high performance thin layer chromatography (hptlc)mazums.ac.ir/dorsapax/userfiles/file/hptlc.pdf ·...
TRANSCRIPT
Dr. Seyed Mohammad AbtahiDr. Sahar Bazrafkan
Medical Entomologists
23 May 2013
In the name of GOD
High Performance Thin Layer Chromatography (HPTLC)
Table of Contents History
Importance
Chromatography
Classification
TLC, HPTLC
Applied HPTLC
Other Chromatographic Methods
Dr. M. Abtahi2
History
Mikhail Tswett, Russian, 1872-1919
Botanist
In 1906, to separate plant pigments
He called the new technique chromatography
Because:
The result of the analysis was 'written in
color' along the length of the adsorbent
column
Chroma means “color” and graphein means to “write”Dr. M. Abtahi3
Importance
Chromatography has application in every branch
of the physical and biological sciences.
12 Nobel prizes were awarded between 1937 - 1972 alone
for work in which chromatography played a vital role.
Dr. M. Abtahi4
Chromatography
A technique used to separate the components of a mixture,
OR
A physical method of separation in which the components to
be separated are distributed between two phases:
stationary phase
mobile phase
The process occurs due to differences in the distribution
constant of the individual sample components.
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Chromatography
Chromatography can be used for both
qualitative and quantitative analysis.
It can also be used to determine the identity of
a substance.
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Different components of a mixture will be attracted
(adsorb to) the stationary and mobile phases to
different extents.
Chromatography uses these differences in attraction
to separate the components.
The mobile phase passes over the stationary phase.
The components of the mixture are separated
according to their relative attractions to the mobile
and stationary phases.
How does chromatography work?
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The rate of movement of each component
depends mainly upon:
How strongly it adsorbs onto (is attracted to) the
stationary phase.
How readily it dissolves in the mobile phase.
How does chromatography work?
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Classification
There are many different methods of
chromatography, but all methods have:
A stationary phase
A mobile phase (or moving phase)
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Classification
According to the mobile phase:
1- Liquid chromatography: mobile phase is a liquid.
2- Gas chromatography: mobile phase is a gas.
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Classification
According to the stationary phase:
1- Paper chromatography (PC): the stationary phase is a
thin film.
2- Thin layer chromatography (TLC): the stationary
phase is a thin layer supported on plates.
3- Column chromatography (CC): stationary phase is
packed in a column.
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Classification
According to the force of separation:
1- Adsorption chromatography
2-Partition chromatography
3-Ion exchange chromatography
4-Gel filtration chromatography
5-Affinity chromatography
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MechanismMobile phaseStationary phaseMode or type
Solutes move at different rates according to the forces of attraction to the stationary phase.
Liquid or gasSolid that attracts the solutes
Adsorption Chromatography
Solutes equilibrate between the 2 phases according to their partition coefficients.
Liquid or gasThin film of liquid formed on the surface of a solid inert support
Partition Chromatography
Solute ions of charge opposite to the fixed ions are attracted to the resin by electrostatic forces & replace the mobile counterions.
Liquid containing electrolytes
Solid resin that carries fixed ions & mobile couterions of opposite charge attached by covalent bonds
Ion Exchange Chromatography
Molecules separate according to their size:1. Smaller molecules enter the pores of the gel, and need a larger volume of eluent. 2. Larger molecules pass through the column at a faster rate.
Liquid Porous gel with no attractive action on solute molecules
Molecular Exclusion Chromatography
Special kind of solute molecules interact with those immobilized on the stationary phase.
Liquid or gasSolid on which specific molecules are immobilized
Affinity Chromatography
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THIN LAYER CHROMATOGRAPHY
(TLC)
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The beginning: Paper chromatography
Deposition of a drop of colour
Deposition of a drop of solvent
Deposition of more solvent
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Evolution: Vertical paper chromatography
Sample is deposited at the bottomline of the paper.
Paper is placed in a tank filled withsolvent.
Solvent migrates on the paper and elutes the solutes.
The solute migrate depending on their affinity for the solvent.
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THIN LAYER CHROMATOGRAPHY
• The modern version of paper chromatography.
• For identification and quantitation of mixtures of organic compounds.
• Paper is replaced by a layer of stationary phase deposited on a plate.
• A plastic, glass or aluminum sheet is coated with a thin layer of silica gel,
alumina or florosil.
