Thin Layer Chromatography (With Diagram) 164756

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Thin Layer
Chromatography
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Let us learn about Thin Layer

Chromatography.
Introduction to Thin Layer

Chromatography:
Chromatography is used to separate
mixtures of substances into their
components. All forms of
chromatography work on the same
principle. They all have a stationary
phase (a solid, or a liquid supported on
a solid) and a mobile phase (a liquid or
a gas).
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The mobile phase flows through the
stationary phase and carries the
components of the mixture with it.
Different components travel at
different rates. Thin layer
chromatography is done exactly as it
says-using a thin, uniform layer of silica
gel or alumina coated onto a piece of
glass, metal or rigid plastic.
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The silica gel (or the alumina) is the

stationary phase. The stationary phase
for thin layer chromatography also
often contains is substance which
fluoresces in UV light. The mobile phase
is a suitable liquid solvent or mixture of
solvents.
Producing the Chromatogram:

The chromatogram can be produced by
dipping the chromatographic plate (TLC
plate with silica and mixture of
different spots to be separated) in the
mixture of various solvent systems (for
e.g., n-Butanol, Glacial Acetic Acid and
Distilled Water) in a specific ratio for
separating amino acids using standard
amino acid kit with known Rf values.
A pencil line is drawn near the bottom

of the plate Fig. 22.2 and a small drop of
solution
Electric Insect of the
and Pest Reject dye mixture is placed on
it.Read
AnyNextlabeling
Story on the plate to show the
original position of the drop must also
be in pencil. If any of this was done in
ink, dyes from the ink would also move
as the chromatogram developed.
When the spot of mixture is dry, the

plate is stood in a shallow layer of
solvent in a covered beaker, it is
important that the solvent level is
below the line with the spot on it.
The reason of covering the beaker is to

make sure that the atmosphere in the
beaker or the chromatographic
chamber is saturated with solvent
vapour. To help this, the beaker is often
lined with some filter paper soaked in
the solvent. Saturating the atmosphere
in the beaker with vapour stops the
solvent from evaporating as it rises up
the plate.
As the solvent slowly travels up the

plate, the different components of the
dye
Electric Insect andmixture
Pest Rejecttravel at different rates
Read Next Story
and the mixture is separated into
different spots.
The diagram shows the plate after the

solvent has moved about half way up it
Fig. 22.10.
The solvent is allowed to rise until it

almost reaches the top of the plate fig.
22.11.
That will give the maximum separation

of the dye components for this
particular combination of solvent and
stationary phase.
Measuring Rf Values:
If and
Electric Insect all Pest
youReject
wanted to know is how many
Read Next Story
different dyes made up the mixture,
you could just stop there. However,
measurements are often taken from the
plate in order to help identify the
compounds present. These
measurements are the distance
traveled by the solvent, and the
distance traveled by individual spots.
When the solvent front gets close to the

top of the plate, the plate is removed
from the beaker and the position of the
solvent is marked with another line
before it has a chance to evaporate.
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These measurements are then taken:
The Revalue for each dye is then

worked out using the formula:
Rf = distance travelled by component

/distance travelled by solvent
For example, if the one component

travelled 170mm from the base line
while the solvent had traveled 50mm,
then the Rf value for that dye is:
If you could repeat this experiment

under exactly the same conditions, then
the Rf value for each dye would always
be the same. For example, the Rf value
for the above dye would always be 3.4.
Electric Insect However if anything changes (the
and Pest Reject
temperature,
Read Next Story the exact composition of
the solvent, and so on), that is no longer
true.
What if the substances you are

interested in all colourless ?
There are two simple ways of getting

around this problem:
Using Fluorescence:
In case of mixtures of generating
colourless spots in stationary phase on
a thin layer plate often a substance is
added to it which will fluoresce when
exposed to UV light. That means that if
you shine UV light on it, will glow fig.
22.12.
That glow is masked at the position

where the spots are on the final
chromatogram-even if those spots are
invisible to eye. That means that if you
shine UV light on the place, it will all
glow apart from where the spots are.
The spots show up as darker patches.
Read Next Story
While the UV is still shining on the plate
you obviously have to mark the
position of spots by
drawing a pencil circle around then. As

soon as you switch off the UV source,
the spots will disappears again.
Showing the Spots Up Chemically:

