Pharmaceutical Chemistry.

1. TOPIC: HPLC and The Basic
Terminologies Of Chromatography.
By
Sayyad Ali
Reg.# FA11-R60-002/ATD
03/12/15 To Prof. Jamshed Iqbal 1

2. HIGH PERFORMANCE LIQUID CHROMATOGRAPHY
(HPLC)
03/12/15 To Prof. Jamshed Iqbal 2

3. HIGH PERFORMANCE LIQUID CHROMATOGRAPHY
• HPLC stands for “High-performance liquid
chromatography”(sometimes referred to as High-pressure liquid
chromatography).
• High performance liquid chromatography is a powerful tool in
analysis, it yields high performance and high speed compared to
traditional columns chromatography because of the forcibly
pumped mobile phase.
• HPLC is a chromatographic technique that can separate a mixture
of compounds
• It is used in applied science and chemistry to identify, quantify and
purify the individual components of a mixture.
03/12/15 To Prof. Jamshed Iqbal 3

4. • Chromatography is a physical method in which separation of
components takes place between two phases a stationary
phase and a mobile phase
• Stationary phase : The substance on which adsorption of the
analyte (the substance to be separated during
chromatography) takes place . It can be a solid, a gel, or a
solid liquid combination
• Mobile phase : solvent which carries the analyte (a liquid or a
gas)
03/12/15 To Prof. Jamshed Iqbal 4

5. Chromatographic techniques are divided into different types based on:
1.The type of chromatographic bed used, i.e. column chromatography
(gas chromatography) and planar chromatography (paper and thin
layer).
2.The physical state of mobile phase i.e. gas chromatography and
liquid chromatography
3.The separation mechanism i.e. ion-exchange and affinity
chromatography.
HPLC is a type of liquid chromatography where the sample is forced
through a column that is packed with a stationary phase composed of
irregularly or spherically shaped particles by a liquid (mobile phase)
at high pressure.
03/12/15 To Prof. Jamshed Iqbal 5

6. PRINCILPE
Liquid chromatography is a separation technique that involves:
•Putting or injecting of a small volume of liquid sample into a
tube packed with porous particles (stationary phase).
•Where individual components of the sample are transported
along the packed tube (column) by a liquid moved under the
effect of gravity.
The main principle of separation is adsorption .
To understand the principle of HPLC , we must first look at the
principle behind liquid chromatography
03/12/15 To Prof. Jamshed Iqbal 6

7. • When a mixture of components are introduced into the column.
various chemical and physical or both types interactions take
place between the sample molecules and the particles of the
column packing .
• They travel according to their relative affinities towards the
stationary phase. The component which has more affinity
towards the adsorbent, travels slower.
• The component which has less affinity towards the stationary
phase travels faster.
• Since no two components have the same affinity towards the
stationary phase, the components are separated
03/12/15 To Prof. Jamshed Iqbal 7

8. HPLC is a separation technique that involves:
•The injection of a small volume of liquid sample into a tube packed
with tiny particles (3 to 5 micron (μm) in diameter called the stationary
phase)
•Where individual components of the sample are moved down the
packed tube (column) with a liquid (mobile phase) forced through the
column by high pressure delivered by a pump.
• These components are separated from one another by the column
packing that involves various chemical and/or physical interactions
between their molecules and the packing particles.
• These separated components are detected at the exit of this tube
(column) by a flow-through device (detector) that measures their
amount. The output from the detector is called a chromatogram
In principle, LC and HPLC work the same way except the speed ,
efficiency and sensitivity of operation .
03/12/15 To Prof. Jamshed Iqbal 8

9. TYPES OF HPLC
BASED ON MODE OF SEPERATION
1.Normal phase chromatography:
stationary phase is polar (hydrophilic) and mobile face is non-
polar (hydrophobic).
2.Reverse phase chromatography:
stationary face is non-polar (hydrophobic) and mobile face is
polar (hydrophilic).
•Polar-Polar bonds and Non Polar-Non Polar bonds have more
affinity than Polar-Non Polar bonds.
•Reverse phase chromatography is more commonly used as
drugs are usually hydrophilic
03/12/15 To Prof. Jamshed Iqbal 9

10. BASED ON PRINCIPLE OF SEPERATION
Absorption Chromatography
• In the Absorption Chromatography solute molecules bond
directly to the surface of the stationary phase
• The component which has more affinity towards mobile phase
elutes first & the component which has less affinity towards
stationary phase elutes later.
No two components have same affinity towards mobile phase &
stationary phase.
03/12/15 To Prof. Jamshed Iqbal 10

11. Ion-exchange chromatography
•Ion exchange chromatography is a process that allows the separation of
ions and polar molecules based on their charge.
•It can be used for almost any kind of charged molecule including large
proteins, small nucleotides and amino acids.
•Retention is based on the attraction between solute ions and charged
sites bound to the stationary phase. Ions of the same charge are excluded.
•The use of a resin (the stationary solid phase) is used to covalently
attach anions or cations onto it. Solute ions of the opposite charge in the
mobile liquid phase are attracted to the resin by electrostatic forces.
03/12/15 To Prof. Jamshed Iqbal 11

12. 03/12/15 To Prof. Jamshed Iqbal 12

13. 03/12/15 To Prof. Jamshed Iqbal 13
Selectivity and retention can be adjusted by changing the pH of the mobile
phase. This occurs because such a change in pH modifies the character of both
the ion exchange medium and the acid–base equilibrium as well as the degree of
ionization of the sample.

