CHROMATOGRAPHY
PAPER CHROMATOGRAPHY, TLC, GLC, MEC, HPLC
WHAT IS CHROMATOGRAPHY
• Chromatography is a method used by scientists for separating organic and
inorganic compounds so that they can be analyzed and studied. By analyzing
a compound, a scientist can figure out what makes up that compound.
Chromatography is a great physical method for observing mixtures and
solvents.
• The word chromatography means "color writing" which is a way that a
chemist can test liquid mixtures. While studying the coloring materials in
plant life, a Russian botanist invented chromatography in 1903. His name
was M.S. TSWETT.
HOW CHROMATOGRAPHY WORKS
• In all chromatography there is a mobile phase and a stationary phase. The
stationary phase is the phase that doesn't move and the mobile phase is the
phase that does move. The mobile phase moves through the stationary phase
picking up the compounds to be tested. As the mobile phase continues to travel
through the stationary phase it takes the compounds with it. At different points in
the stationary phase the different components of the compound are going to be
absorbed and are going to stop moving with the mobile phase. This is how the
results of any chromatography are gotten, from the point at which the different
components of the compound stop moving and separate from the other
components.
HOW CHROMATOGRAPHY WORKS
• In paper and thin-layer chromatography the mobile phase is the solvent. The
stationary phase in paper chromatography is the strip or piece of paper that
is placed in the solvent. In thin-layer chromatography, the stationary phase is
the thin-layer cell. Both these kinds of chromatography use capillary action to
move the solvent through the stationary phase.
WHAT IS THE RETENTION FACTOR, RF?
• The retention factor, Rf, is a quantitative indication of how far a particular
compound travels in a particular solvent. The Rf value is a good indicator of
whether an unknown compound and a known compound are similar, if not
identical. If the Rf value for the unknown compound is close or the same as
the Rf value for the known compound then the two compounds are most
likely similar or identical.
WHAT IS THE RETENTION FACTOR, RF?
• The retention factor, Rf, is defined as
• Rf = distance the solute (d1) moves divided by the distance traveled by the
solvent front (d2)
• Rf = d1 / d2 where d1 = distance that color traveled, measured from center
of the band of color to the point where the food color was applied d2 = total
distance that solvent traveled
WHAT IS THE RETENTION FACTOR, RF?
• The retention factor, Rf, is defined as
• Rf = distance the solute (d1) moves divided by the distance traveled by the
solvent front (d2)
• Rf = d1 / d2 where d1 = distance that color traveled, measured from center
of the band of color to the point where the food color was applied d2 = total
distance that solvent traveled
TYPES OF CHROMATOGRAPHY
• There are four main types of chromatography.
• These are liquid chromatography, gas chromatography, thin-layer chromatography,
and paper chromatography.
• Liquid chromatography is used around the world to test water samples and look for
pollution in lakes and rivers. It is used to analyze metal ions and organic compounds
in solutions. liquid chromatography uses liquids that may incorporate hydrophilic,
insoluble molecules.
• Gas chromatography is used in airports to detect bombs and is used in forensics in
many different ways. It is used to analyze fibers on a persons body and also analyzes
blood found at a crime scene. In gas chromatography, helium moves a gaseous
mixture through a column of absorbent material.
• Thin-layer chromatography uses an absorbent material on flat glass or plastic plates.
This is a simple and rapid method to check the purity of an organic compound. It is
used to detect pesticide or insecticide residues in food. Thin-layer chromatography is
TYPES OF CHROMATOGRAPHY
• Paper chromatography is one of the most common types of chromatography. It uses a
strip of paper as the stationary phase. Capillary action is used to pull the solvents up
through the paper and separate the solutes.
TYPES OF CHROMATOGRAPHY AND WHAT
THEY ARE USED FOR
TYPES OF CHROMATOGRAPHY AND WHAT
THEY ARE USED FOR
PAPER CHROMATOGRAPHY
• In paper chromatography, the sample mixture is applied to a piece of filter paper, the edge of the
paper is immersed in a solvent, and the solvent moves up the paper by capillary action.
