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HPLC- high performance liquid chromatography

? Chromatography is essentially a group of techniques for
the separation of the compounds of mixtures by their
continuous distribution between two phases, one of
which is mobile phase and other is stationary phase.
Example: HPLC, GC

? 14 May 1872 �C 26 June 1919) invented adsorption
chromatography.
? He used liquid-adsorption column
chromatography.(1901)
? Tsvet's work was long ignored for political violence.
? 10 years after his death Austrian biochemist
Richard Kuhn and his student

? Md. Moshiur Rahman 07(2047)
? Mehenaj jahan 02 (2042)
? Afzal hoassain 03 (2043)
? Nipa akter 04 (2044)
? Kaniz fatema 08 (2048)
? Samhan afroz 12 (2052)

? HPLC: High performance liquid chromatography
Definition :It is a chromatographic technique
used to separate component of mixture for the
purpose to identify, quantify or purify the
individual component of mixture.
? It is widely used in the field of analytical
chemistry.

a) Mobile phase: They consist of water, organic solvent
or mixtures of organic solvents. They are continuously
flown ��like river water�� and carried samples. eg:
methanol, acetone,acetonitrile,water.
b) Stationary phase: They consist of a large number of
particles like silica. They are constant in a place and the
separation occurs in this phase.eg:C-18, C-12.

Several apparatus of HPLC
? Solvent reservoir
? Degasser
? Pump
? Mixing unit
? Guard column
? Injector
? column
? Detector
? Recorder
? Waste container

? Lab Solutions (Shimadzu)
? ChemStation (Agilent)
? ChromNAV 2.0 (JASCO)
? TRILUTION (Gilson)
? Chromeleon (JASCO)
? PrepCon5 (SCPA)

? The mobile phase in HPLC refers to the solvent being
continuously applied to the column or stationary phase.
? It acts as a carrier to the sample solution
? It also contains washing solvent to wash HPLC machine
after work.
? A sample is injected into the mobile phase of an assay
through the injector port.

? Degassing is done to prevent air
bubbles in the pump or detector.
? Used to remove the air
bubbles from the mobile phase.

? The role of the pump is to force a liquid (called
the mobile phase) through the liquid
chromatography at a specific flow rate, expressed
in (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).
? Two types of pump are used for HPLC
? Constant pressure pump
? Constant volume pump

? Mixing unit is used to mix solvent in different
proportion & pass through the column.
? Two types of mixing unit
- they are low pressure mixing chamber which
uses helium for degassing solvent.
-High pressure mixing chamber doesn��t require
helium for degassing solvent.

? Guard column is used to remove particle matter and
contamination.
? This is a small column placed before the actual
column/analytical column.
? It protects the column from bubbles, un-dissolved
particles and other harmful substances.
? stationary phase similar to the actual column.

? The injector serves to introduce the liquid
sample into the flow stream of the mobile
phase for analysis
? For a manual injector: the knob is manually
operated to deliver the sample to the column.
? Auto injector :In this system the sample are
injected into the flow lines automatically
through self reading.

? Column is the part of HPLC machine , also called
heart of HPLC . It contain stationary phase &made
by glass or stainless steel.
? Main function of column is separation of compound
from the mixture

? Heart of HPLC
? It contain stationary phase
Made of stainless steel.
Main function of column is
Separation of compound from the
Mixture.
? It is composed of particles like silica.
? It��s length is 10-30cm.
? The HPLC column have fixed length, diameter and particle size.

? Normal phase column
? Reverse phase column
? Ion exchange column
? Size exclusion column

? Column work on the basis of column nature .If the
column is polar the non polar compound elute first &
the polar elute last from the column . If the column is
non polar the polar compound elute first & non polar
elute last from the mixture by the column.

? It��s the rate of flow of the mobile phase or washing
solvent. Its regulated by the pump.
? It is important for the proper separation of
compound from the mixture.
? Flow rate is inversely proportional to
the HETP.

? Column oven is an oven which use to maintain a
desired temperature into the column . because
different temperature is needed to separation of
different component.
? column oven maintain the definite temperature for
proper separation of compound from mixture.

HETP means High Equivalent Theoretical plate
HETP=L/n
Where,
L= Length of column.
n= Number of particles that are responsible
for adsorption.
When the value of HETP is as much lower the quality of the column
is as much better

? Isocratic system means that composition of
mobile phase remains constant throughout the
rum.
? In gradient system pump composition of mobile
phase varies and is not constant .In which pump
can carry different solvents , mixing & carry
according to the reading to produce desired mobile
phase.

? Because it��s fully automated .Here less chance to
error in result .
? In isocratic its controlled manually so there is a
possibility to error in result.

Solvent which used to wash the HPLC before & after the use of
HPLC
Methanol for 10-30 min Washing
H2O (10-30)min
Mobile phase +standard Analysis
-sample
Methanol for 10-30 min Washing
H2O (10-30)min

Retention time: Retention time (RT) is a
measure of the time taken for a solute
to pass through
a chromatography column.
Relative (corrected) retention time
t��R = tR-t0

? The detector can detect the individual molecules that
elute from the column and convert the data into an
electrical signal
? A detector serves to measure the amount of those
molecules
? The detector provides an output to a recorder or
computer that results in the liquid chromatogram
? Detector is selected based on the analyze or the sample
under detection

several kinds of detector
? NMR,IR.
? Electrochemical detector.
? Uv visible detector.
? Fluorescence detector.
? Refractive index.
? Photodiode array detector

? Recorders are used to recorded the
electrical signal from detector.

? Waste container used to collect all the waste
product of HPLC.
? It can reserve waste mobile phase.
? The flow of mobile phase starts on solvent
reservoir and ends on waste reservoir.

? Qualitative analysis
? Checking the purity of a compound
? Presence of impurities
? Quantitative analysis
? Multi component analysis or determination of mixture of drugs
? Isolation and identification of mixture of compound
? Purification of compound
? Environmental application
? Biochemical separation , forensic test.
? Biopharmaceutical & pharmacokinetic studies.
? Isolation & identification of drug.
? Stability studies.

? There is a high cost for equipment needed to conduct
HPLC.
? Its operation can be complex, requiring a trained
technician to operate. Because of the speed of the
process, the equipment has low sensitive.

? Separations fast and efficient (high resolution power)
? Continuous monitoring of the column effluent
? It can be applied to the separation and analysis of very complex mixtures.
? Accurate quantitative measurements.
? Repetitive and reproducible analysis using the same column.
? Adsorption, partition, ion exchange and exclusion column separations are
excellently made.
? Both aqueous and non aqueous samples can be analyzed with little or no
sample pre treatment

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HPLC- high performance liquid chromatography

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