Dr.

Abdur Rahim
M.Sc(DU) M.Phil(BUET) Ph.D(Tohoku University, Japan)
BCS(Education)

Associate Professor
Dept. of Chemistry
GCAHS
Azimpur, Dhaka-1205
Email : a_rahim01@yahoo.com
Topic02_Lec09

High
Performance
Liquid
Chromatography
(HPLC)
 FROM LIQUID CHROMATOGRAPHY TO HIGH
PERFORMANCE LIQUID CHROMATOGRAPHY

• Higher degree of separation!

 Refinement of packing material (3 to 10 µm)
• Reduction of analysis time!
 Delivery of eluent by pump
 Demand for special equipment that can withstand
high pressures

The arrival of high performance liquid chromatography(HPLC)!

 High Performance Liquid
Chromatography (HPLC)
What is HPLC
HPLC is a form of liquid chromatography used
to separate compounds that are dissolved in
solution. HPLC instruments consist of a
reservoir of mobile phase, a pump, an injector,
a separation column, and a detector.
 High Performance Liquid
Chromatography (HPLC)

High Performance Liquid Chromatography

(HPLC) is a form of column chromatography
that pumps a sample mixture or analyte
in a solvent (known as the mobile phase)
at high pressure through a column with
chromatographic packing material (stationary
phase).
 Principle of HPLC
 High Performance Liquid Chromatography
[HPLC] principle is based on adsorption as
well as partition chromatography is
depending on the nature of stationary phase,
if stationary phase is solid principle is based
on adsorption chromatography and if
stationary phase is liquid principle is based
on partition chromatography.
Principle of HPLC contd…
 Compounds are separated by injecting a
sample mixture onto the column.
 The different component in the mixture
pass through the column at differentiates
due to differences in their partition
behavior between the mobile phase and
the stationary phase.
 The mobile phase must be degassed to
eliminate the formation of air bubbles.
Principle of HPLC contd…
 Instrumentation of HPLC
Describing the 5 major components
and their functions
1 – Pump
2 – Injector
3 – Column
4 – Detector
5 – Computer
Instrumentation of HPLC Contd..

1. Pump:
• The role of the pump is to force a
liquid(called the mobile phase) through the
liquid chromatograph at a specific flow
rate, expressed in milliliters per min (mL
/min). Normal flow rates in HPLC are in the
1-to 2-mL/min range.
•Typical pumps can reach pressures in the
range of 6000- 9000 psi (400-to 600-bar).
Instrumentation of HPLC Contd..

•During the chromatographic experiment, a

pump can deliver a constant mobile phase
composition (isocratic) or an increasing
mobile phase composition (gradient).

2. Injector:

•The injector serves to introduce the liquid

sample into the flow stream of the mobile
phase.
Instrumentation of HPLC Contd..
•Typical sample volumes are 5-to 20-
microliter (μL).
•The injector must also be able to withstand
the high pressures of the liquid system.

• An auto sampler is the automatic version

for when the user has many samples to
analyze or when manual injection is not
practical .
 Instrumentation of HPLC Contd..
3. Column:
• Considered the “heart of the chromatograph”
the column’s stationary phase separates the
sample components of interest using various
physical and chemical parameters.
•The small particles inside the column are
what cause the high back pressure at
normal flow rates.
Instrumentation of HPLC Contd..
• The pump must push hard to move the
mobile phase through the column and this
resistance causes a high pressure within
the chromatograph.

4. Detector:

• The detector can see (detect) the individual

molecules that come out (elute) from the
column.
 Instrumentation of HPLC Contd..

•A detector serves to measure the amount

of those molecules so that the chemist can
quantitatively analyze the sample components.
•The detector provides an output to a recorder
or computer that results in the liquid
chromatogram(i.e., the graph of the detector
response).
Instrumentation of HPLC Contd..
5. Computer:
• Frequently called the data system,
The computer not only controls all the
modules of the HPLC instrument but it takes
the signal from the detector and uses it to:
1. determine the time of elution (retention
time) of the sample components
(qualitative analysis) and
2. the amount of sample ( quantitative
analysis) .
 Dr. Abdur Rahim
M.Sc(DU) M.Phil(BUET) Ph.D(Tohoku University, Japan)
BCS(Education)

Associate Professor
Dept. of Chemistry
College of Home Economics
Azimpur, Dhaka-1205
Email : a_rahim01@yahoo.com
SEPARATIONS Injector Chromatogram

Mixer mAU

Pumps

Start Injection time

Column

Detector

Solvents

18
 ADVANTAGES
It is simple, rapid , reproducible.
High sensitivity.
High performance.
Rapid process and hence time saving.
It is having a high resolution and
separation capacity.
Accuracy and Precision.
Stationary phase was chemically inert.
Wide varities of stationary phase.
ADVANTAGES Contd….
Mobile phase was chemically inert.
Less requirement of mobile phase in
developing chamber.
Early recovery of separated component.
Easy visualization of separated components.
It is having Good reproducibility and
repeatability.
ADVANTAGES Contd….
It is analytical technique is important for
validation of product, quality control studies
of product.
 It is important for qualitative and
quantitative analysis.
 It is used for both analytical and
preparative purpose
 HPLC APPLICATIONS

