Paper and Thin Layer

Chromatography
 Instructional Objectives
• Learn the principles and working mechanisms of Paper
Chromatography (PC) and Thin Layer Chromatography (TLC).

• Define the types of mobile and stationary phase used.

• Understand the rate of elution of different polar and non-polar

compounds.

• To calculate the Rf values of TLC and PC.

 Instructional Objectives

• Setting up paper chromatography.

• Understand two-dimensional (two way) paper chromatography.

 What is Chromatography?

• Chromatography is a technique for separating the components of

a mixture on the basis of differences in their affinity for a
stationary phase (static) and a mobile phase (moving).

• Stationary phase is the material inside the column.

• The mobile phase is a solvent that is poured into the column.

 Principles of Chromatography

• Sample is loaded into the stationary phase.

• The mobile phase flows through the stationary phase.
• Acts as a carrier or sample, moves sample across the stationary phase as it
flows.
• Components in sample have different interaction with the mobile and
stationary phase.
• The interaction can be due to polarity, size, charge and chemical
interaction.
• Interaction is reversible → allow the analyte to come out of the column.
6

Principles of Chromatography
- Components that have a stronger interaction with the mobile phase
vs the stationary phase → moves faster and elutes quicker.
- Due to differential mobility, mixtures or molecules with different
properties can be separated.
 Principle of Chromatography
Mobile •Moves over the stationary phase
•Can be gas or liquid
phase
Stationary •Stationary in a column, or on a
support or paper.
phase •Can be solid or liquid that is coated
on the surface of an inert liquid.

Sample or •Mixture of compounds dissolved in solvent or

buffer.
•Carried by mobile phase and travels across the
solute stationary phase. As it travels, it gets separated.

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 Types of Chromatography
Chromatography

Separation
Partition Affinity Adsorption
mechanism

Paper Ion exchange Gel Permeation Liquid

Chromatography (charge of ion) (size exclusion) Chromatography Liquid
Chromatography
Gas
Chromatography
Thin Layer
Gas Gas Chromatography
chromatography chromatography Column
Column Liquid Chromatography
chromatography chromatography

8
 What is Thin Layer
Chromatography?
• Thin Layer Chromatography (TLC) is a technique where
components of a mixture separate by differential migration
through a planar bed of a stationary phase. The mobile phase
flows by virtue of capillary forces.
 Concepts of TLC
• The concepts for TLC and other types of chromatography (HPLC,
Column Chromatography) are essentially the same.

• The molecules of the mixtures have a dynamic and rapid

equilibrium between stationary and mobile phase, i.e. in each
second there are billions of molecules adsorbing to the stationary
phase and billions of molecules desorbing from the stationary
phase into the mobile phase.

• Molecules distribute themselves between stationary and mobile

phase based on the polarity and hydrogen bonding of the
molecule, stationary phase and mobile phase.
 How TLC works?
Select appropriate thin layer plate (stationary phase)

Apply the sample onto the plate

Place plate into TLC chamber containing some developing solvent (mobile phase)

Mobile phase rises up the TLC plate & separation occurs.

Visualization of each analyte (spot) on the TLC plate.

 Select appropriate thin layer plate (stationary phase)

• The TLC plate, made of either glass, plastic or

aluminium, is coated with a thin layer of stationary
phase.

• Types of common stationary phases;

• Silica gel

• Aluminia Use either a forceps to hold TLC

plate at the end or hold the
plate by the sides with gloves.
 Examples of Stationary phase
Silica / Silica gel (SiO2)

- Most widely used in column chromatography &

thin layer chromatography.

- Active sites of silica are the hydroxyl groups

attached to silicon atoms (Si-OH aka silanol group)

- Suitable for a non-polar mobile phase.

 Examples of Stationary phase
Silica / Silica gel (SiO2)

- Often heated to 150°C - 250°C to get rid of water →

water forms a polar film → becomes partition
chromatography (based on solubility).

- Decreasing particle size of the silica particles

increases surface area → increases separation
power!
 Examples of Stationary phase
Alumina / aluminium oxide (Al2O3)

- Either basic, acidic or neutral

- Polar stationary phase (more polar than

Basic Acidic
silica)

- Different adsorption selectively from silica Both acidic and basic groups are polar.

due to its basicity. If it is acidic, it will attract basic groups.