In some cases, it may be possible to
make the spots visible by reacting them
with something which produces a
coloured product. A good example of
this is in chromatograms produces
from amino acids mixtures Fig. 22.13.
The chromatogram is air-dried and is

then sprayed with a solution of
ninhydrin and later allowed to dry (at
about 100° C in oven for few minutes).
Ninhydrin reacts with amino acids to
give coloured compounds, mainly
brown or purple.
Using
Electric Insect Thin
and Pest Reject Layer
Chromatography to Identify
Read Next Story
Compounds:
Suppose you had a mixture of amino
acids and wanted to find out which
particular acids the mixture contained.
For simplicity we, will assume that you
know the mixture can only possibly
contain five of the amino acids.
A small drops of the mixture is placed

on the base line of the thin layer plate,
and similar small spots of the known
amino acids are placed along-side it.
The plate is then stood in a suitable
solvent and left to develop as before. In
the diagram the mixture is M, and the
known amino acids are labeled 1 to 5.
The left hand diagram shows the plate

after the solvent front has almost
reached the top. The spots are still
invisible. The second diagram shows
what is might look like after spraying
with ninhydrin fig. 22.13 and 22.14.
There is no need to measure to Rf

values because you can easily compare
Electric Insect
theand Pest Reject
spots in the mixture with those of
Read Next Story
the known amino acids-both from their
positions and their colours.
How does thin layer

Chromatography Work?
The Stationary Phase-Silica Gel:
Silica gel is a form of silicon dioxide

(silica). The silicon atoms are joined via
oxygen atoms in a giant covalent
structure. However, at the surface of
the silica gel, the silicon atoms are
attached to —OH groups.
So, at the surface of the silica gel you

have Si-O-H bonds instead of Si-O-Si
bonds. The diagram shows a small part
of the silica surface.
The surface of the silica gel is very

polar and, because of the-OH groups,
can from hydrogen bonds with suitable
compounds around it as well as vander
Walls dispersion forces and dipole
attractions.
The other commonly used stationary

phase
Electric Insect is Reject
and Pest alumina-aluminium oxide. The
aluminium atoms on the surface of this
Read Next Story
also have -OH groups attached.
Anything we say about silica gel
therefore applies equally to alumina.
Separation of the Compounds in a

Developing Chromatogram:
As the solvent beings to soak up the
plate, it first dissolves the compounds
in the spot that you have put on the
base line. The compounds present with
then tend to get carried up the
chromatography plate as the solvent
continues to move upwards.
How fast the compounds get carried

up the plate depends on two things:
1. How soluble the compound is in the

solvent. This will depend on how much
attraction there is between the
molecules of the compound and those
of the solvent.
2. How much the compound sticks to

the stationary phase the silica get, for
example. This will depend on how
much attraction there is between the
compound and the silica gel.
Suppose the original spot contained two

compounds-one of which can from
hydrogen bonds, and one of which can
only take part in weaker vander Walls
interactions, the one which can
Read Next Story
hydrogen bond will stick to the surface
of the silica gel more firmly then the
other one. We say that one the
adsorbed more strongly than the other.
Adsorption is the name given to one

substance forming some sort of bonds
to the surface of another one.
Adsorption is not permanent-there is a
constant movement of a molecule
between being adsorbed on to the silica
gel surface and going back into solution
in the solvent.
Obviously the compound can only

travel up the plate during the time that
it is dissolved in the solvent. While it is
adsorbed on the silica gel, it is
temporarily stopped-the solvent is
moving on without it. That means that
the more strongly a compound is
adsorbed, the less distance it can travel
up the plate.
In the example we started with, the

compound which can hydrogen bond
will adsorb more strongly than the one
dependent on van der Walls
interactions, and so won’t travel so far
up the plate. What if both components
of the mixture can hydrogen bond?
It is very unlikely that bond will

hydrogen bond to exactly the same
extent,
Electric Insect and
and Pest be
Reject soluble in the solvent to
exactly the same extent. It isn’t just the
Read Next Story
attraction of the compound for the
silica gel which matters. Attractions
between the compound and the solvent
are also important-they will affect how
easily the compound is pulled back into
solution away from the surface of the
silica.
Few methods of chemical analysis are

truly specific to a particular analyte. It
is often found that the analyte of
interest must be separated from the
myriad of individual compounds that
may be present in a sample. As well as
providing the analytical scientist with
methods of separation,
chromatographic technique can also
provide methods of analysis.
Chromatography involves a sample (or

