Instrumental analysis for Undergraduste level
Kathmandu University 7/26/2019
Planar Chromatography
Planar chromatography includes:
• Thin layer chromatography(TLC)
• Paper chromatography
Stationary phase is flat, relatively thin layer of material that is either self supporting or
is coated on a glass, plastic or metal surface.
The mobile phase moves through the stationary phase by
• Capillary action
• Sometimes assisted by gravity
• Or an electric potential( Slab electrophoresis)
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Instrumental analysis for Undergraduste level
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Thin Layer Chromatography
Scope of TLC:
• Used for developing optimal conditions for separation by column-liquid chromatography.
• Due to speed and low cost it should always precede column experiments.
• Used in drug industry for determination of product purity.
• Used in clinic laboratory for biochemical and biological analysis.
• This can replace liquid column chromatography in many analyses.
Stationary phase = a piece of glass, metal, or plastic coated with a thin, uniform layer of solid
adsorbent. Usually silica gel (SiO2), alumina (Al2O3), or cellulose.
A substance which fluoresces under UV light often incorporated into the stationary phase.
Zinc sulfide
Mobile phase = suitable liquid solvent or mixture of solvents.
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Instrumental analysis for Undergraduste level
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How Does TLC Work?
It’s a combination of interactions which
determine how fast or slow a compound will
move up a TLC plate:
Adsorbent-sample interactions
Solvent-sample interactions
Intramolecular interactions within the sample
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Instrumental analysis for Undergraduste level
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TLC Procedure (Different steps for TLC)
Step 1: Preparing the chamber (Plate Development)
❑ Choose a container that is large enough and can be sealed.
❑ Adding the a few cm of the mobile phase solvent to the chamber.
❑ Sealing the chamber:
• The atmosphere of the chamber should be saturated with the solvent vapors
before running samples.
• You may line part of the inside of your chamber with filter paper to aid in this
saturation process. Allows for better development of the chromatograms.
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Instrumental analysis for Undergraduste level
Kathmandu University 7/26/2019
Step 2: Preparing the stationary phase (TLC Plate)
❑Make slurry of the finely divided solid Adsorbent, Small amount of an inert binder,
Water/solvent
❑Spread a thin layer (no more than a few mm) of the mixture on an non-reactive
support.
❑Dry the plate in air and activate by heating in an oven for approximately 30
minutes at 110˚C.
❑TLC plates are also commercially prepared and can be purchased ready for use.
• Draw a line of origin approximately 0.5cm from the bottom of the TLC plate.
• Indicate where each sample will be added.
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Instrumental analysis for Undergraduste level
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Step 3: Spotting the samples
A) If the sample isn’t in solution, dissolve it in an appropriate solvent.
• As a rule of thumb, a concentration of about 1% (1g in 100ml)
is good. (If the sample is too concentrated = smear, If the The image above shows a sample ran at three different
concentrations. The left plate was ran too concentration
sample is too dilute = no results) and the spots are running together. The other two
plates yielded good separation.
Spot a small amount of sample onto the plate.
• Make sure the sample spot is dry before continuing.
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• Step 4: Developing the chromatograms
• When the sample spot has dried, the TLC plate is placed into the chamber containing the
solvent.
• It is important that the sample spot is above the level of the solvent.
• Allow the solvent to rise until it almost reaches the top of the plate.
• Remove the plate from the chamber and mark the position of the solvent front before it can
evaporate.
• If the sample spots are visible, mark their positions.
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Visualizing
• Step 5: Identify the spots and interpret the data. Colorless
Compounds
A) Use of fluorescence (physical Method)
Substance like ZnS, which can fluoresce under UV light is added to stationary phase.
So, when the TLC plate is exposed to UV light, the entire plate will glow, the glow
will be masked at positions where spots are located.
B) Use chemical methods Using Fluorescence to Visualize Spots
Here analytes are reacted with especial chemical to give a colored product.
• Spraying with a solution of Sulphuric acid
• Placing the plate in a chamber containing Iodine Crystals:
The dried chromatogram is placed into a closed container containing iodine
crystals/iodine vapor. The sample spots will be brownish in color. The spots are
more intense for unsaturated compounds
• Ninhydrin: The dried chromatogram is sprayed with a ninhydrin solution. Reacts
with amino acids to produce a colored product, mainly pink to purple.
• Rhodamine B: Visualization of lipids
• Aniline phthalate: Visualization of carbohydrates
Before After
What if the compounds being separated are colorless? How are the spots visualized? Spot Visualization using chemical
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Performance Characteristic of thin layer chromatography
• Retardation factor: Rf
Distance traveled by the compound dR
Rf = Rf =
Distance traveled by the solvent dM
• The larger the Rf value, the farther the compound traveled up the plate.
• An Rf value is a physical property that can be used for identification purposes.
• It can be difficult to keep all of
• But it does depended on the conditions under which it is measured. these variables constant from
experiment to experiment.
• Rf values are reported as relative values since they can be affected by: • If two substances have the same Rf
• the adsorbent used value they may or may not be the
• the solvent system used same compound.
