PHARMACEUTICAL ANALYSIS I

Chromatography I: Introduction
a. Definitions
b. Types of Chromatography
i. Adsorption
ii. Partition
iii. Ion-exchange
iv. Size exclusion
What is Chromatography?

Derived from the Greek word Chroma meaning

colour, chromatography provides a way to identify
unknown compounds and separate mixtures
Objectives of Chromatography

 To separate a component of interest from a mixture of

components (sample) or
 To separate an analyte from a matrix of interferents
(sample)
 Interferent:
◦ It is a chemical species that causes a systematic error
in an
 analysis by enhancing or attenuating the analytical signal
 Analyte:
◦ It is the component of a sample that is to be
determined
Applications of Chromatography

Forensics

Research
Pharmaceutical industry
 Types of Chromatography…

Thin layer
Paper

Column
HPLC Gas
Classifications of Chromatographic
Techniques
 Chromatographic techniques were classified into two,
based upon the physical means by which the stationary
and mobile phases are brought into contact.
i. Column chromatography
 The stationary phase is packed in a narrow tube
(column) and the mobile phase is forced through
the column under gravity or under pressure.
ii. Planar chromatography
 The stationary phase is on a flat plate or is paper
and the mobile phase moves through the
stationary phase by capillary action or gravity
 Thin layer chromatography & Paper
chromatography
Classification of Column Chromatographic
Methods
 Two basic types:
◦ Liquid Chromatography
◦ Gas Chromatography
 This classification is based upon the nature of the
mobile phase
 The mobile phase in
◦ Liquid chromatography – Liquid
◦ Gas chromatography - Gas
Classification of Column Chromatographic Techniques
General Method Stationary Type of
Classification Phase Equilibrium
Liquid Liquid-Liquid or Liquid adsorbed Partition between
chromatography partition on a solid immiscible liquids
Liquid bonded Organic species Partition between
phase bonded to a solid liquid and bonded
surface surface
Adsorption Solid Adsorption
Ion exchange Ion-exchange Ion exchange
resin
Size exclusion Liquid in Partition/Sieving
interstices of a
polymeric solid
Gas Gas-liquid Liquid adsorbed Partition between
chromatography on a solid gas and liquid
Gas bonded Organic species Partition between
phase bonded to a solid liquid and bonded
surface surface
Gas-solid Solid Adsorption
 Adsorption Chromatography
 It utilizes a mobile liquid or
gaseous phase that is
adsorbed onto the surface
of a stationary solid phase.
 The equilibrium between
the mobile and stationary
phase accounts for the
separation of different
solutes.
 Adsorption just on surface,
partition into thin layer
 Not used as widely as
partition used mainly in TLC
& very small particles in LC
Adsorption Chromatography
 Adsorption chromatography is based on interactions
between the solute and fixed active sites on the
stationary phase.
 It is also known as displacement, liquid/solid
chromatography.
 Stationary phase:
◦ A solid adsorbent packed in a column, spread on a
plate, or on a porous paper.
 Mobile phase:
◦ Usually a liquid solvent/solution
◦ Its role is vital since mobile phase molecules compete
with solute molecules for polar adsorption sites.
 If the interaction between SP & MP is strong, solute will
not be adsorbed onto the SP.
Partition Chromatography
 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.
 Used in GC & LC
 Molecules will partition into
the stationary phase based
upon affinity for stationary
phase & eventually partition
into mobile phase again
 Thin layer is coated onto inside
of GC column or on small
particles on LC column
Partition Chromatography
 Stationary phase:
◦ a film of liquid that is strongly adsorbed to an inert
support (Silica)
 Mobile phase:
◦ a liquid immiscible with stationary phase
 Based on the nature of SP and MP, it is divided into two
kinds
◦ Normal phase
◦ Reversed phase
Normal vs. Reversed Phase
Chromatography

Normal Phase Reversed Phase

Stationary phase Polar Non-polar

Mobile phase Non-polar (organic) Polar (aqueous/organic)

Sample movement Non-polar fastest Polar fastest

Separations based on Different polarities Different hydrocarbon

(Functionality)
Why reversed phase
chromatography is popular?
 It is versatile
 It can separate organic as well as polar
substances present in a mixture
◦ All organic molecules have hydrophobic
regions in their structure, so they can be
separated.
 Mobile phase is polar. So it can separate polar
substances
◦ insoluble in non-polar organic solvents
◦ bind too strongly to the solid support
Problems in Conventional LLC