14. Affinity Chromatography
•This is the most selective type of chromatography employed. It utilizes the
specific interaction between one kind of solute molecule and a second molecule
that is immobilized on a stationary phase.
•It is a liquid chromatographic technique that uses a biologically related agent as a
stationary phase for the purification or analysis of sample components.
•The retention of solutes in this method is based on the specific reversible
interactions that occur in many biological systems, such as the binding of an
enzyme with a substrate or of an antibody with an antigen.
•These interactions are exploited in affinity chromatography by placing one of a
pair of interacting molecules onto or within a solid support and using this
immobilized agent as a stationary phase. This immobilized agent is known as the
affinity ligand and is what gives an affinity column the ability to bind to particular
compounds in a sample.
03/12/15 To Prof. Jamshed Iqbal 14

15. 03/12/15 To Prof. Jamshed Iqbal 15
Steric hindrance: It refers to the loss of
ligand activity due to the presence of a
nearby support or neighboring ligands
that interfere with solute binding. This
effect can be avoided through the use of
a spacer arm or by using stationary
supports that contain a relatively less
dense attachment of the ligand.

16. 03/12/15 To Prof. Jamshed Iqbal 16
BASIC TERMINOLOGIES OF
CHROMATOGRAPHY.
DISTRIBUTION

17. 03/12/15 To Prof. Jamshed Iqbal 17

18. 03/12/15 To Prof. Jamshed Iqbal 18
Movement Through the Column

19. 03/12/15 To Prof. Jamshed Iqbal 19

20. a)
To Prof. Jamshed Iqbal 20
b)

21. 03/12/15 To Prof. Jamshed Iqbal 21
Continue…

22. 03/12/15 To Prof. Jamshed Iqbal 22
Resolution

23. 03/12/15 To Prof. Jamshed Iqbal 23
Theoretical plates:
•As the column efficiency is related to the “number of theoretical plates” or “N”.
The value of “N” is related to the column length “L” and the size or height “H” of
an individual plate by the following equation:
N = L/H
•The value of “L” is usually measured in cm, and can vary from just a few
centimeters for HPLC columns to many meters for GC columns.
•The values of N are usually large, in the thousands or even millions, giving
values of H.
•The size of H affect the efficiency of the column and therefore it is important to
understand what affects the value of H.
•The value of H depends primarily on four factors,
1) the velocity of the mobile phase,
2) multipath diffusion,
3) the diffusion of the compound in the mobile phase, and
4) the transfer of the compound between the stationary phase and the
mobile phase.

24. 03/12/15 To Prof. Jamshed Iqbal 24
Linear Velocity, u
PlateHeight,H
Multipath Term, A
Mass Transfer (both), Cu
Longitudinal diffusion, B/u
A + B/u + Cu
A typical Van Deemter plot

25. 03/12/15 To Prof. Jamshed Iqbal 25
The Multipath Term or constant A:
•This factor is also called Eddy diffusion, but more modern textbooks use
the term multipath.
•In packed columns, peak-broadening is the result of a number of factors.
•As molecules of the analyte move through the column, they take many
different paths around the packed particles.
• Some of these paths are longer than others so as the molecules move
through the column, thus they tend to spread out.
•The amount of spreading is affected by the nature of the column material
and how well the column is packed.
•This factor is generally proportional to the particle size of the packing
material.
1 2
Flow
Direction
Pathways of two molecules
during elution. Distance traveled
by molecule 1 is longer than
that traveled by molecule 2, thus
molecule 1 will take longer to
elute.

26. 03/12/15 To Prof. Jamshed Iqbal 26
The Longitudinal Diffusion Term B/u (m2
/sec)
•Longitudinal diffusion also contributes to peak broadening.
•In this process, analytes diffuse from areas of high concentration to more
dilute area in front of and behind the moving band.
•The concentration of the analyte is less at the edges of the band than at the
center.
•Hence analyte diffuses out from the center to the edges, causing band
broadening.
•If the velocity of the mobile phase is high then the analyte spends less time
on the column, decreases the effects of longitudinal diffusion.
Flow
Flow
Molecules diffuse from areas of high
concentration to areas of low concentration.
Over time….

27. 03/12/15 To Prof. Jamshed Iqbal 27
The Mass Transfer Terms Cu:
•The analyte takes certain amount of time to reach equilibrium between the
stationary and mobile phase.
•If the velocity of the mobile phase is high and the analyte has a strong attraction
for the stationary phase, then the analyte in the mobile phase will move ahead of
that in the stationary phase.
•Like the longitudinal term, the mass transfer term is based on diffusion.
However, longitudinal diffusion takes place parallel to the direction of flow, and
therefore is inversely related to the mobile phase flow rate, while mass transfer
diffusion takes place perpendicular to the flow rate.
•As a result, the faster the mobile phase moves, the less time there is for
equilibrium between the phases and the mass transfer effect.
• peak broadening is directly related to mobile phase velocity or flow rate.

28. 03/12/15 To Prof. Jamshed Iqbal 28