• Temperature • If two substances have different Rf
• the thickness of the adsorbent layer values they are definitely not the
• the amount of sample material spotted same compound.
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Sample Rf Calculation
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Retention Factor Plate
Plate Heightcount
height/Plate
• Relate dR and dM to tR and tM • Approx Plate height can also be
dR d
tM = and t R = M Here equal determined for a given type of
u u distance is packing by TLC measurement.
which gives the capacity factor as, considered
t -t d − dR dM d
N = 16 R
2
k= R M = M dR W
tM dR
d
1− Rf H = R
= N
Rf
2Z 2[(d R ) A − ( d R ) B ]
RS = =
Resolution W A + WB W A + WB
Where the selectivity factor is,
N - 1 k
= k B d M − (d R ) B (d R ) A
4 = = x
1+ k k A d M − (d R ) A (d R ) B
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An Example
Unknown sample containing a mixture amino acids
For simplicity- you know that it can only contain 5 possible amino
acids.
Run unknown mixture with each of the five known amino acids.
Choose the Right Solvent to Use?
The Answer???
TRIAL AND ERROR
Remember: Like dissolves like
Vary the polarity with several trial runs
Ideal solvent system = good sepeartion of components
Non-polar solvents (pentane, hexane):
Polar compounds will not move up the plate much.
Non-polar compounds will travel quite a ways up the plate.
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TLC Applications
• Can be used to determine the number of components in a mixture.
• Can be used to identify the presence of specific compounds/ unknown compounds.
• Can be used to monitor the progress of a reaction.
• Will show if any reactant has disappeared, if any product has appeared, and how many products are present.
• Often used to monitor organic reactions.
• Used to determine which conditions are ideal to use in column chromatography.
• Ex: which solvent system to use
• Quick, fast, and inexpensive
• It is also used to monitor column chromatography.
• Used to quantify the amount of a component present .
• Area of the spot
• Spot extraction, then measure the amount
• Used to determine the purity of a sample.
• Can be used to isolate purified substances
• Scrape the solid support from the region containing the spot with a knife and extract using an appropriate solvent.
• Can then analyze it further ( MS/ IR/ NMR)
• It is often used in forensic drug analysis, explosives analysis, toxicology and analysis of dyes and inks.
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Instrumental analysis for Undergraduste level
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Paper Chromatography
Stationary phase = cellulose paper (water molecules –bound)
Mobile phase = liquid solvent
The solvent travels up the filter paper via capillary
action and carries the sample with it.
The procedure for paper
chromatography is exactly like that
described for TLC.
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Two Way Paper Chromatography
Aids in separating components that have
similar Rf values.
Procedure:
A single spot of sample is
placed towards one end of
the origin line.
Placed into solvent; it is
removed when the solvent
line nears the top.
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Instrumental analysis for Undergraduste level
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Two Way Paper Chromatography
The paper is dried out completely, rotated 90°, and developed once again
using a different solvent.
It isn’t very likely that two components will have similar Rf values in both solvents
Final Chromatogram
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How Does Paper Chromatography Work?
More complex than TLC due to solvent interactions.
paper made of cellulose
The cellulose fibers attract water from the atmosphere as well as
any water present from the sample.
So, there is a very thin layer of water bound to the cellulose
surface.
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• Partitioning: when a compound tends t
time between two immiscible solvent.
How Does Paper Chromatography Work?
− Paper chromatography using a non-po
Using a non-polar solvent: type of partition.
Non-polar molecules in the mixture: Will be attracted to the non-polar solvent / not to the water layer
on the surface of the cellulose paper. Will move far up the paper and have a high Rf value
Polar molecules in the mixture: Will have a high attraction to the water molecules on the surface of the cellulose
paper / not for the polar solvent. Will not move far up the paper and will have a low Rf value
• Using polar solvents:
− If water = mobile phase AND, Water layer on the paper = stationary phase
− You wouldn't think that there would be any meaningful difference between the amount of time spent in
either.
The same must be true for other polar solvents such as alcohols.
Since alcohols are miscible with water– partitioning is not occurring
Most expatiations of paper chromatography don’t address this issue
Describe the interaction as bonding of sample compound directly to the polar cellulose paper.
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Instrumental analysis for Undergraduste level
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Paper Chromatography Applications
Can be used to determine the number of components in a mixture.
Can be used to identify the presence of specific compounds.
Can be used to monitor the progress of a reaction.
Will show if any reactant has disappeared, if any product has appeared, and how many
products are present.
TLC has largely replaced paper chromatography.
Several advantages:
Runs faster
Better separations
Can choose between different stationary phases
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7/26/2019
Solvent Front
4.2
Solute Front (A)
𝑅𝑓(𝐴) = =0.824
5.1
3.6
𝑅𝑓(𝐵) = =0.706
5.1
5.1 cm
1.7
𝑅𝑓(𝐶) = =0.333 Solute Front (B)
5.1
4.2 cm
3.6 cm
Solute Front (C)
1.7 cm
