Principle and DefinitionPrinciple

• The principle of chromatographic separation is very simple.

The process is achieved by distributing the substances to

be separated between a mobile phase and a stationary

phase.

• Those substances distributed preferentially in the moving

(mobile) phase pass through the chromatographic system

faster than those that are distributed preferentially in the

stationary phase.

Theoretical basis of chromatography

These theoretical concepts are applicable to all types of

chromatographic techniques.

I- Distribution equilibria and rate of travel

• During chromatography a given solute finds itself either in the

stationary phase, which acts as a "retarder" or in the mobile

phase, which acts as a "carrier".

• This distribution is based on distribution equilibria and is

expressed by the rate of travel.

1-Distribution equilibria

The distribution of solutes between the two phases is governed

by an equilibrium constant known as the distribution coefficient,

K (or partition coefficient in certain types of chromatography).

This allows quantifying the distribution of a compound between

the stationary and mobile phases:

K = Cstationary / Cmobile

C stationary = concentration of solute in the stationary

phase

Cmobile = concentration of solute in the mobile phase

2-Rate of travel Factors limiting the rate of travel: The rate of travel of a solute in a chromatographic system is limited

by:• The velocity of the mobile phase (or carrier), that is the same for

all components.• The ratio of volume of the stationary phase to the volume of the

mobile phase, that is also the same for all components.• The value of the distribution coefficient that is characteristic for

each component.

Classification of chromatographic techniques

Different systems of classification may be adopted relying on

criteria such as the separation mechanisms, the development

procedure, the method of holding the stationary phase.

i-According to the mechanism responsible for differential

migration:

The components will migrate through the system at different

rates based on competition between the stationary and mobile

phase for the solute molecules. The major types are:

1-Adsorption Chromatography

2-Partition Chromatography

3-Ion Exchange Chromatography (IEC)

4-Molecular Exclusion Chromatography ( Gel permeation or Gel

filtration)

5-Affinity Chromatography

Adsorption Column ChromatographyStationary phaseSelection of the adsorbent The ideal adsorbent should conform to the following

specifications: • It should be insoluble in the solvent used as mobile phase.• It should be inert to the adsorptives (solutes), unless

otherwise required.• It should be colorless, especially when zones which contain

colored substances are to be located visually.• It should have a suitable particle size with great surface area

to allow more efficient adsorption; but, not too fine to avoid slowing of the rate of percolation.

Types of adsorbents and applications of each type.

Mobile phase Selection of the solvent The choice of the suitable solvent (s) depends mainly on

the elution power of the solvent and the relative adsorption of the class of compounds, which are to be separated.

• Strong eluents (more polar solvents) decrease adsorption, while weak eluents (less polar) increase it.

• Elution of the column can, therefore, be carried out with solvents of increasing polarity. A typical series is as follows:

• hexane < cyclohexane < benzene < chloroform < diethylether < ethyl acetate < acetone < ethanol < methanol < pure water.

Types of solvents as mobile phases in adsorption chromatography.

Factors affecting column efficiency and chromatographic

separation1- Particle size of the supporting medium: As the particle size of adsorbent decreases, separation is

improved. However, an adsorbent of very small particle size will offer considerable resistance to flow.

2-Column dimensions: The column efficiency is improved as the length / width ratio of the column is increased.3-Uniformity of packing of the column: If the column is not packed uniformly, uneven and irregular

movement of the solvent front and less uniform zone formation will result.

4-Column temperature: As temperature is increased, the elution speeds up as the

adsorption is generally reduced at higher temperatures.

Packing of the column

This is carried by either the wet or dry packing techniques.

Wet packing:

• A suspension (slurry) of the adsorbent, preferably the first

solvent to be used in the separation, is prepared and gradually

added to the column. The packing is allowed to settle between

additions.

• The suspending liquid is allowed to flow out slowly but the

liquid level in the column is maintained above the packing at all

times.

Wet packing is most commonly used and results in more

homogenous columns.

Dry packing:

• In this method, the solid stationary phase is added in

portions with vibrations between additions. The process is

repeated until the column is adequately filled.

• The column is then washed carefully with the first solvent

• Sample application• Development• Elution• Detection.

Applications

Adsorption Column Chromatography has been used for:

• Separation of alkaloids of Cinchona, ergot, opium and nux

vomica, and cardiac glycosides from Digitalis and

anthraquinones from senna species.

• Isolation and purification of vitamins and hormones.

• Examination of vegetable oil and pharmaceutical

preparations.