• A very small amount of a solution of the substance to be analyzed is
applied in a small spot with a capillary tube.
• The TLC is developed in a chamber containing the mobile phase.
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Thin Layer Chromatography (TLC)
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High Performance Thin Layer
Chromatography
Current evolution: High-Performance TLC (HPTLC)
Controlled size of stationary phase particles
Modified stationary phases
Automated procedures
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TLC - Procedure
A solution of the sample to be analysed is made up.
As small a spot as possible is place onto the end of the
chromatography plate.
The plate is then placed into a container with the edge of the
plate submerged in solvent.
As the solvent rises up the plate the components of each
sample separate.
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The different components
of a mixture will move at
different rates depending
on relative strength of
attractions to the stationary
and mobile phases.
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TLC - Procedure
Cover
Plate(stationaryphase)
● As the mobile phase rises up by capillary
action, the components dissolve in the
solvent and move up.
● Individual components move up at
different rates, depending on
intermolecular forces between:
Component and stationary phase
Component and mobile phase
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● Stationary phase SiO2, very “polar”
● It is capable of strong dipole-dipole and
H-bond donating and accepting interactions
with the “analytes” (the components being analyzed).
● More polar analytes move slowly up the plate.
● By comparison, the mobile phase is relatively nonpolar and is capable of
interacting with analytes by stronger London forces, as well as by dipole-
dipole and H-bonding.
More nonpolar analytes move higher up the plate.
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● Solvent front should be marked.
● The solvent is allowed to evaporate.
● The spots are visualized using a UV lamp, nevertheless Several methods
exist to visualize the spots.
√ A fluorescent compound, usually Manganese-activated Zinc Silicate, is
added to the adsorbent that allows the visualization of spots under a
blacklight (UV254).
√ The adsorbent layer will fluoresce light green by itself, but spots of
analyte quench this fluorescence and appear as a dark spot.
● Once visible, the Rf value of each spot can be determined.
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Chromatogram of 10 essential oils, Stained with vanillin reagent.
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Solvent front
Application lineContains “origin spots”
Interpreting chromatograms of TLC
A chromatogram is the pattern of bands or spots
formed on the plate in TLC.
The identity of the chemicals in the mixture can
be identified in two ways:
1. running standards of known chemicals on the same
chromatogram as the unknown sample.
2. calculating Rf values of the samples.
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Rf values
Always less than one.
The component most strongly adsorbed onto the stationary phase moves the shortest distance and has the lowest Rf value.
By comparing the Rf values of components of a mixture with the Rf values of known substances under identical conditions, the compounds present in a mixture can be identified.
Rf = Distance moved from origin by componentDistance moved from origin by solvent
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The Rf is defined as the distance the center of the spot moved divided by
the distance the solvent front moved (both measured from the origin).
A B CUx xx x
Solvent Front
Origen
Distance solvent migrated = 5.0 cm
Distance A migrated = 3.0 cm
Distance B migrated = 2.0 cm
Distance C migrated = 0.8 cm
0.8 cm
3.0 cm
Rf (A) =
Rf (B) =
Rf (C) =
Rf (U1) =
Rf (U2) =
2. 0 cm5.0 cm = 0.40
= 0.60
= 0.16
= 0.60
= 0.16
3. 0 cm5.0 cm
0. 8 cm5.0 cm
3. 0 cm5.0 cm
0. 8 cm5.0 cm
Dx
Rf (D) = = 0.80 4. 0 cm5.0 cm
4.0 cm
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Calculation of Rf’s
The Rf is defined as the distance the center of the spot moved divided by
the distance the solvent front moved (both measured from the origin).
A B CUx xx x
Solvent Front
Origen
Distance solvent migrated = 5.0 cm
Distance A migrated = 3.0 cm
Distance B migrated = 2.0 cm
Distance C migrated = 0.8 cm
0.8 cm
3.0 cm
Rf (A) =
Rf (B) =
Rf (C) =
Rf (U1) =
Rf (U2) =
2. 0 cm5.0 cm = 0.40
= 0.60
= 0.16
= 0.60
= 0.16
3. 0 cm5.0 cm
0. 8 cm5.0 cm
3. 0 cm5.0 cm
0. 8 cm5.0 cm
Dx
Rf (D) = = 0.80 4. 0 cm5.0 cm
4.0 cm
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Calculation of Rf’s
Retardation Factor
● Rf values can be used to aid in the identification of a substance by
comparison to standards.