Bioscience
Chemical proteins
peptides
polystyrenes nucleotides
dyes
phthalates

tetracyclines
Pharmaceuticals corticosteroids
antidepressantsConsumer Products
barbiturates lipids
antioxidants
sugars
Environmental Clinical
polyaromatic hydrocarbons amino acids
Inorganic ions vitamins
herbicides homocysteine
APPLICATION HPLC

Drug Discovery
Clinical Analysis
Proteomics
Forensic Chemistry
Drug Metabolism study
Environmental chemistry
Diagnostic studies
APPLICATION HPLC Contd…
Cosmetic analysis
Determination of Green Florescent
Protein
Structural Determination
Pharmaceutical Applications
Identification of Bile Acid Metabolite
Clinical Applications
Biochemical Genetics
qualitative and quantitative analysis
Therapeutic Drug Monitoring
SEPARATIONS Separation in based upon differential
Injector
migration between the stationary and
mobile phases.
Mixer Stationary Phase - the phase which
remains fixed in the column, e.g. C18,
Silica

Pumps Mobile Phase - carries the sample

through the stationary phase as it
moves through the column.
Column

Detector

Waste
Solvents

High Performance Liquid Chromatograph

25
SEPARATIONS Injector Chromatogram

Mixer mAU

Pumps

Start Injection time

Column

Detector

Solvents

High Performance Liquid Chromatograph

26
SEPARATIONS Injector Chromatogram

Mixer mAU

Pumps

Start Injection time

Column

Detector

Solvents

27
SEPARATIONS Injector Chromatogram

Mixer mAU

Pumps

Start Injection time

Column

Detector

Solvents

28
SEPARATIONS Injector Chromatogram

Mixer mAU

Pumps

Start Injection time

Column

Detector

Solvents

30
SEPARATIONS Injector Chromatogram

Mixer mAU

Pumps

Start Injection time

Column

Detector

Solvents

31
SEPARATIONS Injector Chromatogram

Mixer mAU

Pumps

Start Injection time

Column

Detector

Solvents

32
SEPARATIONS Injector Chromatogram

Mixer mAU

Pumps

Start Injection time

Column

Detector

Solvents

33
SEPARATIONS Injector Chromatogram

Mixer mAU

Pumps

Start Injection time

Column

Detector

Solvents

35
SEPARATIONS Injector Chromatogram

Mixer mAU

Pumps

Start Injection time

Column

Detector

Solvents

36
SEPARATIONS Injector Chromatogram

Mixer mAU

Pumps

Start Injection time

Column

Detector

Solvents

37
SEPARATIONS Injector Chromatogram

Mixer mAU

Pumps

Start Injection time

Column

Detector

Solvents

38
SEPARATIONS Injector Chromatogram

Mixer mAU

Pumps

Start Injection time

Column

Detector

Solvents

39
SEPARATIONS Injector Chromatogram

Mixer mAU

Pumps

Start Injection time

Column

Detector

Solvents

40
SEPARATIONS Injector Chromatogram

Mixer mAU

Pumps

Start Injection time

Column

Detector

Solvents

41
SEPARATIONS Injector Chromatogram

Mixer mAU

Pumps

Start Injection time

Column

Detector

Solvents

42
WHAT IS AC?

 Affinity chromatography (AC) is a technique

enabling purification of a biomolecule with
respect to biological function or individual
chemical structure.

 AC is designed to purify a particular

molecule from a mixed sample.
 Affinity chromatography
Affinity chromatography is a separation process used to purify molecules or a group
of molecules that are in a biochemical mixture. It employs two phases; a stationary
phase and a mobile phase. Specific molecules from the moving phase will bond to
the stationary phase based on their properties whilst the rest of the solution passing
through unaffected.
The process is often used to purify
biomolecules such as enzymes,
antibodies, and recombinant
proteins. It can also help to remove
harmful substances such as
pathogens through the same
principles.
For example, a protein in a solution
can be purified by passing it
through a column that has another
molecule attached to it (the ligand)
that has an affinity for the protein.
 Affinity chromatography

When they bind, this will allow the unreactive,

non-bound solution to pass through the
column. The target molecule is then eluted
from the ligand by a change made in the buffer
conditions so that the protein can be removed
from that surface.

The process usually involves sets of molecules

that interact with their cognate partner such
as enzymes and substrates, antigens and
antibodies, and ligands and receptors. Thus
the purification requires knowledge of how the
two different sets of molecules are likely to
interact.
 Size-exclusion chromatography
INTRODUCTION
Size-exclusion chromatography (SEC) is a chromatographic
method in which molecules in solution are separated by their size,
and in some cases molecular weight

It is usually applied to large molecules or macromolecular

complexes such as proteins and industrial polymers

Fig 1. A size exclusion column

Size-exclusion chromatography