Chromatography

Ahmed Mohammed Hashem

Introduction for Chromatography:
○ Definition of Chromatography:
Technique for separation of mixture solutes. In which separation
achieved by differential movement of individual solutes through
stationary phase under flow of mobile phase.

○ Principle of Chromatography :
1. Process is achieved by distributing individual solutes through
stationary phase and mobile phase.
2. Each individual solute has an affinity towards both stationary
phase and mobile phase :
The more affinity towards stationary phase: The more time to
take before migration.
The more affinity towards mobile phase: The less time to take for
migration.
3. Individual solutes are eluted from column in inverse order of
their distribution coefficients with respect to stationary phase.
○ Theoretical Basis of Chromatography:
• During chromatography individual solutes finds itself either:
In stationary phase: This acts as a retarder.
In mobile phase: This acts as a carrier.
• This distribution based on equilibrium and expressed by rate of
travel:
1. Distribution equilibrium:
 Known as distribution coefficient or partition coefficient in
some cases.
 Can be calculated by the following equation:
K=Cstationary/Cmobile.
 K value would determine relative population at the two
phases i.e.:
1. If large K value means more time spent in stationary
phase.
2. If small K value means more time spent in mobile
phase.
1. Rate of travel:
 Factors limiting Rate of Travel:
1. Velocity of mobile phase, that`s the same for all
components.
2. Ratio of volume of stationary phase to the volume of
mobile phase, that`s the same for all components.
 3. Value of distribution coefficient which is characteristic
for each component.
 Determination of travel rate:
1. Measuring distance after a fixed time as in planar
chromatography.
2. Measuring time after a fixed distance as in columnar
chromatography.
 Rate of travel is not a consistent measure of the relative
affinity of a compound for stationary and mobile phases,
it varies with flow rate, temp. etc….

Classification of Chromatographic methods:

○ Classification Criteria:
1. The separation mechanisms.
2. The development procedures.
3. The method of holding the stationary phase.
4. The purpose of use of the chromatographic techniques.
○ According to the mechanism responsible for differential
migration:
• Relative affinity of individual solutes for stationary phase is
influenced by difference in their physical and chemical
characters, therefore components will migrate through system at
different rates based on competition between the stationary and
mobile phase for solute molecules.
• The major types are:
1. Adsorption Chromatography:
• Mobile phase: may be gas or liquid.
• Stationary phase: solid (to adsorb on the surface via
charged interaction).
2. Partition Chromatography:
• Mobile phase: may be gas or liquid.
• Solid phase: liquid (a thin formed film on surface of inert
solid support).
3. Ion Exchange Chromatography:
• Mobile phase: only liquid.
• Solid phase: solid (as ion exchange resin that attracts
cations and anions the mobile phase applied should be
bounded to the solute with a larger affinity to exclude
the stationary phase).
 4. Affinity Chromatography:
• The most selective type.
• Based on having a certain binder at the stationary
phase that is selective for one of the components of
solute.

○ According to the development procedures:

1. Frontal analysis: used for separation of a single component
of mixture by having 1 selective solvent (using polarity)
2. Elution analysis: A small amount of mixture is introduced to
a column and eluted with mobile phase (which has lower
affinity towards the stationary phase than the solute
mixture)
3. Gradient analysis:
• A modification of elution method where the elution
power is gradually increased by using conc. , pH ,
polarity , ionic strength.
• Reduces tailing and narrow the zones.
4. Displacement analysis: elution is carried by a solvent,
which has greater affinity for the stationary phase than the
solute mixture.
○ According to method of holding the stationary phase:
1. Planar or plane chromatography.
2. Columnar or column chromatography.
○ According to purpose of use:
1. Analytical chromatography: quantitative or qualitative.
2. Preparative chromatography: isolation of sample
components.
 Adsorption Chromatography:
○ Principle:
1. Sample of mixture is applied to the stationary phase (called
sorbent) so a charge interaction occurs between the active gps
of adsorptive and the sorbent.
2. Conc. a t interface between solid stationary phase and mobile
phase is higher than surrounding medium.
3. Small difference in adsorption behavior lead to is utilized to
achieve separation.
4. More strongly adsorbed pigments (xanthophylls, chlorophylls)
are trapped at the top of the column but the less adsorbed
pigments (Carotene) is found at the bottom of column i.e.
eluted first.
5. Strength of adsorption of polar gps on polar support increases
as follow:
Olefins<
ether<esters<ketones<aldehyds<amines<phenol<water
○ Chromatographic techniques based on adsorption:
1. Liquid solid chromatography commonly known as adsorption
column chromatography.(LSC)
2. Gas solid chromatography.(GSC)
○ Chromatographic techniques based on adsorption under
certain conditions:
1. Thin layer chromatography (TLC)
2. Paper chromatography (PC)
3. High performance liquid chromatography (HPLC) also known as
High pressure liquid chromatography:
• Used for small molecules.
• Needs high pressure.
○ Adsorption Column chromatography:
• Methods of column package:
1. Sprinkling Method: solid stationary phase sprinkled on liquid
Wet method mobile phase.
2. Slurry Method: solid stationary phase mixed with liquid
mobile phase in a beaker then solute mixture is added.
3. Dry method: liquid mobile phase is applied directly on solid
stationary phase.
• Stationary phase:
 Selection of stationary phase material should be based
on:
1. Chemically inert i.e. don`t interact with solutes.
2. Insoluble at the mobile phase (solvent).
3. Large surface area but not very fine to avoid
percolation.
4. Colorless especially in dyestuff mixtures.
• Types of Stationary phases:
1. Alumina: widely used in chromatography due to:
✔ Large capacity it has.
✔ Availability.
✔ Insoluble.
✔ Chemically inert.
Prepared by heating Aluminum trihydrate at 700
C° that will give us three products:
* Neutral alumina (pH7-7.5)
*Basic Alumina: treated with NAOH and dist. H2O (pH=10)
Cationic exchanger.
*Acidic Alumina: treated with 2N HCL and dist. H2O (pH=4)
Ionic exchanger
2. Silica, Silica gel, Silica acid
✔ It`s adsorption power obtained from OH gps that
attract unsaturated and polar compounds by H-
bond.
✔ Those OH gps may be : blocked by water that may
reactivated by using heat at 150-250 C° that will
eliminate water without effecting OH.
✔ Widely used at thin layer chromatography.
3. Charcoal.
4. Kieselguhr (Diatomaceous earth).

