1. Seminar presentation on:
HPLC
(High Performance Liquid Chromatography)
PRESENTED BY: SIRAJUDDIN MOLLA
1ST SEM, M.PHARM
DEPARTMENT OF PHARMACOLOGY
SPER, JAMIA HAMDARD
3. Chromatography is an analytical method in which separation of active
constituent occurs in complex mixture.
It is technique is used for separation, purification, Identification and
INTRODUCTION
4. The basic principle of chromatography is based on Adsorption
and partition chromatography.
Adsorption chromatography - The affinity of molecules towards
stationary phase is known as Adsorption chromatography.
Partition chromatography - The molecule can moves in two
of liquid is known as partition chromatography.
• It is important for qualitative and quantitative analysis.
Contd…
5. A. Based on modes of chromatography
1. Normal phase mode
2. Reverse phase mode
B. Based on principle of separation
1. Adsorption chromatography
2. Ion exchange chromatography
3. Ion pair chromatography
4. Size exclusion(or)Gel permeation chromatography
5. Affinity chromatography
6. Chiral phase chromatography
TYPES OF CHROMATOGRAPHY
6. C. Based on elution technique
1. Isocratic separation
2. Gradient separation
D. Based on the scale of operation
1. Analytical HPLC
2. Preparative HPLC
E. Based on the type of analysis
1. Qualitative analysis
2. Quantitative analysis
Contd…
8. The different component in the
mixture pass through the column and
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
HPLC instruments consist of a reservoir of mobile phase, a
pump, an injector, a separation column, and a detector.
Compounds are separated by injecting a sample mixture onto the
column.
Contd…
9. High Performance Liquid Chromatography is a form of liquid
chromatography, modified from of gas chromatography.
It is applicable for both Volatile as well as Non volatile compound and
it is now one of the most powerful tools in analytical chemistry (having a
high resolution and separation capacity).
It has the ability to separate, identify, and quantify the compounds that
are present in any sample that can be dissolved in a liquid.
HPLC is the most accurate analytical methods widely used for the
quantitative as well as qualitative analysis of drug product.
HPLC is characterized by the use of high pressure to push a mobile
phase solution through a column of stationary phase allowing separation
of complex mixtures with high resolution.
Contd…
10. Other terms of HPLC:
High Performance Liquid Chromatography
High Pressure Liquid Chromatography
High Patience Liquid Chromatography
High Priced Liquid Chromatography
High Precision Liquid Chromatography
Contd…
11. The principle of separation in normal phase mode and
reverse phase mode is adsorption.
A solution of the sample is injected into a column of a
porous material (stationary phase) and a liquid (mobile
phase) is pumped at high pressure through the column.
The separation of sample is based on the differences in
the rates of migration through the column arising from
different partition of the sample between the
and mobile phase.
Depending upon the partition behaviour of different
components, elution at different time takes place.
principle
Mobile
Phase
Stationary
Phase
Solven
t
Bonded
Phase
Separation is based on
the analyte's relative
solubility between two
12. The High Performance Liquid Chromatography is more versatile than
gas chromatography since
(a) It is not limited to volatile and thermally stable samples,
and
(b) The choice of mobile and stationary phases is wider.
When a mixture of components are introduced into a HPLC column,
they travel according to their relative affinities towards the
stationary phase.
The sample compound with the greater affinity to the stationary
layer will travel slower and for a shorter distance in comparison to
compounds with less affinity which travel faster and for a longer
distance.
Contd…
14. The principle of HPLC are based on Van Deemter equation which
relates the efficiency of the chromatographic column to the particle
size of the column, molecular diffusion and thickness of stationary
phase.
The Van Deemter Equation is given as
H or HETP = A + B + Cv
where, A= represents eddy diffusion
B= represents molecular diffusion
C= represents rate of mass transfer
u= represents flow rate
Contd…
height equivalent to a theoretical plate (HETP)
15. 1. Normal Phase: Separation of polar analytes by partitioning onto a
polar, bonded stationary phase.
2. Reversed Phase: Separation of non-polar analytes by partitioning
onto a non-polar, bonded stationary phase.
3. Adsorption: In Between Normal and Reversed. Separation of
moderately polar analytes using adsorption onto a pure stationary phase
(e.g. alumina or silica)
4. Ion Chromatography: Separation of organic and inorganic ions by
their partitioning onto ionic stationary phases bonded to a solid support.
5. Size Exclusion Chromatography: Separation of large molecules
based in the paths they take through a “maze” of tunnels in the stationary
phase.
TYPES OF HPLC SEPARATIONS
16. Stationary Phase:
It is polar or non-polar and the
stationary solid surface is coated with a
2nd liquid (the Stationary Phase) which
is immiscible with mobile phase.