sample extract) being dissolved in a
mobile phase (which may be a gas, a
liquid or a supercritical fluid). The
mobile phase is then forced through an
immobile, immiscible stationary phase.
The phases are chosen such that
components of the sample have
differing solubilities in each phase.
A component which is quite soluble in

the stationary phase will take longer to
travel through it than a component
which is not very soluble in the
stationary
Electric Insect phase
and Pest Reject but very soluble in the
Read Next Story
mobile phase. As a result of these
differences in mobilities, sample
components will become separated
from each other as they travel through
the stationary phase.
Techniques such as H.P. L.C. (High

Performance Liquid Chromatography)
and G.C. (Gas Chromatography) use
columns-narrow tubes packed with
stationary phase, through which the
mobile phase is forced. The sample is
transported through the column by
continuous addition of mobile phase.
This process is called elution. The

average rate at which an analyte moves
through the column is determined by
the time it spends in the mobile phase.
Distribution of Analytes between

Phases:
The distribution of analytes between
phases can often be described quite
simply. An analyte is in equilibrium
between the two phases;
The equilibrium constant, K, is termed

the partition coefficient; defined as the
molar concentration of analyte in the
stationary phase divided by the molar
concentration
Electric Insect and Pest Reject of the analyte in the
mobile phase.
Read Next Story
The time between sample injection and
an analyte peak reaching a detector at
the end of the column is termed the
retention time (tR). Each analyte in a
sample will have a different retention
time. The time taken for the mobile
phase to pass through the column is
called fM.
A term called the retention factor, K is

often used to describe the migration
rate of an analyte on a column.
It is also called the capacity factor.

The retention factor for analyte A is
defined as:
K’A = tR − tM / tM
tR and tM are easily obtained from a

chromatogram. When an analytes
retention factor is less than one, elution
is so fast that accurate determination of
the retention time is very difficult. High
retention factors (greater that 20) mean
that elution takes a very long time.
Ideally, the retention factor for an
Read Next Story
analyte is between one and five.
We define a quantity called the
selectivity factor, α , which describes
the separation of two species (A and B)
on the column;
α = k’B /k’A
When calculating the selectivity factor,

species A elutes faster the species B.
The selectivity factor is always greater
than one.
The Theoretical Plate Model of

Chromatography:
The plate model supposes that the
chromatographic column is contains a
large number of separate layers, called
theoretical plates Fig. 22.16. Separate
equilibrations of the sample between
the stationary and mobile phase occur
in these “plates”. The analyte moves
down the column by transfer of
equilibrated mobile phase from one
plate to the next.
It is important to remember that the

plates do not really exist; they are a
figment
Electric Insect and Pest of the
Reject imagination that helps us
understand the processes at work in
Read Next Story
the column. They also serve as a way of
measuring column efficiency, either by
stating the number of theoretical plates
in a column. N (the more plates the
better), or by stating the plate height;
Height Equivalent to a Theoretical Plate
(the smaller the better).
If the length of the column is L, then

the HETP is:
HETP = L / N
The number of theoretical plates that a

real column possesses can be found by
examining a chromatographic peak
after elution;
where w1/2 is the peak width at half-

height.
As can be seen from this equation,

columns behave as if they have
different number of plates for different
solutes in a mixture.
The Rate Theory of Chromatography:

A more realistic description of the
processes at work inside a column takes
account of the time taken for the solute
toand
Electric Insect equilibrate between the stationary
Pest Reject
and mobile phase (unlike the plate
Read Next Story
model, which assumes that
equilibration is infinitely fast).
The resulting band shape of a

chromatographic peak is therefore
affected by the rate of elution. It is also
affected by the different paths available
to solute molecules as they travel
between particles of stationary phase.
If we consider the various mechanisms
which contribute to band broadening,
we arrive at the Van Deemter equation
for plate height.
HETP = A + B / u + Cu
where u is the average velocity of the

mobile phase. A, B, and C are factors
which contribute to band broadening.
A-Eddy Diffusion:
The mobile phase moves through the

column which is packed with stationary
phase. Solute molecules will take
different paths through the stationary
phase at random. This will cause
broadening of the solute band, because
different paths are of different lengths.
B-Longitudinal Diffusion:
The concentration of analyte is less at
the edges of the band than at the
center. Analyte diffuses out from the
center to the edges. This causes band
Read Next Story
broadening. If the velocity of the
mobile phase in high then the analyte
spends less time on the column, which
decreases the effects of longitudinal
diffusion.
C-Resistance to Mass Transfer:

