Modern Separation
Technique
Chroma tography
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Modern methods/chromatographic methods
Chromatography is the collective term for a family of laboratory
techniques for the separation of mixtures.
The key to the separation is the differing affinities among
analyte, stationary phase, and mobile phase.
It is used by scientists to analyze, identify, purify and quantify
components present in a sample.
Pharmacognosy
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Cont ’d…
❑ Discovered by Mikhail S. Tswett (Tsvet) in 1900s.
❑ Color writing
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Cont’d…
Technique for separating the components of a mixture on the basis of the
relative amounts of each solute distributed between a moving fluid
stream, called the mobile phase, and a contiguous stationary phase.
The mobile phase may be either a liquid or a gas, while the stationary phase
is either a solid or a liquid
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Cont’d…
Stationary Phase Mobile phase
❖ The phase that doesn't move
❖ Solid or liquid supported on a solid. ❖ The phase that does move
❖ Components of the mixture with ❖ Either liquid or gas
similar properties to the stationary ❖ Components of the mixture with
phase elute later. similar properties to the mobile
❖ Column or planar chromatography phase elute earlier.
❖ Liquid or gas chromatography
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Principle
❖ 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.
❖ Because of differences, some components of the mixture stay longer in the
SP, and they move slowly in the chromatography system, while others pass
rapidly into MP, and leave the system faster.
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Principle
❑ Column
❑ SP
❑ Crude extract
❑ MP
❑ Elution
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Classification
1. Purpose (Analytical or preparative)
2. Mechanism of separation
3. Stationary phase ( Column or Planar)
4. Nature of mobile phase (Gas or Liquid)
Pharmacognosy 8
Based on Purpose
Analytical Pr epar a tive
❑ SP coated on very thin layer □ Relatively thicker layer of SP
supporting materials coated on supporting materials
❑ Deals with analytes in micrograms □ Deals with analytes in
quantity milligrams or large qty
❑ Chemical profile (composition) of a □ isolation of compounds
sample
□ purify and collect one or more
❑ Check the purity of the
compound components
□ eg. PTLC; Prep. HPLC; Prep GC
❑ for screening purpose eg. TLC;
HPLC; GC
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Based on Mechanism
Adsorption chromatography
Partition chromatography
Size exclusion chromatography
Ion exchange chromatography
SEC
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IEC
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Adsorption Chromatography
□ One of the oldest types of chromatography
around. It utilizes a mobile liquid or gaseous phase
□ that is adsorbed onto the surface of a stationary
solid phase.
□ used mainly in TLC & CC packed with silica
gel/alumina
Pharmacognosy 13
Adsorptionvs. Absorption
❖ Adsorption is a surface phenomena where interaction takes
place only on the surface of one substance.
❖ In absorption one substance penetrate in to the bulk of
another substance.
Pharmacognosy 14
Adsorption Chromatography
❑ Adsorbents:
❖ It is a solid stationary material where it is either coated or
packed on solid supporting materials
❖ The most common are Silica gel & Alumina in which the
interactions with solute molecules is due to OH groups
present on their surface.
Pharmacognosy
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Normal Silica gel phase
Pharmacognosy 16
Pharmacognosy 17
Pharmacognosy 18
Adsorption strength of Solutes
❑ The adsorption strength of compounds increases with increasing polarity
of functional groups, as shown below:
❑ As stationary phase SiO2 is polar
-CH=CH, 2 -X, -OR, -CHO, -CO2R, -NR2, -NH2, -OH, -CONR, -CO
2 H. 2
(weakly adsorbed) (strongly adsorbed)
(nonpolar) (more polar)
Olefins < Ethers < Esters < Lactones < Aldehydes < Amines <Phenols < Acids.
Pharmacognosy 19
Thin layer chromatography (TLC)
❑ Thin layer chromatography (TLC) is a chromatographic technique used to
separate the components of a mixture using a thin stationary phase
supported by an inert backing.
❑ stationary phase is a thin layer supported on glass,
plastic or aluminum plates.
❑ identifying substances and testing the purity of compounds.
❑ TLC is a useful technique because it is relatively quick and requires
small quantities of material.
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Backing
Glass Aluminum and Plastic
□ Iner t □ Can be cut with scissors
□ Withstand heat and □ Al- may not stand reactive stains
reactive stains □ Plastic:- doesn’t withstand high heat
□ Brittle and difficult to cut □ Both are flexible:- flaking of SP
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Stationary phase
Silica gel Alumina
□ Acidic □ basic and it will not separate
□ deterioration of acid- sample sizes as large as silica
labile molecules gel
□ chemically reactive
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Mobile Phase
❑ Determining the best solvent may require a degree of trial and error.
❑ A common starting solvent is 1:1 hexane:ethyl acetate.
❑ Changing solvents if there is no separation (Changing the PH, Ratio,
polarity of solvents)
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Principle
❑ Separations in TLC involve distributing a mixture of two or more substances
between a stationary phase and a mobile phase.
❖ adsorb on the stationary phase versus how much they dissolve in the mobile
phase.