Silica, Alumina, cellulose
Very small particle size Large surface
area High resolution
pressure maintained 40MP
HPLC CONTAIN
Mobile Phase:
A polar or non-polar mobile phase
the most preferred mobile phase
solvent is acetonitrile(ACN) because it
has optimal viscosity, good UV
transparency, and adequate elution
strength.
Other Me-OH, WATER + BUFFER.
17. Solvent/ mobile phase reservoirs
Gradient controller and mixing unit
De-gassing of solvents
Pump
Pressure gauge
Pre-column
Sample introduction system
Column
Detector
Recorder
INSTRUMENTATION OF HPLC
19. Glass or stainless-steel containers capable of holding up to1
litter mobile phase (pure organic solvents or aqueous
solutions of salts and buffers)
Inert to a variety of aqueous and non aqueous mobile
phases.
Stainless steel should be avoided for use with solvents
containing halide ions.
SOLVENT/ MOBILE PHASE
RESERVOIRS
20. Tubing:
Should be inert.
have ability to withstand pressure.
able to carry sufficient volume.
DEGASSING & FILTRATION OF
MOBILE PHASE
In many cases, aqueous solvents & some organic solvents are
degassed prior to use
Degassing is done to prevent formation of gas bubbles in the pump
or detector ( Mobile phases are degassed by stirring of the mobile
phase under vacuum, sonication or sparing with helium gas)
The mobile phase are filtered to remove particulate matter that may
clog the system
21. The solvents or mobile phase must be passed
through a column at high pressures at up to 6000
psi (Ib/in2) or 414 bar.
As the particle size of stationary phase is smaller
(5 to 10µ) the resistance to the flow of solvent
will be high.
That is, smaller the particle size of the stationary
phase the greater is the resistance to the flow of
solvents.
• Hence high pressure is recommended.
PUMP
22. Several injector devices are available either for manual or auto
injection of the sample.
1. Septum Injector
2. Stop Flow Injector
3. Rheodyne Injector
SAMPLE INJECTOR SYSTEM
1. Septum injector
These are used for injecting the sample through a
rubber septum.
This kind of injectors cannot be commonly used,
since the septum has to withstand high pressures.
23. 2. Stop Flow injector:
In this type the flow of mobile phase is stopped for a while &
the sample is injected through a valve.
3. Rheodyne injector:
It is the most popular injector and is widely used.
This has a fixed volume of loop, for holding sample until its
injected into the column, like 20uL, 50uL or more.
Through an injector the sample is introduced into the
column.
The injector is positioned just before the inlet of the column.
Contd…
24. 1. Pre-column
It contains a packing chemically identical to that in
analytical column.
Mainly used to remove the impurities from the solvent
and thus prevents contamination of the analytical
column, it can protect analytical column.
It is also called as guard column or protective column.
It contains large particle size.
It is having short length of 2 to 10 cm, so does not affect
separation.
Stainless steel
Can withstand
pressure up to 50 MP
5-25 cm lenth
4.5mm diameter
Flow rate:1-3 ml/min
COLUMN
25. 2. Analytical column
The success or failure of analysis depends upon choice of
column.
Actual separation is carried out here.
Stainless-steel tube
Length 25 to 100 cm
Internal diameter 2 to 4.6 mm
Column is filled with small particles 5-10 micron. The solid
support can be silica gel, alumina.
The separation is result of different components adhering to
or diffusion into the packing particles when the mobile
phase is forced through column.
Contd…
26. C8 and C18 columns are considered as examples of reversed phase liquid
chromatography (RP).
The stationary phase here is seen as a thin film of non-polar liquid phase
that has been designed to be chemically similar to an inert material (Silica gel
particles).
The non-polar layer is chemically linked to the silica particles surface by reaction
with the polar silanol groups on the stationary phase surface and so rendering
them less polar or non-polar.
The difference between the two columns will be in the length of the carbon-
chain attached to the silica surface.
Accordingly C8 hplc columns have packing material composed of silica
particles attached to C8 carbon units.
C18 will, of course, have packing materials coated with C18 hydrophobic units.
Categorically both are reversed phase but C18 columns will definitely be more
hydrophobic rather than the C8 columns
27. UV/Vis detector and PDA (Photodiode array): Compounds
with chromophores, such as aromatic rings or multiple
alternating double bonds.
IR detector
Refractive index detector (detects the change in turbidity)
Mass spectrometry (HPLC-MS)
Electrochemical detector (ECD): measures current flowing
through a pair of electrodes, on which a potential difference
is imposed, due to oxidation or reduction of solute
Conductivity detector (CDD) (for ions): Charged
compounds, such as inorganic ions and organic acid.
