High performance liquid chromatography

4.  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

6.  14 May 1872 – 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

7.  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)

9.  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.

10. 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.

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

12.  Lab Solutions (Shimadzu)
 ChemStation (Agilent)
 ChromNAV 2.0 (JASCO)
 TRILUTION (Gilson)
 Chromeleon (JASCO)
 PrepCon5 (SCPA)

15.  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.

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

17.  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

18.  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.

19.  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.

20.  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.

21.  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

23.  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.

24.  Normal phase column
 Reverse phase column
 Ion exchange column
 Size exclusion column

26.  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.

27.  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.

28.  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.

29. 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

30.  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.

31.  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.

32. 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

33. 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

34.  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

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

36.  Recorders are used to recorded the
electrical signal from detector.

37.  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.

38.  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.

39.  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.

40.  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