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)