pharmacognosy- 1 phg 222 - psau · mikhail tswett, russian, 1872-1919 botanist in 1906 tswett used...
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Prof. Dr. Amani S. Awaad
Professor of PharmacognosyPharmacognosy Department,
College of Pharmacy Salman Bin Abdulaziz
University,
Al-Kharj. KSA.
Email: amaniawaad@hotmail.com
Pharmacognosy- 1
PHG 222
1- what is Chromatograph
2- classification of chromatography
3- State the definition of TLC
4-Explain the phases used in TLC
5-List the materials & methods used in TLC
6-List the application of TLC
Mikhail Tswett, Russian, 1872-1919
Botanist
In 1906 Tswett used to chromatography to separate plant pigments
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 grapheinmeans to “write”
History
Chromatography
Chromatography has application in every branch of the physical and biological sciences
12 Nobel prizes were awarded between 1937 and 1972 alone for work in which chromatography played a vital role
Chromatography
Chromatography is a physical method of separation in which the components to be separated are distributed between two phases
one of which is stationary (stationary phase) while the other (the mobile phase) moves through it in a definite direction.
The chromatographic process occurs due to differences in the distribution constant of the individual sample components.
Chromatography
IUPAC definition of chromatography
Chromatography
Is a technique used to separate and identify the components of a mixture.
Works by allowing the molecules present in the
mixture to distribute themselves between a
stationary and a mobile medium.
Molecules that spend most of their time in the mobile
phase are carried along faster.
Other definitions
• Use – to separate and identify components of mixtures.
• All use the principle of “partition” - affinity between two phases, to separate mixtures of substances.
• Substances will move with the mobile phase at different rate depending upon their Partition or Distribution coefficient.
• Compounds with greatest affinity for mobile phase travel further.
• The fraction with greater affinity to stationary phase travels slower and shorter while that with less affinity travels faster and longer.
• The separation is based on Differential partitioning between the mobile and stationary phases
The general principle
Chromatography
Components:
Chromatography
supporting medium: a solid surface on
which the stationary phase is bound or
coated
stationary phase: a layer or coating on the
supporting medium that interacts with the
analytes (a solid or a liquid)
mobile phase: a solvent that flows
through the supporting medium (a gas or a
liquid)
Chromatography cont.…
Classification
Different methods
were attempted for
classification of
chromatography
Chromatography cont.…
Classification I-Classification according the techniques :
In this method they classified it according to the methods for
holding the stationary phase
A- Planar chromatography
In this type of chromatography the stationary phase is used in
the form of layer. Plane chromatography is further classified
into:
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.
B- Column chromatography (CC)
The stationary phase is held in to a tube made of glass or metal
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.
3- Column chromatography (CC): stationary phase is packed in a glass column.
Chromatography cont.…
Classification
II-Classification according to the packing of the stationary phase:
Chromatography cont.…
Classification
III-Classification according to mechanism of separation
The mechanism of separation depends
mainly on the nature of the stationary
phase. Based on separation mechanisms
chromatography can be classified into:
1- Adsorption Chromatography
It is the oldest and most common type of
chromatography. The stationary phase is a solid
with adsorption power. Mixture components will
be adsorbed on the surface of the stationary
phase with different powers and that account for
separation. Silica gel is the most common
stationary phase in adsorption chromatography.
Chromatography cont.…
Classification
III-Classification according to mechanism of separation
2- Partition Chromatography:
The stationary phase is a liquidforming a thin film on an inert solidacts as support. The stationary liquidis usually more polar than the mobileliquid. The two liquids must beimmiscible with each other.
Cellulose powder and wet silica gel areexamples of supports in partitionchromatography that carry film ofwater act as stationary phase. Partitionchromatography is preferable overadsorption when dealing with polarcompounds.
Chromatography cont.…
Classification III-Classification according to mechanism of separation
3- Ion Exchange Chromatography:
It is used for separation of charged molecules.
The stationary phase is an ion exchange resinto which a cationic or anionic groups arecovalently bonded.
Ions of opposite charges (counter ions) in themobile phase will be attracted to the resin andcompete with the components of the mixturefor the charged group on the resin.
Both the mixture components and the mobilephase must be changed.
Mixture of Alkaloids (compounds with positivecharges) can be separated on anionicexchanger, while mixture of organic acids(negative charges) can be separated usingcationic exchanger. Both types are used fordesalination of water.