● The Rf value is not a physical constant, and comparison should be
made only between spots on the same sheet, run at the same time.
● Two substances that have the same Rf value may be identical;
those with different Rf values are not identical.
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Absorption of Solutes:The adsorption strength increases with increasing polarity of functional groups
-CH=CH2, -X, -OR, -CHO, -CO2R, -NR2, -NH2, -OH, -CONR2, -CO2H(weakly adsorbed) (strongly adsorbed)
(nonpolar) (more polar)
Rf value mainly depends on:
Elution Strength of Mobile Phase (e)Elution strength is generally considered to be equivalent to polarity. A solvents elution strength depends on Intermolecular Forces between the solvent and the analytes and between the solvent and the stationary phase.
A more polar (or more strongly eluting solvent) will move all of the analytes to a greater extent, than a less polar, weakly elution solvent.
For example, the elution strength of hexane is very low; e = 0.01the elution strength of ethyl acetate is higher; e = 0.45the elution strength of ethanol is even higher; e = 0.68
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Solvent MF MW
Bp (oC) Density (g/mL)
Hazards* Dipole Elution Stength
() Hexane CH3(CH2)4CH3
C6H14 86.17
68.7 0.659
Flammable Toxic
0.08 0.01
Toluene C6H5CH3
C7H8 92.13
110.6 0.867
Flammable Toxic
0.31 0.22
Diethyl ether CH3CH2OCH2CH3
C4H10O 74.12
34.6 0.713
Flammable Toxic, CNS Depressant
1.15 0.29
Dichloromethane CH2Cl2
CH2Cl2 84.94
39.8 1.326
Toxic, Irritant Cancer suspect
1.14 0.32
Ethyl Acetate CH3CO2CH2CH3
C4H8O2 88.10
77.1 0.901
Flammable Irritant
1.88 0.45
Acetone CH3COCH3
C3H6O 58.08
56.3 0.790
Flammable Irritant
2.69 0.43
Butanone CH3CH2COCH3
C4H8O 72.10
80.1 0.805
Flammable Irritant
2.76 0.39
1-Butanol CH3CH2CH2CH2OH
C4H10O 74.12
117.7 0.810
Flammable Irritant
1.75 0.47
Propanol CH3CH2CH2OH
C3H8O 60.09
82.3 0.785
Flammable Irritant
1.66 0.63
Ethanol CH3CH2OH
C2H6O 46.07
78.5 0.789
Flammable Irritant
1.70 0.68
Methanol CH3OH
CH4O 32.04
64.7 0.791
Flammable Toxic
1.7 0.73
Water HOH
H2O 18.02
100.0 0.998
1.87 >1
Solvent Properties and Elution Strengths
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Resolution The separation between two analytes
on a chromatogram can be expressed as
the resolution and can be determined
using the following equation:
Rs = (distance between center of spots) (average diameter of spots)
In TLC, if the Rs value is greater than
1.0, the analytes are considered to be
resolved.
x x
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Improving Resolution
І To Improve Rs, change the elution strength of the solvent to optimize Rf’s
• change eonet , all compounds will be effected similarly
• Alter the composition of the solvent system so that the components affinity for
the mobile phase vs. the solid phase are differentially changed.
• Changing the chemical nature of the solvent system, such as changing a hydrogen
bonding solvent to a solvent which cannot hydrogen bond to the analyte, is often the
most effective.
Π Improve Rs by decreasing the diameter of the analyte spots. This can be
achieved by applying smaller and less concentrated spots.
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Applied
HPTLC
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Sample preparation (after sampling)
1. Extraction
2. Partition and Clean- up (co-extracts)
3. Concentration
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Deposit is thiner
Better resolutions
Sample deposition
Sample volume: a few nL to a few μL
Preferably using an automated apparatus
Dr. M. Abtahi37 Handle the plates only by their edges.
A B CU D
A B CU
filter paper
D
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● Develop solvent (sometimes experimentally)
● In a saturated chamber
● Filter paper (more quick development)
● Pre-saturation (often preferable)
Spot development
Leave the development chamber uncovered for as little time as possible.
Solvent front migrates less rapidly
Better separations can be achieved
Allows pre-saturating the
layer with solvent vapors
prior to development
Dr. M. Abtahi39When plates are removed from chamber, quickly trace the solvent front.
