Scribd
Upload
33 views

Lecture Chromatography I

Uploaded by

shethvraj4623
Copyright
© © All Rights Reserved
Available Formats
Download as PPTX, PDF, TXT or read online on Scribd
33 views

Lecture Chromatography I

Uploaded by

shethvraj4623
Copyright
© © All Rights Reserved
Available Formats
Download as PPTX, PDF, TXT or read online on Scribd
You are on page 1/ 27

Chromatography I

Pharmaceutical Analytical Chemistry Lecture # 9

Dr. Habibullah
Department of Pharmaceutical
Chemistry & Pharmacognosy
Chromatography
In 1903 M.S. Tswett described a
technique for the separation of
plant pigments.

He called this technique


‘Chromatography”
derived from the Greek word which
means: colour writing
Chroma: colour
& tography: writing
Chromatography

Chromatography is an analytical method

that is widely used for:

 Separation,

 Identification &

 Determination of Chemical

components in complex mixtures.


It is a physical separation technique in

which components of a mixture are

separated between two immiscible phases:

1- A Stationary phase

2- A mobile phase : liquid or a gas.

Separation is based on the rates at which

components are carried through stationary

phase by gaseous or liquid mobile phase .


A: Stationary phase (adsorbent):

 Solid porous media,

 Rigid porous particles, usually silica

• May exist in a variety of forms

(e.g., bed, layer, column ).


B- Mobile phase (eluent):

It may be either a gas (i.e., GC) or a


liquid solvent (i.e. LC) or mixture of
solvents

It is moving through stationary phase


or chromatographic column &
carrying analytes.
Principle of chromatography:
When individual component of a mixture
favours stationary phase, it is held longer &
moves more slowly through stationary phase
(column).
If velocities and hence distribution
coefficients Kd of samples components are
different, mixture could be resolved.
If Kd is large i.e Solute is retained more
strongly by stationary phase
If kd is small i.e Solute is eluted more rapidly
from column
Column chromatography

Oldest form of liquid chromatography is the


column chromatography

In classical column chromatography columns


were open tubes (e.g., burettes), which were
individually packed with coarse material.

Mixture to be separated is loaded onto top


of column followed by more solvent.
Column chromatography
Mechanism of separation of the
mixture

1. Different components in sample mixture pass


through column at different rates due to differences in
their partitioning behaviour between mobile phase &
stationary phase

2. Compounds are separated by collecting aliquots of


column effluent as a function of time. components
were collected as coloured fractions

3. Flow of mobile phase was achieved by gravity


feeding , while in HPLC it move under pressure
2-High pressure liquid
chromatography

 In the 1960’s researchers began developed


high performance liquid chromatography
(HPLC).

 HPLC: means high performance liquid


chromatography, which refers to high speed,
high-resolution separation.
Classification of
chromatographic
methods
1-According to the states of the two phases:

Usually mobile phase is named first

(a) Liquid-solid chromatography (LSC).

(b) Liquid-Liquid chromatography (LLC)

(c) Gas- solid chromatography (GSC).

(d) Gas- Liquid chromatography (GLC)


2-According to the shape of stationary bed:

a. Plane or flat–bed chromatography:

Stationary bed is coated on a flat surface.


e.g.: paper chromatography (PC) & thin layer
chromatography (TLC).

b. Column chromatography:
Stationary bed is contained in a column.
e.g.: Open-column chromatography (OCC),
Gas chromatography (GC) and
High pressure liquid chromatography (HPLC)
3-According to the mechanism of separation
of chromatographic techniques

1) Adsorption chromatography

2) Partition chromatography

3) Ion Exchange

4) Size Exclusion chromatography


1) Adsorption chromatography:

Stationary phase: is a solid on which sample


components are adsorbed.
● Mobile phase may be:
* a liquid e.g. (liquid-solid chromatography)
* or a gas e.g. (gas-solid chromatography);

Mechanism of separation: depends on distribution


of sample components between two phases
through a combination of sorption and desorption
processes.
Thin-layer chromatography (TLC) is a special
example of adsorption chromatography.
1) Adsorption chromatography

Sorption & desorption process


Normal & Reversed phase
chromatography

Two modes are defined depending on relative


polarity of two phases .
These are:
1-Normal phase chromatography
2-Reversed phase chromatography
1-Normal phase: st. phase polar(silica),
Mobile phase nonpolar (such as n-hexane or
tetrahydrofuran).
polar Sample will be retained on the stationary
phase more than less polar
1- The normal phase chromatography:
Stationary bed is strongly polar (e.g., silica
gel),
Mobile phase is non polar (such as n-
hexane or tetrahydrofuran).
Polar samples are thus retained on polar
surface of column packing longer than less
polar materials.
2- The reversed-phase chromatography:
which is the inverse of normal phase.
Stationary bed is non polar (hydrophobic): eg
octadecyl
Mobile phase is a polar liquid,such as mixtures of
H2O & MeOH or ACN.
Non Polar samples are thus retained on non polar
surface of column packing longer than polar
materials.

Reversed phase
chromatography

Fig. Highly polar, Moderately polar


and Non polar component
Reversed phase
column
Normal phase
column

Fig.3: Normal and reversed phase chromatography:


Circles represent types of compounds present in sample, their relative
position relative to direction of mobile phase
2) Partition chromatography:

Stationary phase is a liquid supported on an inert


solid.
● Mobile phase may be:
a liquid (liquid-liquid partition chromatography)
or a gas (gas-liquid chromatography, GLC).

● Mechanism of separation : depends on partition


of sample components between the two phases

● Paper chromatography is a type of partition


chromatography in which stationary phase is a
layer of water adsorbed on a sheet of paper.
(3) Ion exchange chromatography
(IEC)

 Stationary phase is an ion exchange resin


.
 Anions (such as –SO3) or cations (such as
–N (CH3)3+ are covalently attached to
stationary phase, usually called a resin.
 Stationary bed has an ionic charged
surface of opposite charge to sample ions.
 Solute ions of opposite charge are
attached to stationary phase by
Types of stationary phase (resin)

Cation resin: Resins containing the active group -


SO3  are called cation exchangers, Since only
cations can be attracted to it.

Anion resin: Resins containing quaternary


ammonium ions are called anion exchangers,
Since only anions can be attached to it.

Mobile phase is an aqueous buffer, where both


pH and ionic strength are used to control elution
time.
4) Size Exclusion chromatography

Stationary phase : a sieve like structure.

Mechanism of separation: Solvated molecules are


separated according to their size by their ability to
penetrate a sieve like structure ( stationary phase).

There is no attractive interaction between the


stationary phase and the solute.
Mechanism of separation:

Stationary phase is hydrophilic & therefore capable of


absorbing water, resulting in swelling open structure (gel)

Solvated sample molecules larger than pore of swollen gel


cannot penetrate gel particles therefore

 pass straight through column through spaces between


individual particles.

While smaller molecules can penetrate inside pores to a


varying degree depending on their size and shape.  i.e.
retarded & eluted in order of decreasing molecular size.

N.B.: If the Mobile phase is aqueous it called gel filteration


& If the Mobile phase is organic solvent it called gel
permeation
1- Fractionation (molecular weight distribution) of proteins,
enzymes nucleic acids, hormones

You might also like