CHROMATOGRAPHY
CHROMATOGRAPHY
INTRODUCTION
Modern pharmaceutical formulations are complex mixtures including in addition to one or more medicinally active ingredients, a number of inert materials such as diluents, disintegrants, colors and flavors. To ensure quality and stability of the final product, the pharmaceutical analyst must be able to separate these mixtures into individual components prior to quantitative analysis.
Moreover, comparison of the relative efficacy of different dosage forms of the same drug entity requires the analysis of the active ingredient in biological matrices such as blood urine and tisssue.
CHROMATOGRAPHY
Defined as the process in which a solution of a mixture containing inert
materials, drug principles and
impurities is separated into its
components while moving through a
bed of fixed porous solid having
different affinities for the substance
being separated.
CHROMATOGRAPHY
The separation of substances comes about because each component of the mixture possesses a different mobility by
reason of differences in:
Adsorption
Partition
Solubility
Vapor pressure
Molecular size
Ionic charge
The drug principle so separated on a porous solid may be removed from the solid by means of a flowing solvent (elution)
or by simple solvent extraction and assayed to any suitable
analytical method appropriate for the scientific drug.
PRINCIPAL OBJECTIVES:
1. Resolution of mixtures into constituents parts
2. Determination of homeogeneity
3. Comparison of substance suspected to be
identical.
4. Purification
5. Concentration of substances from dilute
solutions
6. Identification and control of technical products
7. Quantitative separation from complex mixtures
8. Indication of molecular structure
LAWS AND THEORIES GOVERNING
CHROMATOGRAPHY Two theoretical approaches have been developed to describe
processes involve on the passage of solutes through
chromatographic system.
PLATE THEORY
Based on the work of Martin and Synge
Considers chromatographic system as a series of discrete layers of theoretical
plates. At each of these, equilibrium of the
solute between the mobile phase and
stationary phases occurs. The movement
of the solute is considered as a series of
stepwise transfer from plate to plate.
PLATE THEORY Entire length of the
chromatographic column is considered to be composed of many small identical cells called theoretical plates. Each cell contains a pair of immiscible solvents (Ex: ether and water). The lower phase is called the stationary phase and the upper phase the, the mobile phase. A mixture of solutes is introduced into the first cell. Equilibrium is established, and the upper phase of each cell is then transferred to the next cell down the column.
RATE THEORY
Discussed in the book by Giddings, considers the dynamics of solute particles
as it passes through the void spaces
between the stationary phase particles in
the system as well as its kinetics as it
transferred to and from the stationary
phase.
Rf Value Chromatographic system achieves their ability to separate
mixtures of chemicals by selectively retarding the passage
of mixtures of chemicals through the stationary phase while
permitting others to move more freely. Therefore, the
chromatogram may be evaluated qualitatively, by
determining the Rf value, or retardation factor, for each of
the eluted substances.
The Rf is the measure of the fraction of its total elution time that any compound spends in the mobile phase.
Because the solute particle proceeds down the column only when it is in the mobile phase, the Rf value is related
directly to the fraction of the total amount off solute that is
in the mobile phase.
TECHNIQUES OF
CHROMATOGRAPHY Basic principles upon which chromatographic depends.
A. ADSORPTION
CHROMATOGRAPHY
Brings about the separation of a mixture through a competitive process in which
the molecules of the mobile phase
compete with the analyte molecules for
polar adsorption sites on the adsorbent.
The process is known as liquid-solid chromatography (LSC).
B. PARTITION
CHROMATOGRAPHY
The mobile and the stationary phases are liquids and is referred to as the liquid-
liquid chromatography (LLC)
EXAMPLE : Paper Partition
Chromatography
The basic principle of separation in partition chromatography is that the differences in partition coefficients of substances between two immiscible liquids, one of which is a stationary phase supported on a solid adsorbent with the other the mobile phase flowing through it.
If the solid adsorbent is filter paper, the process is referred to as paper partition chromatography.
In this process, the mobile phase, usually an organic solvent moves slowly over the stationary phase, usually water, which is held in place by the fibers of the filter paper. Under such conditions, different substances move over the paper at different rates, depending upon the relative solubilities in the immiscible solvents resulting in separation by partition.
EXAMPLE : Paper Partition
Chromatography
In this case, the Rf value is computed as the ration of the distance traveled over the paper sheets by a given compound to the distance traveled by the front of the mobile phase, from the foint of application to the test substance.
3 METHODS OF PAPER PARTITION
CHROMATOGRAPHY
DESCENDING PAPER PARTITON
CHROMATOGRAPHY
Mobile phase flows downward
Ascending Paper Partition
Chromatography
Mobile phase flows upward
RADIAL PAPER PARTITION
CHROMATOGRAPHY
Mobile phase moves out in concentric
circles from the center
of a circular piece of
paper.
ION EXCHANGE
CHROMATOGRAPHY
Uses either cation-or anion exchange resin that are insoluble in water and as the
name suggests, exchange cations or
anions in solution in the mobile phase
which comes in contact with the active
sites.
ION EXCHANGE
CHROMATOGRAPHY
MOLECULAR EXCLUSION
CHROMATOGRAPHY
Known as GEL FILTRATION or GEL PERMEATION CHROMATOGRAPHY
Separation is based on the differential migration of the solute molecules
according to molecular size.
MOLECULAR EXCLUSION
CHROMATOGRAPHY
AFFINITY CHROMATOGRAPHY
This technique makes use of a specific ligand (such as antibody bound to the inert stationary phase) which has been immobilized by being bound to chemically to an insoluble matrix, to adsorb reversibly a single molecular species (like antigen) from a mixture of solutes. This method differs from other modes of chromatography because rather than attempting to separate a mixture of solutes for qualitative and quantitative analysis, it is concerned with removing a single species from the mixture. It achieves a highly specific purification technique for biological molecules.