Vertical Development
1. Solvent in Liquid-Vapour equilibrium
2. Solvent in Vapour adsorbs on the layer
3. Solvent migrating in the layer vaporizes
Effect of gravity
In pre-saturated chamberIn non saturated chamber
Analysis time
Migration distance
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1. HPTLC plate (layer facing down) 2. glass plate for sandwich configuration 3. reservoir for developing solvent4. glass strip5. cover plate 6. conditioning tray
Horizontal Development
No effect of gravity
Migration speed is constant
Better resolutions can be achieved
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Horizontal Development
Better control of the operating conditions(saturation, evaporation)Possibility to develop both sides of the plate= Twice more samples
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Spotting the plate 1st elution 2nd elution90° rotation
Different mobile phases= different principles of separation
2D separation
Analogy: 2D-gel electrophoresis used in biotechnology
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When large numbers of substances are to be separated on a single chromatogram.
Detection of the analytes
● Coloured analytes
● UV cabinet
Densitometry with UV scanner
● Other thechniques
UV light beam
Reflectedbeam
Detector
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● UV visualizing:
Many organic compounds can be seen using this technique, and many
commercially made plates often contain a substance which aids in the
visualization of compounds.
● Some other visualizing agents:
Alkaloids: Dragendorff’s reagent
Cardiac glycosides: Antimony trichloride
Sugar: Aniline phthalate
Amino acids: Ninhydrin
Detection of the analytes
● Absorption of UV radiation is proportional to concentration.
● Quantification is possible.
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Solvent front
Starting line
dsolvent
dsolutesolvent
solutef d
dR
Totally retained solute
Totally unretained solute
Reading the TLC
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Rf(s) often depend on temperature and solvent used in the experiment.
Using software
Importing data
Scaning plate (wavelenght)
Integration
Calibration
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Quantification (TLC Scanner)
Some aspects and tips● Ignore concentration on your mind
● Recovery rate
● Experimental determination of the develop solvent proportion and the wavelength
● Multiple development
● Standard preparation
● Edge effect
● Pollution
● Safety
● Thoroughness
● Faults replication (from sampling up to the end)
● Choosing appropriate chromatographic method
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Summary A TLC plate is a sheet of glass, metal, or plastic, coated with a thin layer
of a solid adsorbent.
A small amount of the mixture to be analyzed is spotted near the bottom
of this plate.
The TLC plate is then placed in a shallow pool of a solvent in a developing
chamber.
This liquid, or the eluent, is the mobile phase, and it slowly rises up the
TLC plate by capillary action.
As the solvent moves past the spot that was applied, an equilibrium is
established for each component of the mixture between the molecules of
that component which are adsorbed on the solid and the molecules which
are in solution.
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In principle, the components will differ in solubility and in the
strength of their adsorption to the adsorbent and some components
will be carried farther up the plate than others.
When the solvent has reached the top of the plate, the plate is
removed from the developing chamber, dried, and the separated
components of the mixture are visualized.
If the compounds are colored, visualization is straightforward.
Usually the compounds are not colored, so a UV lamp is used to
visualize the plates.
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Summary
Other Chromatography Techniques
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Column Chromatography Solid stationary phase, packed into column.
Sample is applied to the top of the packing and
(mobile phase) is dripped on to the column.
A tap at the bottom of the column allows the
solvent to leave the column.
There are two techniques based on column
chromatography:
High Performance Liquid Chromatography
Gas Chromatography
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HPLC and GC
HPLC and GC chromatograms represent the qualitative
and quantitative aspects of chromatography.
Number of peaks – mixture separated into three different
compounds.
Retention time – identifies each component – qualitative analysis.
Area under each peak – relative amount of each compound –
quantitative analysis.
Retention time (Rt)
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HPLC
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GC The most sensitive chromatographic technique Capable of detecting as little as 10-12g Limited to some compounds
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Interpreting HPLC and GC Chromatograms
The time a component takes to pass through the column is
called the retention time, Rt.
The same compound will give the same retention time if the
conditions (temp, mobile phase, stationary phase, flow rate,
pressure etc) remain the same.
Each component forms one peak, however it is possible for a
number of peaks to coincide and be indistinguishable.
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A Chromatogram
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Thanks for your attention
Dr. M. Abtahi59