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Chromatographic analysis
» Principles and theory.
» Definition.
» Mechanism.
» Types of chromatography.
» Uses of Chromatography.
Outlines of Lecture
• In 1906 Mikhail Tswett used to chromatography to
separate plant pigments as chlorophylls, xanthophylls
and carotenoids by using calcium carbonate as
adsorbent and petrol ether/ethanol mixtures as eluent.
Mikhail TswettRussian Botanist (1872-1919)
•He called the new technique
chromatography because the
result of the analysis was
'written in color' along the
length of the adsorbent
column
•Chroma means “color” and
graphein means to “write”
•Definition:
• Is a technique for separating mixtures into their
components in order to analyze, identify, purify,
and/or quantify the mixture or components, by
distribution of its components between a mobile and
stationary phase over time
Separate
• Analyze
• Identify
• Purify
• QuantifyComponentsMixture
•Concepts:
•Mobile phase (Solvent or Eluent or carrier): The mobile
phase can be a gas (GC), a liquid, or a supercritical fluid
(SCFC), that carries the components.
•Stationary phase (Adsorbent): It is a part that does not
move with the sample
•Concepts:
•The sample (solute): The sample
then has the opportunity to
interact with the stationary
phase as it moves past it.
Samples that interact greatly,
then appear to move more
slowly. Samples that interact
weakly, then appear to move
more quickly. Because of this
difference in rates, the samples
can then be separated into their
components.
•Classification of chromatography according to mobile
phase:
• Liquid chromatography (LC): mobile phase
is a liquid (water or organic solvents).
• Gas chromatography (GC): mobile phase is
a gas.
• Supercritical fluid (SCFC): mobile phase is a
supercritical fluid (CO2(
Types of Chromatography
•Classification according to the packing of
the stationary phase:
1.Thin layer chromatography (TLC): the
stationary phase is a thin layer
supported on glass, plastic or
aluminum plates.
2.Column chromatography (CC):
stationary phase is packed in a glass
column.
3.Paper chromatography (PC): the
stationary phase is a thin film of
liquid supported on an inert support.
Types of Chromatography
•Classification according to the force of separation:
1.Adsorption chromatography:
Types of Chromatography
Separation based on the relative
differences in adsorption of
components to the solid
stationary phase.
Stationary phase is solid: applies
to only solid-liquid or solid-gas
chromatography
For polar non-ionic compounds
Ex; CC, TLC.
•Classification according to the force of separation:
2. Partition chromatography:
Solute are separated based on their partition between a liquid
mobile phase and a liquid stationary phase coated on a solid
support.
Stationary phase and mobile phase are liquid in nature.
Types of Chromatography
Phase 2
Phase 1
Phase 2
Phase 1
Reverse – analyte is polar
organic; stationary phase
LESS polar than the mobile
phase
Ex : PC, HPLC, HPTLC, GC
Normal – analyte is nonpolar organic; stationary phase
MORE polar than the mobile phase
•Classification according to the force of separation:
3. Ion exchange chromatography: Use ionic stationary phase
ions separated on the basis of their tendency to displace counter
ions adsorbed on stationary phase (Depends on charge, hydration,
“solubility”…)
Stationary phase: is a resin or gel matrix consisting of agarose or
cellulose beads with covalently bonded charged functional groups
Mobile phase: buffer, pH and salt concentration-opposite charged
solute ions attracted to the stationary phage by electrostatic force
For ionic compounds: large proteins, small nucleotides and amino
acids
Ex : CC, HPLC
Types of Chromatography
•Classification according to the force of separation:
3. Ion exchange chromatography:
Types of Chromatography
Anionic exchange chromatography:
stationary phase is anion (Q-resin, a
Quaternary amine; and DEAE resin,
DiEthylAminoEthane), used for
cation separation
Cationic exchange chromatography:
stationary phase is cation (S-resin,
sulfate derivatives; and CM resins,
carboxylate derived ions), used for
anion separation
•Classification according to the force of separation:
4. Size Exclusion Chromatography:
Separation is a result of “trapping” of molecules in the
pores of the packing material
Very large molecules can’t get into the pores –
unretained
Types of Chromatography
Very small molecules get
hung up in to pores for a long
time - most retained –
longest retention time
stationary phase is a porous
matrix
Ex: CC, HPLC
Uses of Chromatography
• Chromatography is used by scientists to:
• Analyze: Examine a mixture, its components,
and their relations to one another
• Identify: Determine the identity of a mixture or
components based on known components
• Purify: Separate components in order to isolate
one of interest for further study
• Quantify: Determine the amount of the a
mixture and/or the components present in the
sample
Uses of Chromatography
• Real-life uses of chromatography:
• Pharmaceutical Company: Determine amount of
each chemical found in new product
• Hospital: Detect blood or alcohol levels in a patient’s
blood stream
• Law Enforcement (forensic): To compare a sample
found at a crime scene to samples from suspects
• Environmental Agency: Determine the level of
pollutants in the water supply
• Manufacturing Plant: To purify a chemical needed to
make a product
Thin layer chromatography (TLC)
• Definition of TLC:
•TLC: is a method for identifying substances
and testing the purity of compounds.
•The stationary phase: is a thin layer of
adsorbent (usually silica gel) coated on a
plate (glass, metal, or plastic).
•The mobile phase: is a developing liquid
(A solvent of varying polarity), which
travels up the stationary phase, carrying
the samples with it.