If it is basic, it will attract acid groups

- Used to separate aromatics from olefins.

 Apply sample onto the plate

• Draw a start line, ~ 1cm from the end of the plate,

lightly with pencil.

• Mark spots for sample to be applied at the start line

to avoid confusion.

• Apply a small amount of sample in each small spot

using a capillary tube.
 Apply sample onto the plate

Common Problems?!

Start line too low / Large sample spots /

Over concentrated
solvent level too too close to each
sample.
high other.
•Solvent dissolves •Merged together, •Difficult to obtain
the sample before difficult to see → the Rf value
separation occurs! overlapping spots! •Tailing spots
detected on TLC
plate!
 TLC Errors
Tailing spots Large spots

Overlapping
 Place plate into TLC chamber containing some
developing solvent (mobile phase)

• Pour some mobile phase into the TLC chamber.

• Place a piece of filter paper inside the chamber at the

side, partly soaked in the eluant (mobile phase).

• This ensures that the chamber is saturated with

eluant vapours.

• Cover the chamber with a lid to prevent solvent from

evaporation.
 Mobile phase rises up the TLC plate & separation
occurs
• Mobile phase rises up the TLC plate by capillary action.

• Analytes in the sample dissolves in the solvent and moves up the TLC
plate.

• Each analyte may move at different rates, depending on the

intermolecular forces between the analyte with the stationary phase;
and the analyte with the mobile phase.

NOTE: Put the plate in gently, do not allow mobile phase to splash onto the TLC. DO not
move the chamber while the TLC is developing! → prevent the separation from being
affected!!!!
 How TLC works?
• Stationary phase = SiO2

• Stationary phase is very polar.

• Polar analytes interact more strongly with the stationary phase.

• Move very slowly up the plate.

• Non-polar analytes interact less strongly with stationary phase and more
strongly with less polar mobile phase, hence moves higher up.
 How TLC works?

Order of elution of functional groups for SiO2 stationary phase

-CH=CH2-. –X, -OR, -CHO, -CO2R, -NR2, -OH, -CONR2, -CO2H

Non polar More polar

 Separating a mixture
Example: mixture with components A and B
 Visualisation of each analyte (spot) on the TLC plate.

• Remove the TLC plate from the chamber when the

mobile phase reaches ~1cm from the top of the
plate.

• Quickly mark the solvent front (where the solvent

stops) with a pencil.

• Place the plate in the fumehood to evaporate the

solvent.
 Visualisation of each analyte (spot) on the TLC plate.

• Coloured analyte can be seen immediately.

Circle them with a pencil.

• Colourless spots can be visualised using

visualizing agents.

• Common techniques → UV lamp, iodine stain.

• Other techniques → KMnO4, ninhydrin,

dinitrophenylhydrazine
 Visualisation of each analyte (spot) on the TLC plate.

UV lamp

• For compounds that absorbs UV light, aka has

chromophore.

• TLC plates usually contain a fluorescent

indicator which makes it glow green under UV
light of wavelength 254nm.

• Analytes that absorb UV light will quench the

fluorescence, hence giving dark purple or
bluish spots on the plate. TLC plate under UV light
 Visualisation of each analyte (spot) on the TLC plate.

UV lamp

• Chromophores are compounds that contains

π-conjugated systems, for example, aromatic
group, conjugated double bond.

• Some compounds (eg. alkanes, alcohols and

ethers) do not absorb UV light sufficiently to
quench the green fluorescence π-conjugated systems
(example)
 Visualisation of each analyte (spot) on the TLC plate.

Iodine stain

• For either UV active or inactive compounds.

• Expose the plate to iodine vapour in a sealed

chamber. (Pour in iodine power or chips)

• Ensure that the solvent on the TLC plate has

evaporated before placing the plate into the iodine
chamber.
 Visualisation of each analyte (spot) on the TLC plate.

Iodine stain

• Organic analytes will develop dark brown spots.

Outline the brown spots right after the spots are
developed as they will soon disappear as iodine
sublimes away.
 Visualisation of each analyte (spot) on the TLC plate.

Ninhydrin

• The stain is specific for amino acids, primary and

secondary amines. It is especially very sensitive to
amino acids.

• It forms spots that are shades of deep blue/purple.

