chromatography introduction types of chromatography method seperation on chromatography detail about planar chromatography

1. Presenter: DR ANURAG YADAV
Moderator: DR AVINASH S S
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2. The word derived from Greek:
Initial described by Mikhail Tswett in 1903.
• colorChroma
• To writegraphein
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3. CHROMATOGRAPHY
“Chromatography is a technique for separating
mixtures into their components in order to analyze,
identify, purify, and/or quantify the mixture or
components.”
Separate
• Analyze
• Identify
• Purify
• Quantify
ComponentsMixture 3
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4. TERMINOLOGIES:
Chromatograph - equipment that enables a sophisticated
separation
EX. Gas chromatography or Liquid chromatography
Eluent - Fluid entering column/ solvent that carries the analyte.
Eluate - Mobile phase leaving the column.
Stationary phase - Immobilized phase
 Immobilized on the support particles or on the inner wall of
the column tubing.
 Examples : Silica layer - Thin Layer Chromatography
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5. TERMINOLOGIES:
Mobile phase
Moves in a definite direction. Liquid (LC), Gas (GC).
 The mobile phase moves through the chromatography column (the
stationary phase) where the sample interacts with the stationary phase
and is separated.
Retention time : Time takes for a particular analyte to pass through the
system (from the column inlet to the detector) under set conditions.
Sample (Anylate) :Substance analyzed in chromatography.
Solvent : Any substance capable of solubilizing another
substance.
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6.  Chromatogram
 Visual output of the chromatograph.
 Separation - Different peaks or patterns on the chromatogram
correspond to different components of the separated mixture.
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7. CLASSIFICATION:
1. Based on supporting medium
2. Based on mobile & stationary phase
3. Based on mechanism of separation
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8. 1. BASED ON SUPPORTING MEDIUM
Supporting
medium
planar column
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9. 2. BASED ON MOBILE & STATIONARY PHASE:
MOBILE AND STATIONARY
PHASE
GC
GLC GSC
LC
LLC LSC
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10. DEPENDING ON PHYSICAL PROPERTIES OF
STATIONARY PHASE
 LC – flat methods ( Paper chr , HPTLC , TLC )
- Column methods(open column LC, HPLC)
 GC – packed column
- wall coated column ( capillary )
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11. 3. BASED ON MECHANISM OF SEPARATION:
MECHANISM
ION
EXCHANGE
PARTITION
ADSORPTION
AFFINITY
SIZE
EXCLUSION
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12. PLANAR CHROMATOGRAPHY:
 Based on principle of the partition
Chromatography.
“The differential distribution of solute between two
immiscible liquids on plane.”
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13.  partition chromatography a process of separation of solutes utili
zing the partition of the solutes between two liquid phases, namely
the original solvent and the film of solvent.
When substance mixed
with immiscible solvent,it
will distribute such that,
At equilibirum the ratio of
its conc In two phase is
constant
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14.  This ratio is termed as partition coefficient & is
characteristic of a particular substance for a given
pair of solvent.
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15. TYPES OF PARTITION CHROMATOGRAPHY
 Normal phase
 Reverse phase: Ion suppression & Ion pair
chromatography
 Normal phase LC, stationary phase is polar & mobile
phase is non-polar. water is the stationary phase; hexane,
benzene, chloroform or butanol form the mobile phase.
 Reverse phase LC, stationary phase is non-polar (eg.
octadecyl silane packing in a column) and mobile phase
is polar (solvents like methanol, acetonitrile used in
column mode of chromatography). 15
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16. ION-SUPPRESSION CHROMATOGRAPHY
 Ionic character of weakly acidic/basic→suppressed(by
modification of mobile phase PH →solutes become less
polar →interact with nonpolar stationary phase →reverse
phase chromatography
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17. ION-PAIR CHROMATOGRAPHY
 Counter ion of analyte → added to mobile phase →ionic
pair with analyte →neutralysed analytes are separated by
reverse phase chromatography
 Uses : separation of therapeutic drugs & metabolites.
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18. PAPER CHROMATOGRAPHY :
 Paper chromatography is a variant of partition
chromatography procedure in which the cellulose
support is in the form of a sheet or paper
 Cellulose contain a large amount of bound water even
when extensively dried
 Partitioning occurs between the bound water and the
developing solvent
 In paper chromatography the mixture to be separated is
spotted onto the paper and dried
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19. TYPES :
 Paper chromatography.
