Transcript Types of Paper Chromatography

PAPER
CHROMATOGRAPHY
CHROMATOGRAPHY
• Chromatography is a family of analytical
chemistry techniques for the separation of
mixtures.
• It was the Russian botanist Mikhail Tsvet
(Mikhail Semyonovich Tsvet) who invented
the first chromatography technique in 1901.
CHROMATOGRAPHY
• The separation of molecules depends on
differences of
1- size
2- shape
3- mass
4- charges
5- solubility
6- adsorption.
■ Types of Chromatography:
1. Adsorption chromatography.
2. Partition chromatography e.g. paper
chromatography
3. Gel-filtration chromatography.
■ Uses of Chromatography:
* Government laboratories used to check
• for approved dyes in food
• that vegetables contained tiny amounts of
pesticides and herbicides
■All types of chromatography involving
interaction between:
1. The mixture to be separated.
2. The stationary phase.
3. The mobile phase.
Principle of Paper Chromatography:
• Method of separating and identifying both
colored and colorless mixtures.
• Mixtures can be solids, liquids or gases but
their components must be able to dissolve in the
same solvent to different extents.
* Generally involves 2 phases:
1. stationary phase solid support e.g. water on
paper
2. mobile phase solvent or a gas
Principle of Paper Chromatography:
• Test
mixture
is
applied
onto
the
chromatography paper and a solvent is then
allowed to pass over the paper. As the solvent
does so, the components of the mixture travel
along with it.
• The stationary phase retards the passage of the
components of the sample. When components
pass through the system at different rates they
become separated in time.
•The solvent used depends on the substance to be
separated
The components will travel at different
rates over paper depending on:
1. their solubility in the solvent
2. how well the dyes adsorb on the
chromatography paper
Generally, the more soluble the component is in
the solvent and the less it adsorb onto the
chromatography paper, the faster it would move
with the solvent on the paper and hence the spot
appears further up the paper
• Result of chromatography is known as the
Chromatogram.
Types of Paper Chromatography
There are 3 types of paper chromatography:
1. Ascending Paper
Chromatography:
Solvent running up the
paper by capillary
action.
Types of Paper Chromatography
There are 3 types of paper chromatography:
2.
Descending
Paper
Chromatography:
Solvent running down the
paper by both capillary
action and gravity.
•Advantage
of
the
descending method over the
ascending method:
1.Good for long pieces of
paper thus better separation.
2.Aided by gravity thus
faster.
■ Stationary Phase
• In paper chromatography, cellulose in the form
of paper sheets makes an identical support
medium. WHY?
Because it has the ability to adsorb water
molecules between cellulose fibers and forms a
stationary hydrophilic phase.
• Paper: Watman No. 1 of high quality is the
paper most frequently used for analytical
purposes.
■ Mobile Phase
• In paper chromatography, mobile phase is a mixture
of solvents.
• The choice of solvent depends on the mixture
investigated:
1- If the compounds move close to solvent (A) front
>>> these compounds are highly soluble in solvent A
2- If the compounds are crowded around the origin >>>
these compounds are not sufficiently soluble in
solvent B.
• Therefore, a suitable solvent for separation would be
an appropriate mixture of both solvent A & B. As a
result R f values of the components of the mixture are
spread across the length of the paper.
Retention Factor (R f ):
• The retention is measured as the retention factor Rf,
the run length of the compound divided by the run
length of the solvent front:
• Unknown substances could be identified by the Rf
values
Rf = distance moved by the substance
distance moved by the solvent
Retention Factor(R f ):
• The Rf of a compound often differs considerably
between experiments and laboratories due to
variations of
- the solvent,
- the stationary phase,
- temperature, and
- the setup.
• It is therefore important to compare the retention of
the test compound to that of one or more standard
compounds under absolutely identical conditions.
Retention Factor (R f ):
Detection of Spots:
• After development, the spots corresponding to
different compounds may be located by their
color
• However, most compounds are colorless and
are visualized by:
1- Spraying the paper with specific reagents.
2- Dipping the paper in a solution of the reagent
in a volatile solvent.
3- Fluorescent substances can be visualized by
ultraviolet (UV) light.
SEPARATION OF AMINO ACIDS
BY PAPER CHROMATOGRAPHY
SEPARATION OF AMINO ACIDS BY
PAPER CHROMATOGRAPHY
• Separation and identification of amino acids are
operations that must be performed frequently by
biochemists.
• The 20 amino acids present in proteins have similar
structures. However, each amino acid is unique in
polarity and ionic characteristics.
• In this experiment, we will use paper chromatography
to separate and identify the components of an
unknown amino acid mixture.
SEPARATION OF AMINO ACIDS BY
PAPER CHROMATOGRAPHY
• The solvent mixture contains several
components,
- one of which is usually water and
- another of which is a more non-polar solvent.
• As the solvent mixture moves up the paper by
capillary action, the water in the mixture binds
to the hydrophilic paper (cellulose) and creates
a liquid stationary phase of many small water
droplets.
• The non-polar solvent continues to move up the
paper forming a liquid mobile phase.
SEPARATION OF AMINO ACIDS BY
PAPER CHROMATOGRAPHY
• Since amino acids have different R-groups,
they also have different degrees of solubility in
water vs. the non-polar solvent.
SEPARATION OF AMINO ACIDS BY
PAPER CHROMATOGRAPHY
• An amino acid with a polar R-group will be more
soluble in water than in the non-polar solvent, so it
will dissolve more in the stationary water phase and
will move up the paper only slightly.
• An amino acid with a hydrophobic R-group will be
more soluble in the mobile non-polar solvent than in
water, so it will continue to move up the paper.
• Different amino acids will move different distances
up the paper depending upon their relative solubilities
in the two solvents, allowing for separation of amino
acid mixtures.
Applications
• Materials:
1- Filter paper: Watman No.1.
2- Solvent system: Butanol: glacial acetic acid:
water.
3- Ninhydrine reagent.
4- Standard amino acids and mixture of
unknown.
Applications
• Procedure:
1- Draw a light pencil line 1-2cm from the bottom of the
paper.
2- Place a single drop of compound at intervals 2cm.
3- Dry with hair dryer.
4- Dip the paper in the jar with one of the edges of the
paper to which the sample of the spot is adjacent into
the solvent.
5- Allow to run.
6- Remove the paper.
7- Determine the solvent front.
8- Dry.
9- Spray the paper with ninhydrin.
10- Dry the paper.
1.Arginine
2.Tyrosine
3.Mixture
1
2
3
Applications
• Procedure:
1- Draw a light pencil line 1-2cm from the bottom of the
paper.
2- Place a single drop of compound at intervals 2cm.
3- Dry with hair dryer.
4- Dip the paper in the jar with one of the edges of the
paper to which the sample of the spot is adjacent into
the solvent.
5- Allow to run.
6- Remove the paper.
7- Determine the solvent front.
8- Dry.
9- Spray the paper with ninhydrin.
10- Dry the paper.
SEPARATION OF AMINO ACIDS BY
PAPER CHROMATOGRAPHY
After some time