Visualisation of each analyte (spot) on the TLC plate.
Ninhydrin

• It forms spots that are shades of purple.

 Visualisation of each analyte (spot) on the TLC plate.

Dinitrophenylhydrazine (DNPH)

• The stain is specific for aldehydes and ketones.

• It forms yellow to orange spots, which are the

corresponding hydrazones.
Visualisation of each analyte (spot) on the TLC plate.
Dinitrophenylhydrazine

• It forms yellow to orange spots, which are the corresponding

hydrazones.
 Visualisation of each analyte (spot) on the TLC plate.

KMnO4

• It is excellent for functional groups which are

sensitive to oxidation. For example alkenes, alkynes,
amines.

• It forms bright yellow/ brown spot on a bright purple

background.
Visualisation of each analyte (spot) on the TLC plate.

KMnO4

• Some compounds will form the coloured spot readily after

immersion into the stain, however, some will require gently
heating of the TLC plate following immersion into the stain.
 Visualisation of each analyte (spot) on the TLC plate.

Summary of reagents….
Reagent Compound
UV lamp Chromophores
Iodine stain UV active / inactive
Ninhydrin Amino acids
2,4,DNPH Aldehydes, Ketones
KMnO4 Any compound that can be
oxidised.
Rhodamin B Lipids
Trinitrofluorenone Phenols
 Order of Elution

• When the structure of a compound is known, its polarity can be

estimated.

• In most cases of TLC development, a combination of two solvents

is the best choice.
 Order of Elution
• The elution order for some functional groups with a polar stationary phase
(eg. Silica).
 Order of Elution
• The elutropic series of the commonly used solvent (mobile phase)
 Analysis by Rf values
• Rf stands for retention factor.

• Used to identify substances by comparing against standards.

• Not a physical constant, comparisons can only be made between

spots on the same sheet, ran at the same time.

• May have false positives → Same Rf values may be identical

substance (does not mean that it is 100% of that particular
component), but different Rf values are definitely not the same.
 Analysis by Rf values
• Be consistent in way of measurement → usually measure from
start line to center of spot.

• Rule of thumb;
Smallest Rf value → most polar. Largest Rf value → least polar.

• Formula to calculate Rf; (no units!)

 Analysis by Rf values
Conclusion

• D has most non-polar analyte

in it (highest Rf)

• U contains analytes B and C

(the dyes match B and C in
terms of Rf)

• C has the most polar analyte

in it (smallest Rf)
 Analysis by Rf values
What happens if the Rf = 1 or close to 1 and if SiO2 TLC plate is used?

PROBLEMS:

• The sample is too non-polar OR the solvent is too polar.

• A high Rf value means that the spot is too high up or the

analyte travelled too fast for a good separation to occur.

• Note: Typical effect Rf value is 0.30 – 0.70

 Analysis by Rf values
What happens if the Rf = 1 or close to 1 and if SiO2 TLC plate is used?

SOLUTIONS:

• Adjust the solvent to retain the sample → can be a mixture of

solvents in different ratio.

• i.e. 70 : 30 ethyl acetate : acetic acid.

 TLC Applications
Reaction Monitoring

Identification

Purity
Check
 Why do we use TLC?
• To determine the number of components in a mixture. (Ingredient
of a pill, extract from a plant substances etc.)
 Why do we use TLC?
• To monitor the progress of a reaction. (Sampling the reaction
mixture at a regular intervals to check if reaction has completed.)
 Why do we use TLC?
• To determine the effectiveness of purification. (distillation,
recrystallization, extraction etc.)
 Why do we use TLC?
• To determine the appropriate conditions for a column
chromatography. TLC can rapidly determine the correct solvent to
use for column chromatography.
 Why do we use TLC?
• To monitor column chromatography. Column chromatography
results in many small flasks/ fractions.

• To analyse which fractions have the desired compound.