• Purification of tincture of alkaloids from pigments before

determination of their alkaloidal content.

Partition Chromatography

The separation of the components of a mixture is, as in counter-

current extraction, dependent on differences in the partition

coefficients of the components between an aqueous and an

immiscible organic liquid.

The liquid stationary phase is adsorbed on an inert support, which

may be either packed in a chromatographic tube (Column Partition

Chromatography) or layered on a glass plate (TLC) or in the form of

sheets of paper (PC).

Paper Chromatography

The technique was introduced as a method of partition chromatography for the analysis of amino acid mixtures and has extended to all classes of natural products. It remains the method of choice for the fractionation of some groups of substances, e.g. flavonoids

Principle and mode of action• It is a type of planar chromatography in which the stationary

phase is a specified type of paper. • The paper is formed of cellulose, which has a great affinity for

water and other polar solvents and holds them through formation of hydrogen bonds thus forming a polar stationary phase. Distribution of the solutes between the two phases is therefore mainly by partition.

Criteria for ideal PC separation

Efficient separation is achieved by:

1-Using analytical grade chemicals and distilled water for

preparation of the mobile phase (solvent-system).

2-Maintaining the composition of the mobile phase

constant during development by using a sufficient

amount of solvent and carrying the process in a well-

closed chamber (chromatographic jar).

3-Maintaining a suitable and constant temperature through out

the process.

4-Selecting the suitable solvent in which the components of the

analyzed sample have a low but definite solubility. Highly

soluble substances will appear at or very close to the solvent

"front". Substances of very low solubility will remain near the

point of application or " start line".

Procedure

Steps followed to perform paper chromatography are:

1-Sample desalting

• The presence of inorganic salts generally causes streaking

(tailing, trailing), discoloration and other distortions on the

finished chromatogram.

• The removal of these salts can be achieved by ion exchange or

precipitation.

2-Sample application (Loading)

• Spots are applied with micropipettes, at a suitable distance

from each other, on the start line. They are kept as small as

possible (less than 5 mm in diameter).

• In case of repeated applications, spots should be completely

dried after each application.

• When large quantities of material are to be chromatographed,

the sample is applied as a streak (e.g. in preparative paper

chromatography (PPC) and quantitative chromatography).

3-Development of the chromatogram

Several techniques may be adopted. These include the

usual (or general) techniques such as the ascending,

descending, radial and horizontal methods. Special

techniques have also been devised in order to

improve resolution, examples are the stepwise and

multiple development, the continuous development

and the two-dimensional development.

4-Drying of the chromatogram

After development, the paper is withdrawn from the jar. The

front of the solvent is marked immediately with a pencil and

dried by hanging in open air at room temperature or by a

current of cold or hot air (using a hair dryer).

5-Spot detection (location or visualization)

Several methods are used for detection of the separated spots

corresponding to the components of the analyzed sample.

They include:

Thin Layer Chromatography (TLC)

• This is simple and fast and has now achieved a remarkable

success in the separation of mixtures of all classes of natural

products.

• Thin layer chromatograms often serve to identify drugs, plant

extracts and biochemical preparations, or to detect

contaminants or adulterants.

Analogy with Paper Chromatography• Such as paper chromatography TLC is a type of planar

chromatography. • Instead of paper (in PC) a thin layer of finely divided adsorbent

supported on a glass, plastic or aluminum sheet is used.Analogy with Column Chromatography• TLC is also closely related to column chromatography and has

often been called "Open-column chromatography". Both techniques are based on the same mechanisms of

chromatographic separation: adsorption and partition. • The major difference lies in the location of the stationary phase.

In TLC, it is a thin layer spread on a glass plate, and in column chromatography, it is a column held in a glass tube.

Advantages of TLC over PC

• The method is more versatile, it allows the use of wide range of

stationary phases and solvents.

• Fractionation is performed more rapidly, with smaller quantities of

the mixture and is more reproducible.

• The separated spots are usually more compact and more clearly

demarcated from one another.

• Concentrated sulfuric acid and other drastic reagents can be used

for location of separated substance but not for the paper.

Applications

• Like with paper chromatography, TLC can be extended to,

quantitative evaluation and to work that involves radioactive

substances.

• Quantitative analysis can be performed by measuring the size of

the spots with a planimeter or measuring the intensity of the

color by photodensitometry. An alternative method is to scrap

the bands and elute the components with the suitable solvent

followed by colorimetric, spectrophotometric, fluorimetric,

gravimetric or volumetric methods.