The analyte takes a certain amount of
time to equilibrate between the
stationary and mobile phase. If the
velocity of the mobile phase is high,
and the anlyte has a strong affinity for
the stationary phase, then the analyte
in the mobile phase will move ahead of
the analyte in the stationary phase. The
bond of analyte is broadened. The
higher the velocity of mobile phase, the
worse the broadening becomes.
Van Deemter Plots:

A plot of plate height vs. average linear
velocity of mobile phase is called Van
Deemter Plot (Fig. 22.17).

Such plots are of considerable use in
Read Next Story
determining the optimum mobile phase
flow rate.
Resolution:
Although the selectivity factor, α ,

describes the separation of band
centres, it does not take into account
peak widths. Another measure of how
well species have been separated is
provided by measurement of the
resolution.
The resolution of two species, A and

B, is defined as:
Baseline resolution is achieved when

R= 1.5
It is useful to relate the resolution to

the number of plates in the column,
the selectivity factor and the
retention factors of the two solutes:
To obtain high resolution, the three

terms must be maximized. An increase
in N, the number of theoretical plates,
by lengthening the column leads to an
increase in the retention time and
increased band broadening-which may
not be desirable. Instead, to increase
Read Next Story
the number of plates, the high
equivalent to a theoretical plate can be
reduced by reducing the size of the
stationary phase particles.
It is often found that by controlling the

capacity factor, k’ separations can be
greatly improved. This can be achieved
by changing the temperature. (In Gas
Chromatography) or the composition of
the mobile phase (in Liquid
Chromatography)
The selectivity factor, ∝, can also be

manipulated to improve separation.
When ∝ is close to unity, optimizing k’
and increasing N is not sufficient to give
good separation in a reasonable time.
In these case, K’ is optimized 1st, and

then ∝ is increased by one of the
following procedure:
1. Changing mobile phase composition.
2. Changing column temperature.
3. Changing composition of stationary

phase.
4. Using special chemical effects. (Such

as incorporating a spices which
complexes with one of the solutes into
the stationary phase).
Related
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1. Chromatography: Basics, Principles
and Theories
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Let us learn about Thin Layer

Introduction to Thin Layer

The silica gel (or the alumina) is the

Producing the Chromatogram:

A pencil line is drawn near the bottom

When the spot of mixture is dry, the

The reason of covering the beaker is to

As the solvent slowly travels up the

The diagram shows the plate after the

The solvent is allowed to rise until it

That will give the maximum separation

When the solvent front gets close to the

These measurements are then taken:

The Revalue for each dye is then

Rf = distance travelled by component

For example, if the one component

If you could repeat this experiment

What if the substances you are

There are two simple ways of getting

That glow is masked at the position

drawing a pencil circle around then. As

Showing the Spots Up Chemically:

The chromatogram is air-dried and is

A small drops of the mixture is placed

The left hand diagram shows the plate

There is no need to measure to Rf

How does thin layer

Silica gel is a form of silicon dioxide

So, at the surface of the silica gel you

The surface of the silica gel is very

The other commonly used stationary

Separation of the Compounds in a

How fast the compounds get carried

1. How soluble the compound is in the

2. How much the compound sticks to

Suppose the original spot contained two

Adsorption is the name given to one

Obviously the compound can only

In the example we started with, the

It is very unlikely that bond will

Few methods of chemical analysis are

Chromatography involves a sample (or

A component which is quite soluble in

Techniques such as H.P. L.C. (High

This process is called elution. The

Distribution of Analytes between

The equilibrium constant, K, is termed

A term called the retention factor, K is

It is also called the capacity factor.

tR and tM are easily obtained from a

When calculating the selectivity factor,

The Theoretical Plate Model of

It is important to remember that the

If the length of the column is L, then

The number of theoretical plates that a

where w1/2 is the peak width at half-

As can be seen from this equation,

The Rate Theory of Chromatography:

The resulting band shape of a

where u is the average velocity of the

The mobile phase moves through the

C-Resistance to Mass Transfer: