Download Chromatography

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Mikhail Tswett, Russian, 1872-1919
In 1906 Tswett used to chromatography to separate plant
pigments, He called the new technique chromatography
because the result of the analysis was
• Most materials in our surroundings are mixtures of two or
more components.
 Mixtures are either homogeneous or heterogeneous:
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Homogeneous mixtures are uniform in composition, but
heterogeneous mixtures are not.
Air is a homogeneous mixture and oil in water is a
heterogeneous mixture.
Homogeneous and heterogeneous mixtures can be separated into
their components by several physical methods.
• Chromatography is a widely used experimental
technique for the separation of a mixture of compounds
into its components.
• The word chromatography means "separation of colors"
but today chromatography is used for both colored and
colorless substances.
• Chromatography is a physical method of separation in
which the components to be separated are distributed
between two phases, one of which is stationary phase,
while the other the mobile phase moves through it in a
definite direction.
Chromatography
Electrophoresis
Difference in rate of
movement of a solute
through a stationary
phase
Difference in migration
rate in an electrical field
gradient
can be used for liquid,
solid and gaseous
compounds
is generally carried out
on liquid and solid
compounds
partition coefficient of
the element is used to
carry out the
investigations and
separation
the electrical property
of the element is used
 Affinity: natural attraction or force between
things.
 Adsorption: refers to the adhesion or
stickyness of a substance to the surface of
another substance, as opposed to the term
“absorption” which refers to a substance
penetrating into the inner structure of another
substance.
 https://www.youtube.com/watch?v=djIzXvwIz
5U
• The separation process is based on the fact that porous
solids adsorbs different substances to different extremes
depending upon their polarity.
• A mixture to be separated is first applied to an
immovable porous solid (like paper, or alumina, or fine
silica sand) called the stationary phase.
• The components of the mixture then get “washed”
along the porous solid by the flow of a solvent called the
mobile phase.
 Each component of a mixture to be separated will be
attracted differently to the porous stationary phase
depending on its polarity and the polarity of the
stationary phase chosen.
 Remember that “Like dissolve Like”.
 If the stationary phase is polar then polar components
will be attracted or stick more to it but non-polar
components will move across the surface easily.
 If the stationary phase is nonpolar then nonpolar
components will be more attracted to it and the polar
compounds will move along more quickly.
• Chromatography involves the sample being dissolved
in a particular solvent called mobile phase.
• The mobile phase may be a gas or liquid.
• The mobile phase is then passed through another
phase called stationary phase.
• The stationary phase may be a solid packed in a glass
plate or a piece of chromatography paper.
Can separate complex mixtures with great precision. Even very
similar components, such as proteins that may only vary by a
single amino acid.
Can purify basically any soluble or volatile substance if the right
adsorbent material, carrier fluid, and operating conditions are
employed.
Exact quantitative analysis is done even from trace compounds.
Small material consumption.
The quantization has a broad linearity range.
Analyses of several compound can be done during one run.
Chromatography is a fast analysis method.
Stationary
Phase
Separation
Mobile
Phase
Mixture
Components
Components
Affinity to Stationary
Phase
Affinity to Mobile
Phase
• Analyze
Blue
----------------
Insoluble in Mobile Phase
• Identify
Black


Red


Yellow

        
• Purify
•Quantify
Types of
chromatography
Based on
stationary phase
Based on mobile
phase
Column
Liquid
Planar
Gas
1- Liquid chromatography: mobile phase is a liquid.
(LLC, LSC).
2- Gas chromatography : mobile phase is a gas.
(GSC, GLC).
 Planar chromatography: A separation technique in which the
stationary phase serves as a plane.
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The plane can be either a paper (paper chromatography) or a layer of solid
particles sorbent (silica gel, cellulose, aluminum oxide, ion exchange resin)
spread on a support such as a glass- or a plastic- plate (thin layer
chromatography).
THIS allowing to carry out qualitative and quantitative analysis of
chemical components in complex mixtures.
1- Thin layer chromatography (TLC): the stationary phase is a
thin layer supported on glass, plastic or aluminium plates.
2- Paper chromatography (PC): the stationary phase is a thin
film of liquid supported on an inert support.
 For qualitative analysis the different mobilities of substances
are used, the distances passed by different substances are
different. The distance between the starting line and the
center of the spot of substance characterizes the substance.
Retention factor, RF, provides better way to indentify
substances .
 Identification of Unknown Compounds For your initial
analysis, spot your unknown and the colored standard
compounds on one plate. Be sure to note which spot
corresponds to which compound.
 For quantitative determination the intensity of the spot is
used: the bigger the amount of substance in the mixture, the
more intensive is the spot. Also the size of the spot can give
quantitative information – the bigger the spot, the bigger the
content of this compound in the mixture. Intensity of the
spots is evaluated by comparing with the intensities of analyte
spots with known amounts visually or using densitometer.
• Paper chromatography uses paper as the stationary
phase and a liquid solvent as the mobile phase.
• In paper chromatography, the sample is placed on a
spot on the paper and the paper is carefully dipped
into a solvent.
• The solvent rises up the paper due to capillary action
and the components of the mixture rise up at
different rates and thus are separated from one
another.
 Capillary Action: the movement of liquid within the spaces of a
porous material due to the forces of adhesion, cohesion, and surface
tension.
 Cohesion, phenomenon of intermolecular forces holding particles of
a substance together.
 Adhesion, attraction between the surfaces of two bodies.
 The liquid is able to move up the filter paper because its attraction to
itself is stronger than the force of gravity.
 Solubility: the degree to which a material (solute) dissolves into a
solvent.
 Solutes dissolve into solvents that have similar properties. (Like
dissolves like) This allows different solutes to be separated by
different combinations of solvents.
 Separation of components depends on both their solubility in the
mobile phase and their differential affinity to the mobile phase and
the stationary phase.
• Thin layer chromatography is a technique used
to separate and identify compounds of interest.
• A TLC plate is made up of a thin layer of silica
adhered to glass or aluminum for support.
• The silica gel acts as the stationary phase and the
solvent mixture acts as the mobile phase.
• In the ideal solvent system the compounds of
interest are soluble to different degrees. Separation
results from the partition equilibrium of the
components in the mixture.
 Different compounds in sample mixture travel
different distances according to how
strongly
they interact with the stationary phase as compared
to the mobile phase.
 In principle, the components will differ in solubility
and in the strength of their adsorption to the
adsorbent and some components will be carried
farther up the plate than others.
 This liquid, or the eluent, is the mobile phase, and it
slowly rises up the TLC plate by capillary action.
 The specific Retention factor (Rf) of each chemical
can be used to aid in the identification of an
unknown substance.
• Rf values can be calculated and compared with the
reference values to identify the amino acids.
• Highly polar molecules interact fairly strongly with the
polar SiOH groups at the surface of these adsorbents,
and will tend to stick or adsorb onto the fine particles of
the adsorbent while weakly polar molecules are held less
tightly.
• Weakly polar molecules generally tend to move through
the adsorbent more rapidly than the polar species.
Roughly, the compounds follow the elution order given
above.
• Different compound in sample mixture travel different
distance according to how strongly they interact with
the stationary phase as compared to mobile phase. (
)
• The separation depends on several factors:
1.
solubility: the more soluble a compound is in a solvent,
the faster it will move up the plate.(polarity of mobile
phase)
2. attractions between the compound and the silica, the
more the compound interacts with silica, the lesser it
moves, (. activity of stationary phase)
3. structure of substrate
o Eluent: The mobile phase (usually for solvents)
o Elution: Motion of the mobile phase through the
stationary phase
o Elution time: The time taken for a solute to pass
through the system. A solute with a short elution
time travels through the stationary phase rapidly, i.e.
it elutes fast.
o Mobile phase: The part of the chromatography
system that is mobile. Commonly a solvent mixture
(as in column chromatography or thin layer
chromatography or a gas (as in gas chromatography).
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Eluent. [Mix n-butanol, acetic acid (purity 98 – 100 %) and
distilled water in volume ratio 5:1:5. Stir for 10 minutes,
then let the layers separate. Use upper layer as eluent].
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Developing Solution: Dissolve 0.3 g of ninhydrin in 100 ml
n-butanol. Add 3 ml of glacial acetic acid.
