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Planar Chromatography

Filter paper is commonly used as the stationary phase material in paper chromatography. Some key properties of the filter paper that make it suitable include: - Porous structure - The cellulose fibers in filter paper form a porous network that allows capillary action to draw the mobile phase up the paper. This drives the separation process. - Hydrophilic - Filter paper readily absorbs water, forming the stationary aqueous phase. This allows solutes to partition between the mobile organic phase and stationary aqueous phase trapped in the paper. - Inert - Filter paper does not chemically interact with or absorb the analytes, so it acts as an inert support for the separation process. - Thin - Filter paper sheets are thin but strong

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317 views34 pages

Planar Chromatography

Filter paper is commonly used as the stationary phase material in paper chromatography. Some key properties of the filter paper that make it suitable include: - Porous structure - The cellulose fibers in filter paper form a porous network that allows capillary action to draw the mobile phase up the paper. This drives the separation process. - Hydrophilic - Filter paper readily absorbs water, forming the stationary aqueous phase. This allows solutes to partition between the mobile organic phase and stationary aqueous phase trapped in the paper. - Inert - Filter paper does not chemically interact with or absorb the analytes, so it acts as an inert support for the separation process. - Thin - Filter paper sheets are thin but strong

Uploaded by

sanelisofuturemoyo
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© © All Rights Reserved
We take content rights seriously. If you suspect this is your content, claim it here.
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Planar Chromatography

BY SANELISO FUTURE MOYO


Types of planar chromatographic methods include:
1. Thin layer chromatography (TLC)
2. Paper Chromatography (PC)
3. Electrochromatography

• Planar chromatography uses flat, thin layer material, that is either self-supporting
or is coated on a glass, plastic or metallic surface.
• The mobile phase moves through the stationary phase by capillary action, sometimes
assisted by gravity or electrical potential.
• Planar chromatography used to be called two dimensional chromatography.

 Though there are 3 types of chromatography, we shall only discuss TLC and PC.
 In general TLC offers the following advantages over PC:
 It is fast, have better resolution and is more sensitive.
The advantages of TLC over the HPLC

(1) Simultaneous analysis of multiple standards and samples can be carried out under
identical conditions in a time comparable to HPLC.
(2) Strongly retained compounds in comparison to HPLC form the most compact
chromatographic zones and therefore can be detected with the highest sensitivity; in
addition the bands can be removed and purified.
(3) All components can be located, unlike in HPLC, where highly polar materials may
be overlooked as the peaks are very broad and difficult to discern.
Thin layer chromatography (TLC)
is a method for identifying substances and
.testing the purity of compounds

TLC is a useful technique because it is


relatively quick and requires small
.quantities of material

Under what conditions do we use TLC


Separations in TLC involve distributing a mixture of two
or more substances between a stationary phase and a
.mobile phase

:The stationary phase


is a thin layer of adsorbent (usually silica gel or alumina)
.coated on a plate

:The mobile phase


is a developing liquid which travels up the stationary
.phase, carrying the samples with it
Components of the samples will separate on the
stationary phase according to
how much they adsorb on the stationary phase versus
.how much they dissolve in the mobile phase
Thin Layer Chromatography (TLC)
the steps involved in thin layer chromatography (TLC).

TLC can be performed very simply. The steps involved are:


 Selection of a suitable layer.
 Sample application.
 Selection of the mobile phase.
 Development (the name used for the actual running of the
chromatogram).
 Visualization and detection.
 Quantification.

Discuss the solvents that are used in TLC. Comment on why other solvent are not
suitable.
TLC
Preparing the Chamber

To a jar with a tight-fitting lid add enough of


the appropriate developing liquid so that it
.is 0.5 to 1 cm deep in the bottom of the jar
Close the jar tightly, and let it stand for about
30 minutes so that the atmosphere in the jar
.becomes saturated with solvent
Preparing the Plates for
Development
With a pencil, etch two small notches into the adsorbent
.about 2 cm from the bottom of the plate

The notches should be on the edges of the plate, and each


notch should be the same distance up from the bottom of
.the plate

The notches must be farther from the bottom of the plate


.than the depth of the solvent in the jar

Using a drawn-out capillary tube, spot the samples on the


.plate so that they line up with the notches you etched
Developing the Plates
After preparing the development chamber and spotting
.the samples, the plates are ready for development

Be careful to handle the plates only by their edges, and


try to leave the development chamber uncovered for as
.little time as possible

When the plates are removed from the chamber, quickly


trace the solvent front (the highest solvent level on the
.plate) with a pencil
Identifying the Spots (visualization)
If the spots can be seen, outline
.them with a pencil

If no spots are obvious, the most


common visualization technique
is to hold the plate under a UV
.lamp
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
Visualizing Agents
Alkaloids: Dragendorff’s reagent

