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
5.
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
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
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
22.
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
25.
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
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