COO- NH4
+ Resin Resin COO- NH4
+
Counter counter ions ions Anionic exchanger Cationic exchanger
H+
H+
OH-
OH-
Chromatography cont.…
Classification III-Classification according to mechanism of separation
4- Molecular Exclusion ( Size Exclusion )Chromatography:
is a chromatographic method in which molecules in
solution are separated by their size, and in some
cases molecular weight. It is usually applied to
large molecules or macromolecular complexes
such as proteins and industrial polymers.
Typically, when an aqueous solution is used to
transport the sample through the column, the
technique is known as gel-filtration
chromatography,
when an organic solvent is used as a mobile phase
gel permeation chromatography,
Sephadex is an example for stationary phase
Chromatography cont.…
Classification III-Classification according to mechanism of separation
5- Zone Electrophoresis:
In CE methods, analytes migrate
through electrolyte solutions
under the influence of an electric
field.
Analytes can be separated
according to ionic mobility,
additionally they may be
concentrated by means of
gradients in conductivity and pH
Sample application
Bands of negativey
charged compoundsBands of postivey
charged compounds
Chromatography cont.…
Classification III-Classification according to mechanism of separation
6- Affinity Chromatography:
It uses the affinity of
proteins to specific ligands
such as enzymes. The
ligand is attached to
suitable polysaccharide
polymer such as cellulose
- agarose – dextran
7- Chiral Chromatography
In this type we can separate
enantiomers – we used chiral
stationary phase that react with
one enantiomer more then the
other so separation takes place
Chromatography cont.…
Classification IV-Classification according to Mobile phase
1- Liquid Chromatography (LC):
The mobile phase is liquid. In case of
separation by adsorption the stationary phase is
solid so it is called: Liquid-Solid
Chromatography (LSC).
If separation occurs through partition the
stationary phase is liquid so it is called: Liquid
-Liquid Chromatography (LLC).
2- Gas Chromatography (GC)
Where the mobile phase is inert gas nitrogen or
helium. Again if the stationary phase is solid it is
called: Gas–Solid Chromatography (GSC). When
stationary phase is liquid it is called: Gas-Liquid
Chromatography (GLC).
In this regard chromatography is classified into:
Chromatography cont.…
Classification V- According To Purpose Of Use:
1- Analytical Chromatography:
Chromatography can be used for analytical work and also to
obtain pure materials from mixtures
a- Qualitative Chromatography
In this case Chromatography can be used to:
1- Confirm the absence or probable presence of certain
constituent in the sample under investigation
2- Give an idea about the complexity of the mixture and
the least number of compounds present.
3- Check purity and identity of any compound.
4- Establish a (finger print ) pattern for extracts , volatile
oils or pharmaceutical preparations. These finger prints
can be then used to check the identity and purity in the
future.
5- Monitor both column chromatography and organic
chemical reactions.
Chromatography cont.…
Classification V- According To Purpose Of Use:
1- Analytical Chromatography:
Chromatography can be used for analytical work and also to obtain pure materials
from mixtures
b- Quantities Chromatography
The development of modern instruments enable the
use of chromatography to determine the amount of
any component in a mixture as absolute amount or
relative to another component HPLC/ GC/ HPTLC
can be used for there applications.
2- Preparative application:
This was the first and is the main application of
chromatography. The technique was developed
primarily for this purpose. Chromatography is
used to obtain reasonable quantities of pure
compounds from mixtures.
Chromatography cont.…
A- Planar chromatography
Plane chromatography
includes two types:
1- Thin layer
chromatography (TLC)
2- Paper chromatography
(PC).
Chromatography cont.…
Thin layer chromatography (TLC)
TLC is generally used as a qualitative analytical
technique, such as checking the purity of a compound
or determining the number of components in a
mixture or column chromatographic fraction.
In addition.
TLC is useful for determining the best solvents for a
column chromatographic separation.
It can also be used for an initial check on the identity
of an unknown sample (by spotting the plate with a
known compound as well as with the sample).
With calibration, TLC can be used as a quantitative
technique. Preparative work can be carried out with
special thick-layered TLC plat
Chromatography cont.…
Thin layer chromatography (TLC)
Separations in TLC involve distributing a mixture of two or more substances between a stationary phase and a mobile phase.
The stationary phase:
is a thin layer of adsorbent (usually silica gel or alumina) coated on a plate.
The mobile phase:
is a developing liquid which travels up the stationary phase, carrying the samples with it.