• Mobile phase:
 Selection of the solvent: depends on:
1. Elution power of the solvent.
2. Relative adsorption of the compounds to be separated
according to their class.
The more the elution power, the more polar is the solvent
and the less adsorption occurs.
 Types of solvents: they`re classified according to their
function in chromatographic process as follow:
1. Solvents: no separation.
✔ Inert towards stationary phase and solute mixture.
✔ Bring the mixture to be separated on the column.
✔ Mobile phase with low polarity than solute
mixture.
✔ Used for dissolving the solute mixture.
2. Developers: no separation.
✔ Stronger than solvents but less adsorbed on
stationary phase.
✔ Leads to movement of zones in the chromatogram
by competition with the solute mixture for
stationary phase.
✔ Called chromatographic solvents and wash liquids.
✔ Used for forming zones of components.
3. Eluents:
✔ Better eluting power than developers and
removes all adsorptive outside column.
✔ Less adsorbed than the adsorptive.
✔ Used for eluting the mixture components.
4. Displacers:
✔ Strong mobile phases.
✔ More adsorbed than the adsorptive causing
displacement from the adsorbent to the mobile
phase.
✔ Used for eluting most absorbed components.

:
 ○ Factors effecting column efficiency and chromatographic
separation
1. Particle size of supporting medium: when particle size
decreases the separation is improved but fine particles will
offer considerable resistant to flow.
2. Column dimensions: when length\wide ratio increases the
efficiency increases.
3. Uniformity or packing column: will affect uniformity of zones.
4. Column Temperature: adsorption decreases leading to
speeding up the elution.
5. Solvent flow rate: when small particle size obtained that leads
to a uniformed & low flow rate giving a neat separation and
uniformed zones.
6. Constancy of flow: should be continuous.
7. Adsorbent activity: to control retention volumes.
8. Solvents selection: low viscosity solvents will give high
efficiency separation.
9. Effect of conc.: High conc. leads to fast elution.
○ Applications of adsorption chromatography:
1. Separation: of alkaloids of cinchona, ergot and nux vomica. And
cardiac glycosides from digitalis and anthraquinones from senna.
2. Isolation and purification: of vitamins and hormones.
3. Examination: of vegetable oil and pharmaceutical preparation.
4. Purification: of tincture of alkaloids from pigments before
determination of alkaloid content.
Paper chromatography
○ Identification:
1. It`s a planar chromatography used for separating amino acid mixtures and
flavonoids.
2. Stationary phase is a special type of paper made of cellulose.
3. Tested material is applied at the end of the paper.
4. Mobile phase is allowed to flow through chromatogram.
○ Principle:
1. Paper has great affinity towards water and polar solvents and holds them via H-
bond, so it forms polar stationary phase.
2. Separation of sample mixture depends on difference in partition coefficients
between aqueous stationary phase and mobile organic phase.
○ Criteria for ideal PC:
1. Using analytical grade chemicals and dist. Water for preparing mobile phase.
2. Keeping mobile phase composition constant by: a) Using sufficient amount of solvent.
b) Using well closed chamber.
3. Maintaining suitable and constant temperature through the time.
4. Selecting suitable solvent of a low but definite solubility because:
a) ↑ soluble substances will appear very close to solvent “front”.
b) ↓ soluble substances will remain at point of application.
○ Stationary phase: paper solid support.
• Specifications:
1. Highly purified cellulose.
2. ↑ pores sizes leads to ↑ rate of rise.
3. Some of OH groups of glucose may be oxidized during manufacture to
aldehyds, ketones or carboxyl functional groups.
4. Traces of impurities due to manufacture such as inorganic substances
(adsorbed salts or mineral water contained in paper).
5. Stationary phase is aqueous and is held on paper.
• Modifications: we may modify paper used by impregnation.
Impregnate with Examples For

1.Hydrophilic substance Formaimde Separation of polar

compounds

2.Hydrophobic substance Paraffin, Vaseline Separation of lipids known

as reserved phase PC

In this case MP is polar

3.Buffer or specific pH Separation of alkaloids

value
Mobile phase: solvent, solvent system and developer.
Specifications:
1. Partially miscible with water e.g. phenol, butanol and amyl alcohol are
the best solvents can be used.
2.Developer is mainly prepared by shaking organic component with
water in separating funnel till saturation occurs:
a) Organic-rich phase is used as developer (mobile Phase)
b) Water-rich phase is placed in small beaker within
chromatographic chamber to facilate saturation of atmosphere around paper.
3. Paper adsorbs some of vapor to form stationary phase (partition
mechanism).