 Ascending paper.
 Descending paper.
 Ascending-descending.
 Radial paper.
 Two-dimensional paper.
 Thin layer chromatography.
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20. Paper Chromatography has different types or
modes:
Ascending chromatography: As
the name indicates, the
chromatogram ascends. Here the
development of paper occurs due
the solvent movement or travel in
upward direction on the paper.
Descending chromatography: Here the development of paper occurs
due to solvent travel downwards on the paper.
Ascending- descending mode: Here solvent first travels
upwards and then down wards on the paper. 20
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21. Radial mode: Here the solvent travels from center(mid point) towards
periphery of Circular chromatography paper.
Two dimensional chromatography: Here
the chromatogram development occurs in
two directions at right angles.
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22. INSTRUMENTATION
Chromatography jar
Capillary tube
Stationary phase (liquid impregnated paper)
Mobile phase
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23. INSTRUMENTATION
Chromatography jar:
It is made of glass and has a lid on it. Jar maintains
proper environment that is required for separation.
Capillary tube:
It is used to apply sample mixture.
Stationary phase:
liquid impregnated paper
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24. INSTRUMENTATION
Mobile phase:
Mobile phase may be a single liquid or a mixture of
liquids.
Commonly used mobile phases are;
 Methanol
 Ethanol
 Ethyl acetate
 Diethyl ether
 Acetone
 Chloroform 24
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25. HOW TO PERFORM PAPER
CHROMATOGRAPHY?
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26. 1. PREPARING THE PAPER STRIPS
• Cut the filter paper into 5
x4 measurement.
• Draw a line 0.5 cm above
the bottom edge of the
strip with the pencil.
• Label each strip with its
corresponding solution.
•
• Place a spot from each
pen on your starting line.
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27. 2. DEVELOPING THE CHROMATOGRAMS
• Place the strips in the beakers.
• Make sure the solution does not come
above your start line.
• Keep the beakers covered.
• Let strips develop until the ascending
solution front is about 2 cm from the top
of the strip.
• Remove the strips and let them dry.
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28. More polar!
Less polar!
solvent front
origin mixture
solvent front
component B
component A
origin
solvent front
component B
component A
origin
Increasing Development Time
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29. VISUALIZATION OF CHROMATOGRAPHY
If the sample is separated into colored components, then
the location is dried in ordinary light. But in case of
colorless components following are used;
 Uv lamp
 Iodine crystals
 Spraying agents: Ninhydrin for aminoacids and
proteins , sulfuric acid for phospholipids ,
diphenylamine for sugars 29
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30. DOCUMENTATION
Storage of chromatogram.
Calculating Rf values
 Calculate Rf value & interpret.
 Defined : “as the ratio of the distance travelled by
the substance & the distance travelled by solvent
front”
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31. RF VALUE IMPORTANCE:
 Ratio of distance travelled by the solute to the
distance travelled by the solvent
 Rf value is constant for a particular solvent system
at a given temperature
 Spots of the unknown substance can be identified
by comparing those of the pure standards
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32. APPLICATIONS
It is used for separation and identification of;
Amino acids
Carbohydrates
Tannins
Glycosides
Alkaloids etc.
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33. THIN-LAYER CHROMATOGRAPHY (TLC)
“ The technique which involves flowing of mobile phase over a
thin layer of adsorbent, applied on solid support, where
separation of components occur by differential migration
which occurs when solvent flows along fine powder spread on
glass plates, is called thin –layer chromatography.”
 Silica / Alumina layered over a glass plate ----- uniform thin
layer(0.2mm)
 Procedure same as that for paper
chromatography.
 Better resolution
 HPTLC: particle size-4.5μm. 33
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34. Instrumentation:
 Chromatography jar
 Capillary tube
 Thin layer chromatography plate
 Stationary phase
 Mobile phase
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35. Chromatography jar:
 It is made of glass and has a lid on
it.
 Jar maintains proper environment
that is required for separation.
Capillary tube:
 It is used to apply sample mixture
on TLC plate.
TLC plate:
 Borosilicate glass plates are
preferred. Most commonly used
sizes are;
 20 X 20cm
 20 X 10cm
 20 X 5cm
Mobile phase:
Mobile phase may be a single
liquid or a mixture of liquids.