Components of the samples will separate on the stationary phase according to
how much they adsorb on the stationary phase versus how much they dissolve in the mobile phase.
Chromatography cont.…
Thin layer chromatography Cont…
Types supportive surfaces
TLC plate is a sheet of glass, metal(Aluminum), or plastic
which is coated with a thin layer of a solid adsorbent
Coating materials
1-Adsorption:a- silica gel (silicic acid). b- Alumina (Aluminum oxide).
c- Magnesium Silicate (florisil) for Lipids.
2-Partition: Cellulose.
3-Ion Exchange TLC: Cellulose phosphate
4-Reversed – phase partition: a) C-18 silica gel b) C-8 silica gel c) C-4 silica gel
5- Polyamides: E-poly caprolactam &Poly acrylonitrite
6- Gel chromatography (Size exclusion):
Sephadex G25, Sephadex G50 , Sephadex G75, Sephadex G100, Sephadex LH20
Chromatography cont.…
Thin layer chromatography Cont…
Binders:These are materials used to hold the thin layer of
the coating material into the surface of the
supporting plates.
Types of binders:a- CaSO4 (Plaster of Paris) Gypsum (10-15%)
b- Silicon dioxide
c- Starch (1-3 %)
d- Organic polymers e.g. polyvinyl alcohol.
Indicators:These are materials mixed
with the coating material and binder to
help locating the spots on the TLC. The
most common used indicator is the
fluorescent materials (silica gel 60 F254).
Chromatography cont.…
Thin layer chromatography Cont…Developing system:
(Mobile phase – developing solvent)
Using a single solvent (very rare) or mixture of solvents to allow the separation. The type of adsorbent used will affect the choice of the developing system.
Adsorption: Usually mixture of non polar organic solvents are used.
Partition: More polar organic solvents such as butanol- acetic acid –water are used. Buffer solution are also used in partition chromatography.
Ion Exchange: Acidic or basic solutions are used.(HCl, NaOH, NaCl, LiCl)
Reversed phase: Methanol- acetonitril- water- acetone-acetic acid are used as mixtures.
Polyamide: Mixtures of Water – ethanol- acetone can be used.
Gel: Buffer solutions and aqueous acidic or basic solutions can be used
Chromatography cont.…
Thin layer chromatography Cont…Types of developments:
1- Single development: The solvent system is allowed to
move through the stationary phase one time only against
gravity.
2- Repeated developments:a- Multiple developments: The plated are developed more
than one time using the same solvent system. The plates must
be completely dried after each development.
b- Stepwise developments: The plated are developed more
than one time using different solvent systems.
3- Two-dimensional developmentIs used to verify if a given spot on TLC using the above
methods of development (one Dimensional) is one pure
compound or mixture of two closely related compounds. The
spots are applied to one corner and the plate developed as
usual. The plate is then rotated 90 ˚C and then developed
again. This method allow better separation of related
compounds.
A- Ascending:
Chromatography cont.…
Thin layer chromatography Cont…Types of developments:
This method of
development require the use of
Chromatotron. Simply it is
composed of motor rotate in high
speed (about 1000 rpm) to
accelerate the speed of the mobile
phase. Circular plates are used and
the mixture is applied to the center
of the plate. Mobile phase is also
allowed to flow from the plate
center to the edges. The separated
materials will appear as concentric
zones. Chromatotron is used only
for preparative work.
B- Centrifugal (chromatotron):
Chromatography cont.…
Preparing the Chamber
To a jar with a tight-fitting lid add enough of the appropriate developing liquid so that it is 0.5 to 1 cm deep in the bottom of the jar.
Close the jar tightly, and let it stand for about 30 minutes so that the atmosphere in the jar becomes saturated with solvent.
Thin layer chromatography Cont.…
Chromatography cont.…
Thin layer chromatography Cont.…
Preparing the Plates for Development
With a pencil, etch two small notches into the adsorbent about 2 cm from the bottom of the plate.
The notches should be on the edges of the plate, and each notch should be the same distance up from the bottom of the plate.
The notches must be farther from the bottom of the plate than the depth of the solvent in the jar.
Using a drawn-out capillary tube, spot the samples on the plate so that they line up with the notches you etched.
Chromatography cont.…
Thin layer chromatography Cont.…
Developing the Plates
After preparing the development chamber and spotting the samples, the plates are ready for development.
Be careful to handle the plates only by their edges, and try to leave the development chamber uncovered for as little time as possible.
