This document provides an overview of chromatography techniques. It defines chromatography as a laboratory technique used to separate mixtures into individual components. The mixture is dissolved in a mobile phase that carries it through a stationary phase, causing different constituents to separate as they have different affinities for the stationary phase. Several chromatography techniques are described in detail, including paper chromatography, thin layer chromatography, gas chromatography, and high performance liquid chromatography. The principles, processes, and applications of each technique are summarized. Contact information is also provided for the Indian Institute of Chromatography & Mass Spectrometry.

1. CHROMATOGRAPHY
TECHNIQUES SUBMITTED TO :
POST GRADUATE & RESEARCH CENTER
DEPARTMENT OF MICROBIOLOGY
(Government Aided)
M. GANTHIMATHI
II M.SC MICROBIOLOGY
20201232516104
PG AND RESEARCH DEPARTMENT OF MICROBIOLOGY
DR.S.VISHWANATHAN
PG AND RESEARCH DEPARTMENT OF MICROBIOLOGY
SRI PARAMAKALYANI COLLEGE
ALWARKURUCHI

2. Chromatograpgy :
 chromatography is a laboratory technique for the separation of
a mixture into its components.
 The mixture is dissolved in a fluid solvent (gas or liquid) called the mobile
phase, which carries it through a system (a column, a capillary tube, a
plate, or a sheet) on which a material called the stationary phase is fixed.
 Because the different constituents of the mixture tend to have different
affinities for the stationary phase and are retained for different lengths of
time depending on their interactions with its surface sites, the constituents
travel at different apparent velocities in the mobile fluid, causing them to
separate.

3. History:
 Russian botonist first proposed this technique at first, “Michel
Twsett” in 1906.

4. PRINCIPLE :
 In greek, the word “ chromo” means color
 “Graphenin” means “ writing “
 Here the color writing is happenes between the mobile phase and
stationary phase.
 All the components do not have the same type of affinity.
 Stationary phase : Solid phases or a layer of a liquid adsorbed on the
solid support.
 Mobile phase : Gaseous component or liquid. It is a thing which can move
that may be liquid or gas.

5. MOBILE AND STATIONARY PHASE :

8. PAPER CHRONATOGRAPHY:
 We use paper as a “stationary phase “ and solvent act as a “ mobile phase “
in this techninique.
 This techniques was invented by Synge and Martin in the year 1943.
 It is a “Particition chronatography” (divide ) – Here the solvent diffuse itself
between move across the paper. It will distribute themselves between
mobile and stationary phase.
 Each component has a specific affinity to the water.
 Select suitable filter paper (eg: Whattman no. 1 filter paper. The pore size
of the filter paper is also important )

9. Procedure :
 Capillary tube is like a “tip” which is used to load the sample.
 Draw 1 cm line above from the bottom edge of the paper. The sample in
the center or 2 equal distance on the line we drawed.
 Immerse the paper into the solvent.
 Samole should not touch the mobile phase. It should away from the
mobile phase (liquid)
 If sample is immersed, the sample will get dissolved into the liquid and
not able to move upwards and do not get seperation of pigments

10.  The solvent should be move 1/3rd of the paper not the upward edge. (It
takes 10-20mins)
 Dry the paper in a hotair oven.
 Spray Ninhydrin and measure the solvent travel.
 After spraying the ninhydrin the aminoacids travelling point shows as a
“purple colur”
 Each component has a specific affinity to the mobile phase(solvent)
upwards.
 High affinity component move faster and high travel distance.

13.  Retardation factor = Distance travelled by the component
 ------------------------------------------------
 Distance travelled by the solvent.
 = a
 ---
 b
 Eg: 5.5
 ------ = 0.78
 7.0

16. Descending chromatography :

18. Thin Layer Chromatography :
 Here also “Partician principle “ involed like paper chromatography.
 Thin layer of substrate is used here as a “ stationary phase “. Usually silica
gel is used a stationary phase in this type.
 Solvent is act as a “ mobile phase “ (liquid)
 Prepare a silica gel and slury pn a plate and dry it for 1 hour. ( Chemical –
calcium sulphate is added to silica gel to as a binder )
 Rf value must be between “0” to “1”.