Thin layer chromatography (TLC)
• Interpreting the Data:
• If the spots can be seen, outline them
with a pencil.
• If no spots are obvious, use a UV lamp.
• If no spots are obvious with UV, use
iodine solution or Spray plate with
visualizing agents: Alkaloids:
Dragendorff’s reagent, Cardiac
glycosides: Antimony trichloride, Sugar:
Aniline phthalate, Amino acids:
Ninhydrin, flavonoids: Aluminium
chloride
Thin layer chromatography (TLC)
• Interpreting the Data:
•Once visible, the Rf (relative
flow OR retention factor)
value of each spot can be
determined.
•The Rf is defined as the
distance from the center of
the spot moved divided by the
distance the solvent front
moved (both measured from
the origin)• Rf value is constant for a particular compound, solvent
system and insoluble matrix.
Thin layer chromatography (TLC)
• Advantages of TLC:
• Low cost
• Short analysis time
• Ease of sample preparation
• All spots can be visualized
• Uses small quantities of solvents
• Wide choice of materials as sorbents
Paper chromatography (PC)
• Definition of PC:
•PC: A method of partition chromatography using filter
paper as carrier or inert support.
•The factor governing separation of mixtures of solutes
on filter paper is the partition between two immiscible
phases.
•Stationary phase: usually water adsorbed on cellulose
fibers in the paper (bound water).
•Mobile phase: is the organic solvent flows past the
sample on the paper.
Paper chromatography (PC)
• Principles of PC:
1. Compound is placed on
stationary phase
2. Mobile phase passes through the
stationary phase
3. Mobile phase solubilizes the
components and carries the
individual components a certain
distance through the stationary
phase, depending on their
attraction to both of the phases
Paper chromatography (PC)
• Types of PC:1. Mono-dimension PC: Ascending chromatography: The
solvent travel in upward directionon the paper.
Descending chromatography: Thesolvent travel in downwarddirection on the paper. Note: The solvent reservoir is at the top,
The movement of solvent is assisted bygravity besides capillary action.
Radial chromatography: Thesolvent travels from centertowards periphery of paper
Paper chromatography (PC)
• Types of PC:
2. Two dimensional chromatography
When large numbers of substances are to be
separated on a single chromatogram.
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 are used
in each direction.
It is essential that the first solvent be
completely volatile
Paper chromatography (PC)
• Interpreting the Data:
•Spots in paper chromatograms can
be detected in 4 different ways: 1.
By their natural color. 2. By their
fluorescence. 3. By their chemical
reactions. 4. By radioactivity.
•The spots are usually identified by
comparing of standards of known
Rf values.
•By comparing the Rf values of components of a mixture
with the Rf values of known substances under identical
conditions, the compounds present in a mixture can be
identified
Column chromatography (CC)
• CC is an extremely valuable technique for
purification of synthetic or natural products.
The same mechanism as TLC.
• A variety of adsorbents can be used as the
stationary phase (Organic: cellulose,
polyamide, polyethylene or Inorganic: silica
gel, aluminum oxide and magnesium silicate);
silica gel (which is very polar) is most
commonly used in organic chemistry.
• The mobile phase is a moving liquid or gas.
• There are two techniques based on column
chromatography, high performance liquid
chromatography (HPLC) and gas
chromatography (GC).
Gas chromatography (GC)
• This is the most sensitive chromatographic technique
• It is capable of detecting as little as 10-12g of a
compound
• It is limited to compounds that can be easily
vaporised without decomposing.
Gas chromatography (GC)
• Gas chromatography has the following features:
The mobile phase is a gas, usually nitrogen, called
the carrier gas
A small amount of sample is injected into the top of
the column through an injection port
The injection port is heated to a temperature
sufficient to instantly vaporise the sample, which is
then swept into the column by the carrier gas.
The column is in a loop, this is because of the fast
moving gaseous phase. So the column must be
longer than in HPLC to allow for effective interaction
with the stationary phase
The column is mounted in an oven and heated
Gas chromatography (GC)
• Interpreting Chromatograms:
The time a component takes to pass through the
column is called the retention time, Rt.
The same compound will give the same retention
time if the conditions (temp, mobile phase,
stationary phase, flow rate, pressure etc) remain the
same.
Each component forms one peak, however it is
possible for a number of peaks to coincide and be
indistinguishable.
Gas chromatography (GC)
Figure. Chromatogram of a reference sample containing a mixture
of butane, 2-methylbutane, hexane, benzene and 2-methylhexane.
Figure . Gas chromatogram of a petrol sample.
High performance liquid chromatography (HPLC)
• This method is used for pharmaceutical and industrial
analysis.
• It allows extremely sensitive analysis of a wide range
of compounds.
High performance liquid chromatography (HPLC)
• Advantages of HPLC:
High separation capacity, enabling the batch
analysis of multiple components
Superior quantitative capability and reproducibility
Moderate analytical conditions
Unlike GC, the sample does not need to be
vaporized.
Generally high sensitivity
Low sample consumption
Easy preparative separation and purification of
samples
High performance liquid chromatography (HPLC)
• What is HPLC used for?
Food products: Vitamins, food additives, sugars,
organic acids, amino acids, etc.
Environmental samples: Inorganic ions, Hazardous
organic substances, etc.
Organic industrial products: Synthetic polymers,
additives, surfactants, etc.
Biogenic substances: Sugars, lipids, nucleic acids,
amino acids, proteins, peptides, steroids, amines,
etc.
Medical products: Drugs, antibiotics, etc.