PAPER AND THIN LAYER CHROMATOGRAPHY (TLC)
Lab#4
B CH 3 3 3
Chromatography:
• It is a collective term for a set of laboratory techniques that enables the separation, identification,
and purification of the components of a mixture.
• The general principle for all chromatography techniques:
Separation of molecules by distribution between a stationary phase and a mobile phase.
The stationary phase can be solid, gel, or liquid. Also called matrix, resin, or beads.
The mobile phase is the solvent, and it is usually a liquid, but may also be a gas.
(Usually one phase is hydrophilic and the other is lipophilic).
The compounds to be separated are considered solutes.
The separation of materials is based on differential partitioning [retardation] between the mobile
and stationary phases.
Types of Chromatography:
• Paper chromatography.
• Thin-layer chromatography.
• Gel filtration (molecular sieve) chromatography.
• Ion-exchange chromatography.
• Affinity chromatography.
• Gas chromatography.
• Dye-ligand chromatography.
• Hydrophobic interaction chromatography.
• Pseudoaffinity chromatography.
• High-pressure liquid chromatography (HPLC).
Partition Chromatography:
• Is the distribution of a solute between two liquid phases (mobile and stationary phase).
• This may involve direct extraction using two liquids, or it may use a liquid immobilized on
a solid support as in the case of paper and thin layer chromatography.
Originally, partition chromatography involved coating the support with a liquid stationary
phase that was immiscible with the mobile phase.
1. Paper Chromatography (PC):
• Paper chromatography is one of the chromatography types, procedures which runs on a
piece of specialized paper.
• It is a “liquid-liquid” chromatography.
• Separation of solutes depend on the polarity.
• Requires small quantities of material.
• Rapid.
1. Paper Chromatography (PC) cont’:
The system is composed of
The mobile phase: The stationary phase:
[shallow layer of solvents] [Water]
Is a developing solution that Layer of cellulose highly
travels up the stationary phase, Separated molecules saturated with water (the water
carrying the samples with it. trapped between the cellulose
fibres of the paper).
Non-Polar mixture
“Liquid” Polar substance
“Liquid”
1. Paper Chromatography (PC) cont’:
The system is composed of
The mobile phase: The stationary phase:
is a developing solution that is usually a piece of high-quality
travels up the stationary phase, filter paper.
carrying the samples with it. Separated molecules [cellulose].
[shallow layer of solvents]. Polar substance
Non-Polar mixture
Stationary phase
Mobile phase
Figure 1. Ascending Paper Chromatography
Figure 1. Ascending Paper Chromatography
1. Principle of Paper Chromatography (PC):
• The Separation is depending on:
Components of the sample will separate on the stationary phase according to how strongly they
adsorb to the stationary phase versus how much they dissolve in the mobile phase. (how?)
• Explanation:
This paper is consisting of a layer of cellulose highly saturated with water [a polar substance,
stationary phase], as the solvent travel up [non-polar, mobile phase], the compounds within the
mixture travel farther if they are non-polar.
So, more polar substances bond with the hydrated cellulose paper more quickly, and therefore
do not travel as far.
As a result, different compounds in the sample mixture travel at different rates due to:
• Differences in their solubility in the solvent [mobile phase].
• Differences in their attraction to the fibres in the cellulose paper [stationary phase].
Paper Chromatography Modes
Descending: Ascending:
• In this method, the solvent is kept in a • In this method, the solvent moves
trough at the top of the chamber and is upward against gravitational force.
allowed to flow down the paper. • The only force that cause the
• The liquid moves down by capillary motion of solvent and the
action as well as by the gravitational
force. compounds is capillary force, so
• In this case, the flow is more rapid as the speed of the process is slow.
compared to the ascending method.
Figure 1. Descending Paper Chromatography
1. Result of Paper Chromatography (PC):
Chromatogram
2. Thin layer Chromatography (TLC):
• Procedures which runs on a solid adsorbent substance coated on glass plates.
• Rapid, the separation can be completed in less than one hour.
• It is a “solid-liquid adsorption” chromatography. ?
• Separation depend on the polarity.
• Requires small quantities of material.
• Widely used.
2. Thin Layer Chromatography (TLC) cont’:
The system is composed of
The stationary phase (adsorbent):
The mobile phase: Is a thin layer, (0.25 – 0.5 mm) of adsorbent like
Mixture of solvents. silica gel [ polar substance], [aluminium oxide or
Non-Polar mixture magnesium silicate] spread uniformly over the
Separated molecules surface of a flat, inert surface of the glass plastic
plate.
The stationary phase+ support medium should be inert.
Polar substance
2. Principle of Thin Layer Chromatography (TLC):
• Partition of a solute between a moving solvent phase and a stationary aqueous phase.
• The solute moves in the direction of a solvent flow at a rate determined by the solubility of
the solute in the moving phase.