Commonly used mobile phases
are;
Methanol
Ethanol
Ethyl acetate
Diethyl ether
Acetone
Chloroform
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36.  Procedure
 Location Of Separated Components
 Documentation
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37. APPLICATION:
It is used for separation and identification of;
 Amino acids
 Peptides and proteins
 Alkaloids
 Carbohydrates
 Fats and fatty acids
 Antibiotics
 Narcotic analgesics
 Glycosides 37
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38. ADVANTAGES OF TLC
 Simple
 Rapid
 Ability to process large number of samples in minimal
time
 Low cost in terms of reagent & equipment.
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39. APPLICATIONS OF CHROMATOGRAPHY.
• In clinical diagnosis : detection & estimation of amino
acids, metabolites, sugars, mucopolysaccharides in urine
& blood.
• Useful for screening and diagnosis of inborn metabolic
disorders : Aminoacidurias, hemoglobinopathies,
mucopolysaccharidoses, etc.
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40. APPLICATIONS OF CHROMATOGRAPHY
 In clinical diagnosis
Paper chromatography,TLC –qualitative
HPLC, GC –For quantitation
 In clinical diagnosis
Assay of Hormones, drugs, vitamins,
metabolites ---HPLC and GC
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41. APPLICATIONS OF CHROMATOGRAPHY
 Chromatography in protein research –
1) Purification –adsorption, ion exchange ,affinity, gel
filtration chromatography.
2) Sequencing – ion-exchange chromatography.
3) Mol.wt. determination – gel filtration chromatography.
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42. TROUBLESHOOTING
 It seems to be easy procedure, but error do
occur like;
- Compound runs as streak rather than spot
(sample was overloaded)
- Sample run as smear/upward crescent :
compound possess strongly acidic or basic
group- add few drops of ammonium hydroxide
or acetic acid to the solvent.
- Sample runs downward crescent- adsorbent
was disturbed during spotting.
streak
More polar
Cross placed
Less sample
Normal
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43. - Plate solvent front runs crookedly:
either the adsorbent has flaked off the sides
or the side of plate are touching sides of
container.
- Many random spots
- No spots are seen on plate
- Blur blue spots on plate : use of ink pen
instead of pencil to mark the origin.
streak
More polar
Cross placed
Less sample
Normal
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46.  ADSORBENTS:
 “ An adsorbent is a substance, usually porous in nature and with a high surface area that can adsorb substances onto its surface by intermolecular forces.”

 AN IDEAL ADSORBENT:
 The Ideal adsorbent must fulfill the following requirements:

 Insoluble in mobile phase
 Inert to solutes (adsorptive)
 Colorless especially when work with colored mixtures
 Suitable particle size enough to give good separation and reasonable flow rate
 COMMON ADSORBENTS:
 Hydrated silica gel
 Silica gel G
 Silica gel S
 Silica gel GF 254
 Silica gel H
 Silica gel N
 Silica gel HF 254
 Silica gel PF 254
 Modified silica gel
 Alumina
 Kieselghur (Diatomaceous earth)
 Cellulose MN 300
 Cellulose microcrystalline
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47. THIN LAYER CHROMATOGRAPHY (TLC)
 In TLC, any substance that can be finely divided and
formed into a uniform layer can be used.
 Both organic and inorganic substances can be used
to form a uniform layer for TLC.
 Organic substances include: cellulose, polyamide,
polyethylene
 Inorganic: silica gel, aluminum oxide and magnesium
silicate
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48. THIN-LAYER CHROMATOGRAPHY: A
TWO-COMPONENT MIXTURE
More polar!
Less polar!
solvent front
origin mixture
solvent front
component B
component A
origin
solvent front
component B
component A
origin
Increasing Development Time
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49. APPLICATIONS
1. Separation of carbohydrates:
Mobile phase:
acetonitrile : water (85:15)
Detection:
sulfuric acid : methanol (1:3)
heat for 10 min at 110 C to see
brown spots
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50. Separation of Total Lipid into
different Classes
Mobile Phase: hexane: diethyl ether: formic acid (80:20:2)
Cholesteryl esters
TAG
Free fatty acids
Cholesterol
1,3-DAG
1,2-DAG
Monoacyl glycerols
Phospholipids
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51. Separation of Triacylglycerols
Mobile Phase: Pet ether: diethyl ether: acetic acid (90:9:1)
Tristearin
2-oleodistearin
1-stereodiolein
Triolein
Trolinolein
With HUFA
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