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0.02 M solutions of different amino acids (e.g. leucine,
methionine, alanine and serine) in H2Odd.
Chromatographic paper
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Elution chamber, Glass capillaries for spotting the samples.
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Drying oven at ~ 60° C.
1.
Rubber gloves must be used during this work to avoid contamination of
chromatographic paper with amino acids from skin, and for protecting
skin from solvents and ninhydrin while working with the sprayer or
sprayed paper.
2. While the paper is being prepared for chromatographic analysis it should
be kept on a piece of filter paper.
3. Mark the starting line to the paper - 8-9 mm from the edge of the plate with graphite pencil (very slight line!). Also mark the locations where the
samples will be spotted. The distance between neighboring spots should
be about 8 mm and the spots should be at least 5 mm away from the
paper’s edge. Usually the spot of unknown substance is applied to the
center of the starting line.
pencil (do not use a pen, as the dye may separate and run along with your
sample),
Over spotting or inadequte spacing between samples can lead to their
bleeding
into
each
other
during
the
run.
4.
Before applying samples to the paper and filling the elution
chamber fit the length of chromatographic paper with the height
of elution chamber.
5.
The spots of individual amino acids and sample solutions are
applied to the chromatographic paper. Use separate clean and dry
glass capillary for each solution. Dip the capillary into solution –
some solution is drawn into the capillary. With the filled capillary
touch the prepared location on chromatographic paper. The spot
on the paper should not be bigger than 2-3 mm. (You can exercise
spotting on a sheet of filter paper.)
6.
After application of samples let the spots dry. Meanwhile
measure with a graduated test-tube 5 ml of eluent into the
elution chamber. Cover the chamber with lids and let the chamber
atmosphere saturate with eluent vapors for at least 10 min.
7. Elution is stopped when the solvent front has traveled up
the plate until 7-10 mm from the lid.
8. Remove the paper from elution chamber and place it on a
sheet of filter paper. After 2-3 minutes mark the eluent
front with pencil and dry the paper in oven.
When the plates are removed from the chamber,
quickly trace the solvent front (the highest solvent level
on the plate) with a pencil.
Monitor the run.
The time that it takes for the solvent to rise up the plate is
dependent upon your choice of solvent. Generally a run will
take only a few minutes, but you must monitor the solvent
front to be sure that it does not reach the end of the plate.
This could result in your sample being carried to the end of
the plate and an unusable TLC run. A good guideline to
follow is to allow the solvent front to reach a point about 1
cm from the end of the slide.
9. When the paper is dry, take it into the fume hood and
spray it with solution of ninhydrin until the paper is
slightly damp. Chromatographic paper and the paper
supporting it should lie at 45° angle while spraying.
The chromatographic paper is again put in the drying
oven (60 C) for 15 min to speed up the reactions.
10. Remove chromatographic paper from drying in the
oven, draw the contours and centers of the
chromatographic bands. Calculate RF values by the
method described above.
11. Compare retention of standard substances and
components in sample and determine which amino
acids were present in the sample.
1. Adding fluorescence indicators to the sorbent
layer during the process of preparation of the
plates or spraying the plates with fluorescent
solutions and then observing under ultraviolet
lamp.
2. I2 vapor as indicator .
3. Ninhydrin spray.(Visualizing Agents for
amino acids ) Development of Ruhemann’s
purple from ninhydrin and amino acid.
This value is constant for any given
compound under a given set of
conditions and can be used to identify an
unknown compound.
 Is this a pure substance or mixture?
TLC can be used to determine whether a sample is a single
substance or a mixture.
A single pure substance gives a single spot. However, care
should be taken since compounds of similar properties,
isomers for example, may not separate.
A sample that results in many spots after development is a
confirmation of a mixture of different compounds.
In that sense, TCL can also be used to determine the number
of components in a mixture.
 Are these compounds identical?
TLC can be used to confirm whether two or more
compounds are identical or not. The compounds to
be analyzed are spotted on the same plate and the
plate developed. If the compounds have the same Rf
value, this can be taken as an indication that they
have a similar chemical nature.
However, it is important to keep in mind that many
chemical compounds have the same Rf and TLC
cannot be used as the only analytical tool to decide on
the identity of a chemical compound.