecular biology, the study of proteomics and metabolic often involve the use
of various hyphenated chromatographic techniques (the most notable of which being EC-LC-
MS).
 Nucleic acid research is also known to make extensive use of such chromatographic
techniques.
 A specific type of chromatography known as HPLC is widely used in protein separation
applications. This type of chromatography is also useful in enzyme purification, plasma
fractionation, and insulin purification.

32. CONCLUSION
Initially chromatographic techniques were used to separate substances based on their colour 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.
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.

33. REFERENCES
1. Cuatrecasas P, Wilchek M, Anfinsen CB. Selective enzyme purification by affinity
chromatography. Proc Natl Acad Sci U S A. 1968;61:636–43.
2. Porath J. From gel filtration to adsorptive size exclusion. J Protein Chem. 1997;16:463
3. Harris DC. Exploring chemical analysis. 3rd ed. WH. Freeman & Co; 2004.
4. Gerberding SJ, Byers CH. Preparative ion-exchange chromatography of proteins from dairy
whey. J Chromatogr A. 1998;808:141–51.
5. Donald PL, Lampman GM, Kritz GS, Engel RG. Introduction to organic laboratory
techniques. 4th ed. Thomson Brooks/Cole; 2006. pp. 797–817.
6. Harwood LM, Moody CJ. Experimental organic chemistry: Principles and Practice. Oxford:
Blacwell Science; 1989:180–5.

34. THANK YOU