 There is a possibility of light miscibility of the stationary

phase in the mobile phase
◦ This results in slow removal of stationary phase,
called “bleeding”.
 The analyte may adsorb onto the polar sites present in
solid support result in „tailing of the peak‟ in a
chromatogram
 To overcome these problems, the SP may be chemically
bonded to the support material, called bonded phases.
Affinity Chromatography
Common for all types of affinity chromatography is that an affinity ligand
specific for a binding site on the target molecule, is coupled to an inert
chromatography matrix.

Under suitable binding conditions this affinity matrix will bind molecules
according to its specificity only. All other sample components will pass
through the medium unadsorbed.

After a wash step the adsorbed

molecules are released and eluted by
changing the conditions towards
dissociation or by adding an excess
of a substance that displaces the
target molecule from the affinity
ligand.
The development of an
affinity chromatography
method boils down to:

[Link] a ligand specific

enough to allow step
elution.
[Link] conditions for
safe binding and release
within the stability window
of the target molecule and
the ligand.
Separation of specific antibodies from blood serum using
affinity chromatography

 Consider the blood serum consists of antibodies

of our interest (Red), other antibodies (green)
and serum proteins (Yellow)
 An immuno adsorbent is prepared with specified
antigen (blue) in a solid matrix (Agarose,
sephadex, cellulose derivatives etc. )
 The serum is passed over the immuno adsorbent
 Antibodies of other specificities (green) and
serum proteins (yellow) will pass through.
 As long as the capacity of the column is not
exceeded, those antibodies in the mixture
specific for the antigen (shown in red) will bind
(non-covalently) and be retained.
 A buffer solution is passed into the column to
release the antibodies from the immuno
adsorbent.
Ion Exchange Chromatography
 Chromatography in which
separation is based mainly
on differences in the ion-
exchange affinities of the
sample components.
 Anions like SO3- or cations
like N(CH3)3+ are covalently
attached to stationary
phase, usually a resin
 Separation based on relative
strength of ionic bond.
 Anion exchange has cations
on surface
 Used in LC exclusively
Ion-Exchange Chromatography
 It is the most popular method for the purification of
proteins and other charged molecules.
 Two types:
◦ Cation exchange chromatography
 Stationary phase carries mobile cations.
 So cations in the analyte mixture are exchanged
with mobile cations and retain on the SP while
anions move down the column.
◦ Anion exchange chromatography
 Stationary phase carries mobile anions.
 So anions in the analyte mixture are exchanged
with mobile anions and retain on the SP while
cations move down the column.
Ion-Exchange Chromatography

Ion Exchange Resin:

Resins are
manufactured with
ions attached. The
ions present a
certain degree of
positive or negative
charge, depending
on the buffer pH.
 Molecular Exclusion Chromatography
 It is also called molecular
sieve chromatography.
 It is subdivided into 2:
◦ Gel filtration
chromatography: Use
aqueous solvents and
hydrophilic packing
◦ Gel-permeation
chromatography: Use
organic solvents and
hydrophobic packing
 Separation based on size
 Small molecules get trapped
in pores & take longer to get
out
Size Exclusion Chromatography

 No chemical interaction occurs between the

components & the stationary phase.
 The components are separated according to their size.
 Stationary phase:
◦ A chemically inert material such as a gel or a porous
inorganic solid.
 Mobile phase:
◦ Water or an aqueous solution that solely serves as a
carrier for the analyte
Gel-Filtration (Size-Exclusion) Chromatography

Gel Filtration Resin.

When starting protein
purification, technicians
sometimes use a gel-
filtration (size-exclusion)
column first. They know the
molecular weight of their
protein, so they can often
eliminate several
contaminant proteins by a
quick run through a sizing
column.
Gel Permeation Chromatography
Size Exclusion Chromatography
 Basis for separation:
◦ Analyte inclusion in large medium
pores
◦ Smaller analytes fit in small
pores and are retained
more
◦ Larger analytes pass
through
 Used for separation of
polymers based on size
 Variety of packing
geometries can be used
(best if weaker analyte –
stationary phase
interactions)
 Pore size is critical and
affects retention