Chromatography is a method of separating mixtures into individual components based on differences in how they interact with and move through stationary and mobile phases. There are various types of chromatography classified by the separation principle used and phases involved, including adsorption, partition, ion exchange, and size exclusion. Column chromatography is commonly used, involving a solid stationary phase packed into a column with a liquid mobile phase passed through to separate components. Factors like choice of stationary phase, mobile phase, development technique affect the separation achieved.

1. Chromatograhpy
3RD YEAR PHARMACY STUDENTS PRACTICAL
COURSE
BY MOHAMED SAMY, B.SC

2. Chromatography
introduction
• Chromatography, literally "color writing", was first employed by Russian-Italian
scientist Mikhail Tsvet in 1900, primarily for the separation of plant pigments
such as chlorophyll, carotenes, and xanthophylls.
• The word Chromatography is derived from Greek words Chroma=Color and
Graphy=wrting.
Definition:
- 'A method of separating a mixture of components into individual components through equilibrium
distribution between two phases’
- ‘A technique by which a mixture is separated into its components on the basis of relative ability of each
component to be moved along/through a stationary phase by mobile phase’
- The technique of chromatography is based on the differences in the rate at which the components of a
mixture move through a porous medium (called stationary phase) under the influence of some solvent
or gas (called moving/mobile phase).

3. Classification of chromatography

4. I- According to the principle of the separation process:-
1- Adsorption: It uses a mobile liquid phase or gaseous phase that is adsorbed onto the surface of a
stationary solid phase. The equilibration between the mobile and stationary phase accounts for the
separation of different solutes.
2- Partition: based on a thin film formed on the surface of a solid support by a liquid stationary phase.
Solute equilibrates between the mobile phase and the stationary liquid.
3- Ion exchange: employs porous beads of a resin that will exchange either cations or anions. There is one
type of ion on the surface of the resin and these are released when other ions are bound in their place –
e.g. a basic anion exchange resin might remove nitrate ions (NO3–) from a solution and replace them with
hydroxide ions (OH–).
4- Gel filtration: works on the basis of size exclusion, the stationary phase (the gel) typically consists of
particles of a cross-linked polyamide which contains pores, the mixture of solutes is carried through the
column by a solvent.
5- Bioaffinity: It is based on the specific interaction between two molecules. The one is solute molecule
and a second molecule is immobilized on a stationary phase. For example, the immobilized molecule can
be an antibody which interacts on the particular area of protein.

5. II- According to phases between which the
fraction process takes place :
1- liquid chromatography
2- Gas chromatography

6. Commonly used terms in chromatography:
• The analyte is the substance to be separated during chromatography.
• Analytical chromatography is used to determine the existence and the concentration
of analyte(s) in a sample.
• A chromatogram is the visual output of the chromatograph. In the case of an ideal
separation, different peaks or patterns on the chromatogram represent different
components of the separated mixture.
• A chromatograph is an equipment that enables the separation e.g. gas
chromatographic or liquid chromatographic separation.
• The eluate is the mobile phase leaving the column.
• The eluent is the solvent that carries the analyte.
• Effluent is the mobile phase leaving the column.
• An eluotropic series is a list of solvents ranked according to their eluting power.
• Elution is the process of extracting a substance that is adsorbed to another by
washing it with a solvent.

7. • An immobilized phase is a stationary phase that is immobilized on the support
particles, or on the inner wall of the column tubing.
• The mobile phase is the phase that moves over the stationary phase. It may be a liquid
liquid (LC) or a gas (GC). The mobile phase moves through the stationary phase where
the sample interacts with the stationary phase and is separated.
• The retention time (Rt) is the time required for the mobile phase to sweep a
component from the stationary phase.
• The retention volume is the volume of the mobile phase required to sweep a
component through stationary phase.
• The sample is the matter analyzed in chromatography. It may consist of a single
component or it may be a mixture of components.
• The solute refers to the sample components in partition chromatography.
• The solvent refers to any substance capable of solubilizing another substance, and
especially the liquid mobile phase in liquid chromatography.
• The stationary phase is the substance fixed in place for the chromatography
procedure. It may be solid, gel or a liquid. e.g ; silica, alumina, cellulose

8. • The detector refers to the instrument used for qualitative and quantitative
detection of analytes after separation.
• Rf value or Retention factor (Rf) is defined as the ratio of the distance traveled
by the center of a spot (solute) to the distance traveled by the solvent front
(solvent).
Adsorbent is the solid material that serves the stationary phase and has ability
adsorb or desorbs the different components of the mixture
Development when the mobile phase is caused to flow over the adsorbent or
support
Resolution is the degree of separation of the component after development

9. 1. Adsorption chromatography:
The separation depend on different affinities of different compounds
to be adsorbed on the surface of a particular solid adsorbent.
The moving (mobile) phase is liquid and the stationary phase is solid
Examples:-
A. Thin-layer Chromatography
B. Column Chromatography
Two factors are involved in adsorption chromatography
-Forces attracting solutes to adsorbent.
-Forces tending to remove the solute from the adsorbent.

11. Adsorbent (stationary phase)
The adsorbent in chromatography functions as an activated surface that can attract and hold solutes to
a given degree.
The adsorbent must be
Insoluble in the solvent used
If colored substances are to be separated it is desirable for the adsorbent to be colorless.
It must neither react nor catalyze the decomposition of the substance to be separated (Inert).
Particle size must be sufficient small to give large surface area.
Strength of adsorbent
Strength of the adsorbent is usually determined by measuring the rate at which a zone travels in an
elution experiment, the great the rate the weaker the adsorbent.
It can be classified into
weak as sucrose, starch.
Intermediate as calcium carbonate.
Strong as alumina, silica gel.