❑ The stationary phase:
❖ is a thin layer of adsorbent (usually silica gel or alumina) coated on a plate.
❑ The mobile phase:
❖ is a developing liquid which travels up the stationary phase, carrying the
samples with it.
Pharmacognosy
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Preparing the development chamber (DC)
Jar, beaker or flask Sealed well,
Large
Add the mobile phase about 0.5 to 1
cm deep in the bottom.
Place a piece of filter paper into the
developing chamber.
Seal the chamber tightly
Chamber stand until the atmosphere
becomes saturated
Preparing the SP
Cut a piece of paper
Mark a point above the solvent level
on a piece of scratch paper.
Transfer the mark onto the chromatography
paper
It is on this line that all samples will be
spotted
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Loading the sample
Sample should be dissolved in an
appropriate solvent
Then the dissolved samples may be spotted
to the plate by capillary tube.
Allow the solvent to evaporate.
More than one sample can be added
Spots on the chromatogram should be
2 to 2.5 cm
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Developing a chromatogram
Handle the plate only by its edges
Place the TLC plate into
the DC with the spots toward the bottom
Immerse the bottom of the plate in the liquid
(liquid doesn’t touch the spotted samples.
When solvent front approaches, of the
plate, remove the plate from the DC.
Quickly trace the solvent front with a pencil
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Identifying the Spots (visualization)
Three techniques involved
1) If the spots can be seen (visible), outline them
with a pencil.
2) If no spots are obvious, the most common
visualization technique is to hold the plate under
a UV lamp.
3) Derivatization by spraying with reagents
(chemical appropriate treatment)
Pharmacognosy
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Identifying spots…
✓A fluorescent compound, usually
Manganese-activated Zinc Silicate,
✓ is added to the adsorbent
✓ allows the visualization of spots under
a black light (UV254).
✓ The adsorbent layer will fluoresce
light green by itself.
t
✓ Spots of analytes quench
fluorescence and appear as a dark
spot.
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Chromoplates at 254nm
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Visualizing Agents
❑ Alkaloids: Dragendorff’s reagent
❑ Cardiac glycosides: Antimony trichloride
❑ Sugar: Aniline phthalate
❑ Amino acids: Ninhydrin
❑ Iodine
❑ Permanganeate
❑ P-anisaldehyde
❑ Ceric ammonium molybdate
Pharmacognosy 32
Interpreting the Data
❑ The Rf (retention factor) value for each spot should be calculated
Rf = Distance travelled by compound
Dist. trav. by the solvent front (mobile phase).
□ For two closely migrating components, optimum resolutions are usually obtained
when the Rf’s of both compounds are between 0.2 and 0.5
□ It is characteristic for any given compound on the same stationary phase using
the same mobile phase for development of the plates.
□ Hence, known Rf values can be compared to those of unknown substances
to aid in their identifications. 33
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Cont’d….
❑ Rf values often depend on
Type of stationary phase
The solvent used in the TLC experiment
Temperature (to small extent)
❑ The most effective way to identify a compound is to spot known substances
– authentic - next to unknown substances on the same plate.)
❑
In addition, the purity of a sample may be estimated from the
c hromatogram.
❑
An impure sample will often develop as two or more spots, while a pure sample
will show only one spot.
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The process
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❑ Resolution defined as the separation b/n two spots (compounds)
❑ How to improve resolution
❖ Multiple chromatography
❖ Two dimensional chromatography
Pharmacognosy 38
Multiple chromatography
❑ Multiple development: the chromatogram is repeatedly developed in the
same direction and thus the complete resolution of two or more
substances which have Rf values close together can be obtained.
❖ As the mobile phase one can use either
✓ the same solvent system or
✓ different solvent systems.
Pharmacognosy
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Two-dimensional chromatography
This technique is applicable when large numbers of substances
are to be separated on single chromatogram.
Development in a direction perpendicular to the first stand
with a solvent system different from that used initially is
often necessary.
The sample is applied on one corner of a square piece of
chromatogram and after development with the first solvent, the
chromatogram is dried , rotated 90o and developed in the second
direction.
Usually different types of solvents systems are used in each direction.
It is essential that the first solvent be completely volatile.
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Two-dimensional paper chromatography
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Common problems
Over large spots
Uneven Advance of Solvent Front
Spotting
Streaking
TLC Problems: Troubleshooting
Problem Causes Solution
a) Over migration a) Developer too polar a) Reduce polarity
b) Under migration b) Developer too non-polar b) Increase polarity
c) Distorted solvent front c) Developer not equilibrated c) Equilibrate
d) Distorted spots d) Wrong adsorbent d) Change plates
e) Distorted spots e) Spotted too much e) Change concentration
f) No separation f) Wrong developer f) Change developer
g) No separation g) Wrong adsorbent g) Change plate type
h) Tailing h) Spot overloading h) Reduce concentration
i) Tailing i) Component is basic i) Increase basicity
j) Tailing j) Component is acidic j) Increase acidity
k) Tailing/no separation k) Decomposition k) Developer/plate
Pharmacognosy 43