AFFINITY CHROMATOGRAPHY
APPLICATION OF DIFFERENT
MODES OF
CHROMATOGRAPHIC
SEPARATION
GAS CHROMATOGRAPHY
If the materials are volatile and stable in
the gas phase.
LIQUID PARTITION or THIN
LAYER CHROMATOGRAPHY
If it is necessary to isolate eluted
compounds in
quantity.
MOLECULAR-SIZE EXCLUSION
CHROMATOGRAPHY
If the substances have a high molecular
weight, such as
proteins, triglycerides
or polymers.
ION-EXCHANGE
CHROMATOGRAPHY
If the compounds are ionized in solution,
such as amino acids.
PAPER-PARTITION or COLUMN
CHROMATOGRAPHY
If the compounds are highly polar or
hydrophilic of
intermediate
molecular weight,
such as sugars.
LIQUID-ADSORPTION
CHROMATOGRAPHY
If the substances are non-ionizable,
hydrophobic, or non-
polar.
AFFINITY CHROMATOGRAPHY
If it needs highly selective isolation of
certain biological
substances such as
antibiotics or
enzymes.
BASED ON THE TECHNIQUES
EMPLOYED IN HOLDING THE
POROUS SOLID:
The procedure is called:
COLUMN CHROMATOGRAPHY
Separation is based on partition ion exchange and molecular exclusion
properties.
PAPER-PARTITION
CHROMATOGRAPHY
Separation is based on the differences in partition coefficients of substances
between two immiscible liquids.
THIN-LAYER
CHROMATOGRAPHY
Involves spotting of a sample of a mixture of components at one end of an
adsorbent-coated glass plate or other
suitable support followed by passage of a
solvent (developer) through the adsorbent
for the purpose of separating the
components of the sample.
INSTRUMENTATION
THIN-LAYER CHROMATOGRAPHY
THIN LAYER CHROMATOGRAPHY
(TLC)
Thin-layer chromatography is a method of analysis in which the stationary phase, a finely divided solid, is spread as a thin layer on a rigid supporting plate; and the mobile phase, a liquid, is allowed to migrate across the surface of the plate.
It differs from the other techniques of chromatographic separation in that the separation does not take placed in a closed column, but rather on a planar surface; and the mobile phase does not flow under the influence of gravity or high pressure, but is drawn across the plate by capillary action.
TECHNIQUE STATIONARY PHASE MOBILE PHASE
ADSORPTION Silica gel
Alumina
Charcoal
Polyamide
Non-polar or polar
organic solvents
PARTITION Cellulose
Silica gel
Polar organics
Mixed aqueous,
organic solvents
REVERSED PHASE
PARTITION
ODS (Octadecylsilyl)
Silica gel
Coated silica gel
Acetylated Cellulose
Mixed aqueous, polar
solvents
ION-EXCHANGE Ion-exchange resin
DEAE
(Diethylaminoethyl)
CM (Carboxymethyl)
cellulose
Buffered aqueous
solutions
SIZE-EXCLUSION Dextran gels Aqueous buffers
THIN LAYER
CHROMATOGRAPHY
The application of thin-layer chromatography to the separation
of bioactive constituents from plants and chemicals as well as for
biological screening may be illustrated in the following concept
map/
Points to consider about the
Stationary Phase used in TLC
To ensure that the stationary phases adheres firmly to the backing plate and
does not flake off during the development,
binders such as calcium sulfate (gypsum),
starch or carboxymethylcellulose are
added to the adsorbent.
Points to consider about mobile
phase used in TLC
The solvent used as a mobile phase in TLC are identical to those used in in liquid
chromatography.
They must be of high purity, because additives such as ethanol in chloroform or
antioxidants in ethers can affect the
separation and must be removed or their
effects determined.
Points to consider about mobile
phase used in TLC
Changes in viscosity of the solvents due to impurities will alter its rate of travel, as the more viscous the solvent, the more slowly it is drawn up to develop the chromatogram rather than a multi component mixture, because solvents are adsorbed preferentially by the stationary phase, and as the mixture moves up the plate, the composition of the mobile phase is always changing.
Points to consider about mobile
phase used in TLC
Compounds that travel a greater distance up the plate, therefore will be exposed to a different mobile phase than those that are retained strongly.
.If a mixture must be used, the components should measure carefully so that the subsequent experiments will be reproducible; solvents also should be volatile so they can be evaporated from plate after the development is completed.
Special Color-producing
Reagents used in TLC For colored components such as dyes, it is not
necessary to spray a special color-producing reagent in order to detect the location of the spot.
Most components, however, are colorless and must therefore treated with a reagent of some kind or examined under UV radiation in order to make the spots discernible.
CONCENTRATED SULFURIC ACID Used in TLC for purpose if detecting organic
components.
Special Color-producing
Reagents used in TLC IODINE VAPOR
The chromatogram is placed in a closed container holding a few iodine crystals.
The organic components react with iodine vapor and form brown spots.
It is interesting to note that in most cases the reaction between iodine and the organic compounds is physical rather than chemical and is
reversible.
When the chromatogram is placed in ordinary laboratory surroundings, the iodine is released and the brown spots disappear.
UV RADIATION
Many organic compounds fluoresce when examined by UV radiation.
Two UV radiation sources which are widely used and commercially available are UV shortwave (254 nm) and long wave (360 nm).
CALCULATION
Distance traveled by solute (mm)
Rf = --------------------------------------
Distance traveled by solvent (mm)