When the plates are removed from the chamber, quickly trace the solvent front (the highest solvent level on the plate) with a pencil.
Chromatography cont.…
Thin layer chromatography Cont.…
Identifying the Spots (visualization)
If the spots can be seen, outline them with a pencil.
If no spots are obvious, the most common visualization technique is to hold the plate under a UV lamp.
Many organic compounds can be seen using this technique, and many commercially made platesoften contain a substance which aids in the visualization of compounds.
Chromatography cont.…
Thin layer chromatography Cont.…
Identifying the Spots (visualization)
In these methods the materials can be recovered.
• Day light for colour compounds.
• UV light for fluorescentcompounds (conjugated doublebonds).
• I2 vapour for any compoundscontain at least one double bond
• Spray with water where organiccompounds appear as whiteopaque spots.
1- Physical methods (Non –
Destructive methods)
Chromatography cont.…
Thin layer chromatography Cont.…
Identifying the Spots (visualization)
The use of color-forming reactions is another
valuable technique for visualizing spots.
A very specific procedure involves spraying the
plate with a reagent that will react with a
compound on the plate to form a colored
product. For example, an acid-base indicator,
like phenolphthalein, can be used to identify
acidic or basic compounds.
Other specialized sprays contain ferric chloride (to
identify phenols), 2,4-dinitrophenylhydrazine,
DNP (to identify aldehydes and ketones),
ninhydrin (to identify amino acids), and
dragendorff’s reagent (to identify alkaloids).
1-Chemical methods Destructive methods
Chromatography cont.…
Thin layer chromatography Cont.…
Application of TLC
1- Qualitative:
Identification through comparison of the Rf value with that of Reference material.
Determination of Complexity of mixtures. That will be indicated from number of spots.
Determination the purity of materials.
Monitoring the progress of Chemical reactions.
Monitoring of column chromatography.
Development of finger print TLC for extracts, volatile oils or pharmaceutical preparation for future identification and comparison.
In this application plates 5×5, 5×10 cm with thin film of coating material are usually used.
Chromatography cont.…
Thin layer chromatography Cont.…Application of TLC
2- Quantitative:In this case an accurate volume of samples are
applied using syringes. The dimensions of plates
range from 5x10 to 20x20 according to the
number pf spots used.
The plates are developed as usual in the
chromatographic tanks. After development the
concentration of material can be determined by:
Spot area measurement: Which is directly
proportional to the conc. of materials.
Photodensitometry: Measure transmittance,
reflection or fluorescence of spots.
Radioactivity: For radioactive material.
These measurements are done using TLC Scanner
connected to computer that perform all
calculations.
Chromatography cont.…
Thin layer chromatography Cont.…Application of TLC
3- Preparative TLC:
In preparative application
20×20 plates with thick layer of
adsorbent 0,25m are used. The mixture
is apply as bands and a pilot or guide
spots may be used in one side of the
plate to enable the detection of the spots
location.
Chromatography cont.…
Thin layer chromatography Cont.…Interpreting the Data
The Rf (retention factor) value for each spot should be calculated.
It is characteristic for any given compound on the same stationary phase using the same mobile phase for development of the plates.
Hence, known Rf values can be compared to those of unknown substances to aid in their identifications.
Rf = distance moved by substance
distance moved by solvent front
For substances that are very soluble in the
liquid Rf will be close to … 1
For substances that are rather insoluble in
the liquid Rf will be close to…. 0
• The distance that the spot of a particular compound moves up the plate relative to the distance moved by the solvent front is called the retention factor, or Rf value.
Chromatography cont.…
Thin layer chromatography Cont.…
Rf values often depend on the temperature and the solvent used in the TLC experiment.
the most effective way to identify a compound is to spot known substances – authentic - next to unknown substances on the same plate.)
In addition, the purity of a sample may be estimated from the chromatogram.
An impure sample will often develop as two or more spots, while a pure sample will show only one spot
Important note
In some cases instead of getting roundspots a Tailed or comet like spots areobtained leading to overlapping of the spotsand poor resolution.
Chromatography cont.…
Thin layer chromatography Cont.…
Reasons and solution for tailing problem:
1-Ionic characters of acids and bases when they
are chromatographed under neutral conditions.
Solution: add acids or bases to the developing
system.
2-Application of large amounts of material.
Solution: decrease conc. of material.
3-Unproper choice of solvent system.
Solution: change the solvent system.
Tailing in TLC:
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