19. Thin layer chromatography:

25. Gas chromatography :
 Versatile instrument.
 In early 1900s, Gas chromatography was discovered by “Mikhali semenovic
Tsvett.
 A. T. James and P. Martin use this technique to sepaerate fatty acids in
1952.
 Used to analayze the volatile substances in the gas phase.
 Volatile nature – both organic and inorganic nature.
 Gas – mobile phase
 Solid or liquid molecule – stationary phase.
 “Partician principle “- Distribution between mobile and stationary phases.

26.  GCMS – Gas Chromatography Mass Spectrophotometer
 If the component have a high afffinity with the stationary phase they take a
long timee in stationary phase.
 Affinity for the stationary phase is by the intermolecular interaction and
polarity of the stationary phase.
 Inject the sample in a liquid form. They then meet the gas
chromatography.
 Principle mechanism: Gas chromatography, at first the sample is heated
and converted the liquid sample into vapour form. That vapour travels to
the mobile phase.

27. Gas chromatography :

28.  If the component has high affinity towards the mobile phase it will travel
stationary phase and reach detecory very fastly.
 Seperation in the colum : A component that adsorbes most strongly to the
stationary phase will spent the most in the colum. (Retention time )
 We all know that polar attracts polar. This attraction make higher time to
stay in stationary phase.
 Component which do not have high affinity to the stationary phase will
come yo the detector at first than the component which have high affinity
to the stationary phase come last.

32. High Performance Liquid
Chromatography (HPLC)
 We apply high pressure in this method.
 It is a part of colum chromatography.
 Mobile phase – solvent(eluvent)
 Stationary phase – sample
 5, 000 range of pressure
 Analytical technique.
 We can use spectrometry or uv detecor to check the final results.

33. PRINCIPLE:
 “Adsrobtion and Partition” principle is employed here.
 (adsorption principle – when the stationaty phase is solid : partician
principle – when both the stationary and mobile phases are liquid )
 Colum is the heart of the chromatography.
 Mostly silica gel is used as a stationaty phase
 Sample should be placed in the liquid phase and sample component move
at the stationary phase.
 Migration sample on stationary phase is based on the polarity and
hydrophobicity of the sample.

34.  Polar samples only interact with polar sample only not a hydrophobicity.
 Mobile phase is organic in nature
 Stationary phase is polar in nature.
 Non polar component does not attact with polar. So they move faster (first
)with the mobile phase. (1st peak)
 But polar sample bind with each other (large amount or smaller amount )
of the sample. The peak graph is “Chromatograph”
 Non polar sample component “elute” come out first.

38. Iron exchange chromatography :
 Principle : We will separate the molecules based on the “charges “ of the
molecules.
 It is versatile in nature and has high resultion.
 Large amount of the samples can be seperated by using this technique.
 It has a high loading capacity.
 Stationary phase: Charged sample molecules(resign – iron exchanger)
 Mobile phase : Solvents
 Opposite charged molecules get attached each other.

39.  There will be exchange of irons (Target ions – counter ion)between the
stationary phase and ions in the components (mixture )
 There are 2 kinds of exchangers – whuch act as the stationary phase.
 Cationic ion exchanger – Negatuve charged group(acidic ions)
 Anionic ion exchanger – Posituve charged groups (basic ions)

40. Isoelectric point or isoelectric PH: (PI –
electric point )
 Amino acids are charged molecules either positive or negative charged at
different PH.
 There will be a patricular PH, the net charge of the particular protein is “0”
– That is called iso electric point
 In iso electric point protein do not have any charge either posituve or
nnegative.
 At PI = 0
 Abow PI = Negative charge
 Below PI = Positive chage.
 Proteins are active on particular PH only.

47. Indian institute of chromatography :

48. Contact address of indian institute of
chromatography and mass
spectrography:

49.  Indian Institute of Chromatography & Mass Spectrometry (IICMS) offers
post graduate diploma courses to freshers to enhance their job
opportunities in pharmaceutical and allied chemical industries. IICMS
helps post graduate students to carry out their projects in chromatography
& mass spectrometry.

51. References :
 1.Reed.G. - IndustrialMicrobiology,CBSPublishers,AVIPublishingCompany.
 2. A. H. Patel – Industrial Microbiology

53.  https://www.iicms.in/institute.html
 https://youtu.be/ZCzgQXGz9Tg