Components of the mixture are carried along with the solvent up the paper to varying degrees,
depending on the compound's preference to be adsorbed onto the paper versus being carried along
with the solvent. The paper is composed of cellulose to which polar water molecules are adsorbed,
while the solvent is less polar, usually consisting of a mixture of water and an organic liquid. The
paper is called the stationary phase while the solvent is referred to as the mobile phase. Performing
a chromatographic experiment is a three-step process:
• 1) application of the sample
• 2) "developing" the chromatogram by allowing the mobile phase to move up the paper
• 3) calculating Rf values and making conclusions.
PAPER CHROMATOGRAPHY
• In order to obtain a measure of the extent of movement of a component in a
paper chromatography experiment, we can calculate an “Rf value" for each
separated component in the developed chromatogram. An Rf value is a
number that is defined as: the distance traveled by component from the
application point
TLC CHROMATOGRAPHY
• Thin layer chromatography (Tlc) is an important technique used for the
identification and separation of mixture of chemical compounds into its
individual components.
• Tlc is a form of liquid chromatography consisting of two phases: a mobile
phase (liquid) and a stationary phase (solid).
• Differences in the interactions between the solutes and stationary and mobile
phases enable separation.
TLC CHROMATOGRAPHY PRINCIPLES
• Tlc technique involves the distribution of components of a mixture to be
separated between two phases.
• The components of the mixture are partitioned between an adsorbent
(stationary phase), and a solvent (mobile phase).
• Different compounds will have different solubility and adsorption to the two
phases between which they are to be partitioned. •
• In Tlc separation of the individual substances is based on their relative
affinities towards stationary and mobile phases.
TLC CHROMATOGRAPHY METHOD
• Adsorbents used as stationary phase: - Inorganic: silica gel, kieselguhr, aluminum
silicate, bentonite. - organic: cellulose & its acetylates, charcoal & activated charcoal,
dextran gel, polyamides.
• Solvents used as mobile phase: - petroleum ether, benzene, carbon tetrachloride,
hexane, methanol
• Selection of adsorbents and solvents:
The adsorbent should not adhere to the glass plate.
solvents should be of high purity.
selected based on the nature of the compound to be separated (polar or non-polar.)
TLC CHROMATOGRAPHY Rf VALUES
• Rf value indicates the position of migrated spots on the chromatogram.
• In Tlc the results are represented by the Rf value which represents the
migration of solute relative to the solvent front.
• The Rf value is calculated as:-
TLC TECHNIQUE
TLC TECHNIQUE
TLC TECHNIQUE
TLC TECHNIQUE
TLC TECHNIQUE
APPLICATION OF TLC
GAS LIQUID CHROMATOGRAPHY
GAS LIQUID CHROMATOGRAPHY
GAS LIQUID CHROMATOGRAPHY
GAS LIQUID CHROMATOGRAPHY
GAS LIQUID CHROMATOGRAPHY
GAS LIQUID CHROMATOGRAPHY
GAS LIQUID CHROMATOGRAPHY
GAS LIQUID CHROMATOGRAPHY
GAS LIQUID CHROMATOGRAPHY
GAS LIQUID CHROMATOGRAPHY
MOLECULAR EXCLUSION
CHROMATOGRAPHY (MEC)
MOLECULAR EXCLUSION
CHROMATOGRAPHY (MEC)
HIGH PERFORMANCE LIQUID CHROMATOGRAPHY (HPLC)
HIGH PERFORMANCE LIQUID CHROMATOGRAPHY (HPLC)
HIGH PERFORMANCE LIQUID CHROMATOGRAPHY (HPLC)
HIGH PERFORMANCE LIQUID CHROMATOGRAPHY (HPLC)
HIGH PERFORMANCE LIQUID CHROMATOGRAPHY (HPLC)
QUESTIONS ?
Tha
nk
You