12. Common adsorbents
Silica and Alumina are the most widely used adsorbent mainly in C.C.
- There are three forms of alumina
Basic ( PH) 10
It has a wide range of application ,most organic compound except saturated
aliphatic hydrocarbon
Neutral ( PH) 7.5
It is largely used for separation of keto sterols, lactones and dehydration of
solvent
Acidic ( PH) 3.4
It is largely used for the separation of mixture of dicarboxylic amino acids
and acid peptide

13. Column chromatography
• Column chromatography is one of the most useful methods for the separation
and purification of both solids and liquids.
• This is a solid - liquid technique in which the stationary phase is a solid & mobile
phase is a liquid.
PRINCIPLE
• Adsorption
• Mixture of components dissolved in the M.P is introduced in to the column.
Components moves depending upon their relative affinities.
• Adsorption column chromatography, the adsorbent, packed in a glass column,
and a solvent, the mobile phase, that moves slowly through the packed column.
A solvent used as a mobile phase is called an eluent.

15. Column chromatography Cont’d
• A compound attracted more strongly by the mobile phase
will move rapidly through the column, and elute from, or
come off, the column dissolved in the eluent.
• In contrast, a compound more strongly attracted to the
stationary phase will move slowly through the column.

16. Experimental aspects of column chromatography:
• Adsorbents: The usual adsorbents employed in column chromatography are silica,
alumina, calcium carbonate, calcium phosphate, magnesia, starch, etc.,
• Alumina is generally suitable for chromatography of less polar compounds. Silica
gel gives good results with compounds containing polar functional groups.
Adsorbent in C.C should meet following criteria
• Particles should be spherical in shape & uniform in size.
• Mechanical stability must be high.
• They shouldn’t react chemically.
• It should be useful for separating for wide variety of compounds.
• It should be freely available & inexpensive.
(The particle size of the commercially available grade is in the range 50 – 200 µm.)

17. Mobile Phase
They act as solvent, developer & eluent.
The function of a mobile phase are:
• As developing agent.
• To introduce the mixture into the column – as solvent.
• To developing agent.
• To remove pure components out of the column – as eluent.
- Different mobile phases used: ( in increasing order of polarity)
• Petroleum ether, carbon tetrachloride, cyclohexane, ether, acetone,
benzene, toluene, esters, water, etc
• It can b e used in either pure form or as mixture of solvents

19. PREPARATION OF THE COLUMN
• It consists of a glass tube with bottom portion of the column – packed with glass
wool/cotton wool or may contain asbestos pad, » Above which adsorbent is
packed »
The following steps are followed for preparation of the column
1. Packing of the column
2. Application of the sample
3. Development and elution
4. Detection of the components
Method of packing of column
1- Dry method
2- Wet packing method
- First: A piece of cotton is used to plug the lower end of the column

20. Different types of column’s packing
DRY PACKING
• Adsorbent is packed in the column
in dry form
• Fill the solvent, till equilibrium is
reached
- DEMERIT: Air bubbles are
entrapped b/w M.P & S.P→ cracks
appear in the adsorbent layer.
- After filling tapping can be done to
remove void spaces.
WET PACKING
• ideal & common technique The
material is slurried with solvent and
generally added to the column in
portions.
• S.P settles uniformly & no crack in
the column of adsorbent. » solid
settle down while the solvent
remain upward. » this solvent is
removed then again cotton plug is
placed

21. Preparation of the column
• The sample which is usually a mixture of components is dissolved in
minimum quantity of the mobile phase.
• The entire sample is introduced into the column at once and get
adsorbed on the top portion of the column.
• From this zone, individual sample can be separated by a process of
elution.

22. Development technique ( Elution)
• By elution technique, the individual components are separated out from
the column. The two techniques are:
• (i) Isocratic elution technique : in this elution technique , same solvent
composition or solvent of same polarity is used throughout the process of
separation. Example: chloroform only
• (ii) Gradient elution techniques: ( gradient – gradually) Solvents of
gradually ↑ polarity or ↑ elution strength are used during the process of
separation. E.g. initially benzene, then chloroform, then ethyl acetate then
chloroform.

23. DETECTION OF COMPONENTS
• If the compounds separated in a column chromatography
procedure are colored, the progress of the separation can
simply be monitored visually.
• If the compounds to be isolated from column
chromatography are colorless. In this case, small fractions
of the eluent are collected sequentially in labelled tubes
and the composition of each fraction is analyzed by TLC.
Application of column chromatography
Separation and purification of vitamins, hormones
,alkaloids, glycosides and other active constituents.
Examination of vegetable oil and pharmaceutical
preparation.

24. Pros and Cons of column chromatography:-
Pros:
• » Any type of mixture can be separated.
• » Any quantity of mixture can be separated.
• » Wider choice of Mobile Phase.
• » Automation is possible.
Cons:
• » Time consuming
• » more amount of Mobile Phase are required
• » Automation makes the techniques more complicated &
expensive

25. Practical example of column chromatography
•Method of packing: Wet packing
•Stationary phase: Silica gel
•Mobile phase: Methylene chloride then
Ethanol
•Sample: Gentian violet , sudan III
•Development technique: gradient elution.

26. Practical example of column chromatography
•Procedures:
• 1) inset a plug of cotton in the tapering lower end of column
• 2) pack column by wet method
• 3) elute excess solvent till reaching equilibrium.
• 4) after settling of silica layer , apply sample (1ml) from the top of
column
• 5) Place a cotton plug at the top after adding sample
• 6) Add methylene chloride till separation of sudanIII (red layer)
completely (non-polar)
• 7) Change mobile phase(ethanol) to elute gentian violet (polar)