Cardiac glycosides: Antimony trichloride

Sugar: Aniline phthalate

Amino acids: Ninhydrin


Interpreting the Data

The Rf (retention factor) value for each spot


.should be calculated
It is characteristic for any given compound
on the same stationary phase using the same
.mobile phase for development of the plates
Hence, known Rf values can be compared to
those of unknown substances to aid in their
.identifications
Note: Rf values often depend on the temperature and(
.the solvent used in the TLC experiment

the most effective way to identify a compound is to spot


known substances – authentic - next to unknown
).substances on the same plate

In addition, the purity of a sample may be estimated


.from the chromatogram

An impure sample will often develop as two or more


spots, while a
pure sample will show only one spot
Summary
A TLC plate is a sheet of glass, metal, or plastic which is coated
with a thin layer of a solid adsorbent (usually silica or
.alumina)

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 so that only the very bottom of the plate is
.in the liquid

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
High
Performance
Thin
Layer
Chromatography
Introduction of HPTLC

HPTLC is the improved method of TLC which utilizes the conventional


technique of TLC in more optimized way. It is also known as planar
chromatography or Flat-bed chromatography.
THE PRINCIPLE
HPTLC takes place in high-speed capillary flow range of the mobile phase. There are
three main steps HPTLC procedure:
SAMPLE APPLICATION
Sample to analyzed to chromatogram layer, volume precision and exact position are
achieved by use of suitable instrument.
CHROMATOGRAM DEVELOPMENT
Solvent (mobile phase) migrates the planned distance in layer (stationary
phase) by capillary action. In this process sample separated into it’s components.
CHROMATOGRAM EVALUATION
Separation tracks are scanned in densitometer with light beams in visible or uv
region
TLC Plate

HPTLC Plate
Difference between TLC and HPTLC
TLC HPTLC
10 - 25 µm 5 - 7 µm
Plate particle size:
Separation distance: 100 - 150 mm 60 mm
Development time: 30 - 200 min 3 - 20 min
Application: manual automated/semi-

ADVANTAGES OFFERED BY
automated
Development: manual automated
Derivatization: spraying dipping

HPTLC
Analysis data:
Quantitative analysis:
no documentation
no
fully documented
yes
Environment: no control no problems
Resolution: often poor very good
Procedure: flexible fully standardized
Reproducibility: impossible highly attainable
cGMP Compliant: usually not YES!!
What are the advantages of using small particle size in HPTLC.

• Small particles provide fast and efficient separations.


the advantages of HPTLC over TLC

The small particle size and more uniform nature of the particles in HPTLC results in much
greater chromatographic efficiency than found in conventional TLC and enables the same
separations to be achieved much more rapidly and also separations that are not possible by
conventional TLC.

Detection limits in HPTLC are 10 times better than in conventional TLC. Two factors
contribute to this improved sensitivity: the layer itself in HPTLC is usually somewhat
thinner and, more importantly, the surface is more uniform than that of earlier TLC plates.
More uniform surfaces reduce background noise with instrumental detectors. The second
factor is the more compact spots on high performance layers as a result of the reduced band
spreading per plate length.

In summary the advantages of HPTLC over TLC, including increased resolution, greater
sensitivity, better reproducibility, greater number of samples per plate, combined with
performance approaching that of HPLC have not been obtained without cost.
STEPS OF THE HPTLC PROCEDURE

Wincats
Paper Chromatography
A method of partition chromatography using filter
.paper strips as carrier or inert support

The factor governing separation of mixtures of solutes


on filter paper is the partition between two
.immiscible phases

One is usually water adsorbed on cellulose fibres in


.the paper (stationary phase)

The second is the organic solvent flows past the


.sample on the paper (stationary phase)
What material is used to make the special paper for paper chromatography and state its
characteristics.

• These papers are prepared from specially purified cellulose (98-99% a-cellulose, 0.3-
1.0% f3-cellulose, 0.4-0.8% pentosans and <0.01% mineral ash).

• They provide a highly purified and reproducible surface with respect to porosity,
thickness and arrangement of cellulose fibres.

• Chromatography papers are characterized by their thickness, weight per unit area
and flow-rate index.
Partition occurs between the mobile phase and the
.stationary aqueous phase bound by the cellulose

The isolation depends on partition coefficient of the


.solute

c( stationary )
K
c( mobile)
General Procedure
Describe the steps involved in paper chromatograph

.Choice of paper and solvent to be used -1


.Desalting of sample -2
.Application of the sample -3
.Equilibration of paper -4
.Development -5
.Detection -6
.Identification of substances -7
Techniques of development with various flow
directions
Radial development Ascending development

Descending development
Multiple chromatography
Multiple chromatography includes all
procedures in which the development is
.repeated after one development is completed

A- multiple development: the chromatogram is


repeatedly developed in the same direction
and thus the complete resolution of two or
more substances which have Rf values close
.together can be obtained
As the mobile phase one can use either the same
.solvent system or different solvent systems
:B- two-dimensional chromatography
When large numbers of substances are to be separated
.on a single chromatogram
Development in a direction perpendicular to the first,
and with a solvent system different from that used
.initially is often necessary

The sample is applied on one corner of a square piece of


paper and after development with the first solvent,
the paper is dried , rotated 90o and developed in the
.second direction
Usually, different types of solvents systems are used in
each direction. It is essential that the first solvent be
.completely volatile

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