This is the basic information of High performance liquid chromatography

1. HIGH PERFORMANCE LIQUID CHROMATOGRAPHY
(PART – 1)
Presented by
YENDA MANISHANKAR
M.PHARM ( Pharmaceutical Analysis)
GITAM Institute Of Pharmacy
Submitted to
Dr. M . MATHRUSHREE ANNAPURNA
Professor ( pharmaceutical chemistry)
GITAM Institute Of Pharmacy
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2. CONTENTS
• Principle
• Instrumentation
– Mobile phase reservoirs and solvent pre-treatment systems.
– Solvent delivery pumps
– precolums / guard column
– Solvent injection systems
– Analytical column
– Detectors
– Recorders and integrators
• Pharmaceutical applications
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3. INTRODUCTION
• High performance/ pressure liquid chromatography (HPLC) is a liquid chromatographic
technique in which the separation of the components from a mixture is achieved by
pumping mobile phase at high pressure through a suitably modified column.
• Since the mobile phase is delivered under high pressure by using appropriate displacement
pumps or gas pressure, the technique has derived its name as high pressure.
• Moreover, high performance in the name indicates that HPLC offers improved performance
than other conventional column chromatographic techniques. This is because it utilises a
stationary phase which has a very small particle size (3 – 20 µm) and provides a greater
surface area.
• This in turn increases the elution rate by over 100 folds, thereby rendering the method best
suited for efficient separation of complex mixtures. In simple terms, HPLC technique is an
improved version of conventional column chromatography.
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4. PRINCIPLE
The principle involved in the separation of solute components in HPLC is based upon the type of
technique chosen for the process:-
• Adsorption chromatography :- In this, separation depends upon the relative affinities of the
solute molecules towards the stationary phase. The solute molecules having greater affinity
towards the stationary phase take longer time to get eluted from the column while those
having less affinity elute faster.
• Ion – pair chromatography :- This technique is used for the separation of mixtures
containing wide variety of ionic and non – ionisable molecules. It utilizes a standard reverse
phase column which is converted temporarily into an ion exchange column using ion pairing
reagents ( e.g. : pentane/ hexane / heptane / octane sodium sulphonate, tetra methyl /
tetraethyl ammonium hydroxide ).
• Gel permeation / size exclusion chromatography :- This principle involved in separation is
the difference in the sizes and shapes of the solute molecules. Different types of gels are
used which act as sieves for the separation of molecules of different shapes and sizes under
the influence of steric or diffusion effects.
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5. • Affinity Chromatography :- In this technique, separation of components from a mixture
takes place depending upon the affinity of the solute molecules towards specific stationary
phase. It is widely employed in the field of biotechnology, microbiology etc., for the
separation of enzymes, nucleic acids etc.
• Chiral Phase Chromatography :- This technique is employed for the separation of optical
isomers using chiral stationary phases. The principle involved in separation differs with the
type of stationary phase used and the solute molecules to be separated.
• Ion – exchange Chromatography :- The principle involved in separation is the reversible
exchange of ions ( functional groups) which takes place between ion exchangers present on a
solid support and ionic solute molecules. This techniques is used to separate the mixtures
containing similar charged ions. The cation exchange resin is used for the separation of
cations and an anion exchange resin is used for the separation of anions from the solute
mixture.
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6. INSTRUMENTATION
Components of HPLC instrumentation units are:
• Mobile phase reservoirs and solvent pre-treatment systems.
• Solvent delivery pumps
• precolums / guard column
• Solvent injection systems
• Analytical column
• Detectors
• Recorders and integrators
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7. MOBILE PHASE RESERVOIRS & SOLVENT PRE-TREATMENT SYSTEMS
• Mobile phase selected in HPLC may be either a pure solvent or a mixture of two or more
solvents having appropriate eluting power for the sample components.
• The selection is mainly based on the polarity of the stationary phase, the nature of
sample components and upon the chromatographic method.
• Other properties like boiling point, flammability, viscosity, toxicity, and PH and detector
compatibility of the solvents are also considered.
• Isocratic elution either 1 or a mixture of two or more solvents with fixed composition is
used where is for gradient elution the composition of solvents used is changed
continuously.