Thus a compound with high mobility [less polarity] is more attracted to the moving
solvent [mobile phase] than to the stationary phase.
• Separation depend on the polarity.
Figure 1.Traveling of the solvent in TLC via capillary action.
2. Result of Thin layer Chromatography (TLC):
Chromatogram
Factors affect the resolution of separation:
1. Ion exchange effect: Any ionized impurities in the support medium will tend to bind or
attract oppositely charged ions and will therefore reduce the mobility of these solutes
[resulting in bad resolution].
2. Temperature: Since temperature can affect the solubility of a solute in a given solvent
temperature is also an important factor and often a chromatography laboratory has a
fixed temperature for optimum results [increased temperature increase solubility].
3. Composition of the solvent: Since some compounds are more soluble in one solvent than
in the other the mixture of solvents used affect separation of the compounds.
4. Concentration and volume of the sample.
Sample Analysis
Sample Analysis:
1. Visualization:
• Amino acids Specific color reagents are sprayed onto the plate or the paper [ninhydrin].
• Sugars Spraying the plate or the paper with [aniline diphenylamine].
*The paper or plate remaining after the experiment is known as the Chromatogram.
Sample Analysis:
2. Expression of the results:
Relative flow [Rf]:
• Also known as relative mobility.
• Used to express the performance of a solute in a given solvent system/stationary medium.
• Rf value may be defined as: the ratio of the distance moved by a compound to that moved by
the solvent.
• Rf value is constant for a particular compound, solvent system and stationary phase.
• Its value is always between zero and one “remember it is a ratio value”.
Rf = Distance of migration of solute
Distance moved by solvent
Relative flow [Rf] value:
Rf = Distance of migration of solute
Distance moved by solvent
If Rƒ value of a solute is closer to zero The solute has more attraction to stationary phase.
If R ƒ value of a solute is closer to 1 Then the solute has more affinity for the mobile
phase and travels further.
• The final chromatogram can be compared with other known mixture chromatograms to
identify sample mixes, using the Rf value in an experiment.
Example:
Comparison between PC and TLC:
TLC Paper chromatography.
It has better separations than paper
Separation Separate the sample.
chromatography
Wide choice between different adsorbents
Stationary phase Hydrated Cellulose “water”
(stationary phase).
Principle Based on adsorption Based on partition.
Resolution It has better resolution. Low resolution.
Quantification Allow for quantification. Allow for quantification.
Expanded zonal spread
Compact zonal spread
(not concentrated for quantification analysis
Zonal spread (concentrated for quantification analysis in
in need).
need).
Application of TLC and PC:
1. Identifying unknown compounds present in a given substance.
2. Qualitative and semi quantification tests.
3. Separation of compounds in a sample.
4. Identify the purity of the sample.
5. Diagnosis.
Using of TLC and PC in diagnosis:
Phenylketonuria:
• Non-functional phenylalanine hydroxylase enzyme.
• This enzyme is necessary to metabolize the amino acid phenylalanine (Phe) to the amino acid
tyrosine.
• Phenylalanine accumulates and is converted into phenylpyruvate (also known as phenylketone),
which is detected in the urine.
Cystinuria:
• Cystinuria is an inborn error of amino acid transport that results in the defective reabsorption
by the kidneys of the amino acid called cystine.The name means "cystine in the urine.“
• When the kidneys do not reabsorbed cystine, this compound builds up in the urine.
OBJECTIVES:
• Using paper chromatography and TLC to diagnose two samples of urine with phenylketonuria
and cystinuria.
METHOD:
• You are provided with silica gel Thin Layer Chromatography [TLC] plate and paper
chromatography[PC]:
1. Draw a sample starting line (base-line) about 2.5-3 cm from the bottom of the TLC plate
and PC, divide it uniformly then apply one small drop (5 μl) of each standard (Phe &
Cys) and sample (1 &2), allow to dry.
Base-line
2.5 cm Standard 1
Standard 2
Sample 1
Sample 2
METHOD:
2. Label your TLC plate and PC, then place them in the solvent chamber. Do not forget to
cover the solvent chamber.
Standard 1
Standard 2
Sample 1
Sample 2
St1 St2 S1 S2
2 cm
3. Leave them for 45 min ( or until the level of the solvent is 2 cm from the top).
St1 St2 S1 S2
METHOD:
4. Remove the TLC plate and PC paper from the solvent chamber draw a line to mark the
solvent front.
Solvent front
St1 St2 S1 S2
METHOD:
5. Spray the TLC plate and PC paper with ninhydrin (wear gloves and be very careful
ninhydrin is carcinogenic).
6. Put the TLC plate and PC paper in the oven at 70 ℃ , until the color develops.
Color develops
St1 St2 S1 S2
RESULTS:
• For TLC and PC record the following:
1. Distance migration by solvent front = ________________ cm.
2. Fill the table:
Distance
Sample Rf
(unit)
Phe standard
Cys standard
Sample 1
Sample 2