 Advantages of TLC
• Low cost • Short analysis time

• Ease of sample preparation • All spots can be visualized

• Sample cleanup is seldom necessary • Adaptable to most

pharmaceuticals

• Uses small quantities of solvents • Requires minimal training

• Reliable and quick • Minimal amount of equipment is needed

 Limitations of TLC
• poor reproducibility

• difficult to quantify spots

Food application: Separation of Chlorophyll

•Chlorophyll can be extracted by polar solvents e.g. acetone,

methanol, ethanol.
o Nonpolar solvents are less effective e.g. hexane or petroleum ether

•Separation method
o Paper chromatography
o Thin-layer chromatography https://youtu.be/J8r8hN05xXk
https://youtu.be/e3lRt9XdV0s
o HPLC (most popular) https://youtu.be/MLoitPJQH3g
Thin Layer Chromatography to separate chlorophyll
Carotenoid (yellow)

Phenophytin (grey)

Chlorophyll a (blue-green)

Chlorophyll b (olive-green)

Xanthophylls (yellow)

Origin of loading

https://www.youtube.com/watch?v=e3lRt9XdV0s
 Thin Layer Chromatography to separate chlorophyll

• Chl b is more polar than chl a

• Normal phase separation (polar stationary phase, non-polar

mobile phase) – chl a will pass more quickly through

• Reverse-phase separation (non-polar stationary phase, polar

mobile phase)– chl b will elute first

o Detection is based on absorption of visible light

• Chl a is blue-green (absorbs at 660.5 & 428.5 nm)

• Chl b is olive-green (absorbs at 642 & 452.5 nm)

 Chlorophyll
• There are two chlorophylls, a and b

• Chlorophyll b differs from chlorophyll a in that the methyl

group (-CH3) on carbon 3 is replaced with an aldehyde group
(-CHO)

• Major light-harvesting pigments

 Paper Chromatography
• An analytical technique that is based on
partition chromatography.

• Usually used for the separation of mixture of

amino acids or coloured pigments in leaves /
ink.
 Paper Chromatography
• Stationary phase: layer of water that is coated on a piece of
chromatography paper.

• Mobile phase: usually one or a mixture of liquid that is immiscible

with the stationary phase.

• Similar to TLC but different stationary phases are used, and hence
separation mechanism is different!
 Question
The stationary phase in partition chromatography is the liquid
coated on a solid support. Can the solid support be soluble in the
liquid? Explain your answer.

No. The solid support must be immiscible with

the liquid.
 Paper Chromatography
• Chromatography paper is made of cellulose fibres.

• Cellulose is a polymer (repeating chains) of glucose.

• Has a lot of –OH groups sticking out, hence attracts water vapour from
surroundings.

• As such, stationary phase is the layer of water that is coated on the paper.
 Paper Chromatography
• Non polar molecules tends to get partitioned into non polar phases and
vice versa.

• If analyte partitions more into the mobile phase, it will move up the
paper more.

• If analyte partitions more into the stationary phase, it will move lesser.

• Hence, this technique depends on distribution (partition or solubility) of

the substances to be separated between the stationary phase (layer of
water coated onto chromatography paper) & the mobile phase
(solvent)
Setting up Paper Chromatography
• Similar to the set-up of thin layer
chromatography.

• Can be ascending (solvent travels up),

descending (solvent travels down), radial or
two dimensional.

• Spot the sample mixture on the start line drawn

using a pencil on the chromatography paper.

• Hang the chromatography paper vertically in a

glass tank that contains a suitable solvent.
Setting up Paper Chromatography
• Solvent and sample migrates up the paper by
capillary attraction.

• The higher the solubility of the analyte in the

solvent, the faster the analyte will move up.

• Remove the paper from the tank when solvent

has moved up near to the top of the
chromatography paper.

• Mark the position of the solvent front before

the solvent evaporates.
Setting up Paper Chromatography

• Each analyte has a characteristic Rf value for a

given mobile phase.

• Hence Rf can be used for identification,

especially in known samples.

• There is no Rf value for column

chromatography.
 Two-dimensional Paper Chromatography

• Usually used to separate complex mixture of

analytes (i.e. amino acids mixture)

• The paper chromatogram is ran twice in different

directions & different solvents → two dimensional.

• A drop of mixture is placed in one corner of a square

of chromatography paper and the first mobile phase
travels up the paper.
 Two-dimensional Paper Chromatography

• The paper is then turned 90° clockwise and immersed in

the separating chamber that is filled with a different
solvent.

• Two dimensional provides a higher degree of separation

as compared to one dimensional paper chromatography.

• Identify of each spot can be determined by the Rf value of

the spot and compared with Rf value of known
substances ran under the same conditions.