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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Dr. M. Abtahi45

● 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