Light scattering detector
DETECTORS
28. High Sensitivity
Universality or predictable specificity
Large linear response range
Low dead volume
Non-Destructive
Insensitive to temperature & mobile phase
Continuous operation
Reliable and easy to use
No single detector fits all these criteria
IDEAL DETECTOR PROPERTIES
29. 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 is chemically inert.
Wide varieties of stationary phase.
ADVANTAGES
30. Mobile phase is 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.
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.
Contd…
31. 1. Coelution
• Because of the speed of HPLC and it reliance on different polarities of compounds, two
compounds with similar structure and polarities can exit the chromatography
apparatus at the same time or nearly the same time. This is known as coelution.
2. Adsorbed Compounds
• HPLC typically uses a glass column filled with beads made of different materials.
• The mixtures being forced through the column have chemicals that bind with different
strengths to the beads.
• The strength of the binding, which depends on the similarity in polarity, determines
how long the chemical will bind to the bead before being released.
• Some compounds bind so strongly that they are essentially never released from the
beads in the column and are never measured in the solution exiting the column.
DISADVANTAGES
32. 3. Cost
Typical laboratory separation techniques involve developing an assay, or method of
separation, and then implementing that assay to separate individual compounds from a
solution. However this usually results in multiple solutions that also need to undergo
procedures, leading to an exponential increase in complexity. Although HPLC can often
simplify and speed up this process, the cost of developing an HPLC apparatus can become
tremendous. Developing an HPLC apparatus, although much more efficient, is much more
costly than developing other assays for separating compounds. This makes it not
financially viable for many small privately owned laboratories.
4. Complexity
HPLC is not only used to separate simple compounds, it is also used to isolate specific
proteins out of a cellular mixture. In this case the beads in the column are usually coated
with an antibody specific to the protein you need to collect. The proteins bind the the
antibodies and the remaining solution is passed through the column, then the proteins are
released using another solution and collected. This requires a highly skilled technician to
monitor the column at all times and make sure that the process is running exactly as
planned.
Contd…
33. Simultaneous analysis
High resolution
High sensitivity
Good repeatability
Moderate analysis condition
Easy to fractionate and purify
Not destructive
WHY HPLC USED
42. The information that can be obtained using HPLC includes
identification, quantification and resolution of a compound.
Preparative HPLC refers to the process of isolation and
purification of compounds.
This differs from analytical HPLC, where the focus is to obtain
information about the sample compound.
APPLICATIONS
43. Quantification of ions in human urine
Analysis of antibiotics in blood plasma.
Estimation of bilirubin and bilivirdin in blood plasma in case of
hepatic disorders.
Detection of endogenous neuropeptides in extracellular fluids of
brain.
2. Clinical applications
Tablet dissolution study of armaceutical dosages form.
Shelf-life determinations of pharmaceutical products
Identification of active ingredients of dosage forms
Pharmaceutical quality control.
1. Pharmaceutical applications
Contd…
44. It is based on the fact that certain compounds have different migration
rates given a particular column and mobile phase, the extent or degree
of separation is mostly determined by the choice of stationary phase
and mobile phase.
3. Chemical Separations
Purification is defined as the process of separating or extracting the
target compound from a mixture of compounds or contaminants.
Each compound showed a characteristic peak under certain
chromatographic conditions.
The migration of the compounds and contaminants through the column
need to differ enough so that the pure desired compound can be
collected or extracted without incurring any other undesired compound.
4. Purification
Contd…
45. Generally assay of compounds are carried using HPLC.
The parameters of this assay should be such that a clean peak of
the known sample is observed from the chromatograph.
The identifying peak should have a reasonable retention time and
should be well separated from extraneous peaks at the detection
levels for which the assay will be performed.
5. Identification
Detection of phenolic compounds in Drinking Water.
Identification of diphenhydramine in sedimented samples.
Bio-monitoring of pollutant.
6. Environmental applications
Contd…
46. Quantification of the drug in biological samples.
Identification of anabolic steroids in serum, urine, sweat, and
hair.
Forensic analysis of textile dyes.
Determination of cocaine and metabolites in blood.
7. Forensic applications
Ensuring the quality of soft drink and drinking water.
Analysis of beer.
Sugar analysis in fruit juices.
Analysis of polycyclic compounds in vegetables.
8. Applications in Foods and Flavours
Contd…
47. Drug Discovery
Proteomics
Drug Metabolism study
Diagnostic studies
Cosmetic analysis
Determination of Green Florescent Protein
Structural Determination
Identification of Bile Acid Metabolite
Biochemical Genetics
qualitative and quantitative analysis
Therapeutic Drug Monitoring
OTHER APPLICATIONS
Contd…