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8. MOBILE PHASE RESERVOIRS
• Glass or stainless steel reservoirs of capacity 500 ml or more are
used in modern HPLC instrument for storing the mobile phase the
material chosen for construction of the mobile phase revolt should
not be reactive towards the mobile phase.
• Mobile phase used should be a pure solvent devoid of any
particulate matter or gases. Degassers and filtration systems are
often provided with reservoirs.
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9. SOLVENT DELIVERY PUMPS
In HPLC the mobile phase is passed through the stationary column under high pressure
by means of palms the terms used in HPLC should possess the following features: -
• They should generate high pressure up to 600 psi
• They should provide a flow rate ranging from 0.1 to 10 ml per minute flow control and
flow reproducibility up to 0.5 %
• All the components of the thumb should be resistant to the solvents employed in must do
a pulse free output
• there should be easy to dismantle and repair
• They should be easy to dismantle and repair they should be adaptable for replacing
calving or changing their proportions in case of gradient elution HPLC.
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10. PRE COLUMN / GUARD COLUMN
• A guard column is placed between the analytical column and sample injector port and is filled
with the same packaging material as that off the analytical column.
• It protects the analytical column from damage or loss of efficiency by strongly the
particulate matter or impurities present in the sample or the solvent
• These are made up of his stainless steel that helps them withstand high pressures of about 500
psi.
• They are usually short in length ranging between 2 to 20 centimeters smaller inner diameter
2 - 20 millimeters of three columns gives good results and helps to increase their working
efficiency.
• They are generally packaged with an inactive material of larger particle size impregnated with
20 to 30% of a suitable silicone face due to the larger particle size the pressure drop across the
free column is negligible when compared with that of the analytical column.
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11. SOLVENT INJECTION SYSTEMS
• In simple HPLC systems, the sample is introduced with an valve where as
sophisticated HPLC systems, an auto sampler provided with a microprocessor is
used.
• The sample is delivered to the column head ensuring that the column packing
material remains undistributed.
• There are three kinds of sample injectors are mostly used in HPLC:-
– Syringe injectors / septum injectors
– Stop flow injectors
– Loop injectors / Rheodyne injectors
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12. ANALYTICAL COLUMN
• Analytical column is the centra; component of HPLC instrument
where the actual separation takes place.
• Therefore , proper selection of the chemical nature and dimensions
of the column is necessary as it affects the resolution, peak
symmetry and the speed of analysis.
• Columns made up of either stainless steel, heavy glass, polyethylene
ether ketone (PEEK) are used.
• Stainless columns are preferred as they can withstand high
pressures. When columns made of heavy glass are used, the system
is maintained at pressures lower than 600 psi.
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13. DETECTORS
• The main function of detectors in HPLC is to monitor the column effluent and also
to detect the amount of sample present in it.
• The detectors emit electrical signals that are directly proportional to a certain
property or characteristic of either the sample or the mobile phase.
• The detectors used in HPLC should possess the following features:-
– They should possess high sensitivity and should be able to detect very low levels of solute.
– They should be cheap, reliable, easy to use, stable and non destructive.
– They should detect all the components in the sample in a wide range of mobile phases.
– They must be unaltered by changes in the temperature and flow rate.
– They should not lead to zone spreading.
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14. RECORDERS & INTEGRATORS
• They record the signals emerging from the detector as deviations from the base line. The electrical
signals obtained from the detector are amplified ( if necessary) and recorded as a function of time
with the help of a potentiometric recorder.
• The responses are obtained in the form of chromatographic peaks from which retention time of the
solute molecules is determined.
• However, the height/ area of the peaks which gives a direct measure of the concentration of a
component in the mixture cannot be determined.
• Integrators are improvised version of recorders. They possess data processing ability and record the
individual peaks with their retention time, height and width peak area, percentage of area etc.
• Therefore , they help to overcome the disadvantages associated with detectors.
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15. PHARMACEUTICAL APPLICATIONS
• Tablet dissolution study of the pharmaceutical dosage form.
• To control drug stability, Shelf-life determination.
• Identification of active ingredients.
• Pharmaceutical quality control.
• Tablet dissolution of pharmaceutical dosage forms.
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16. OTHER APPLICATIONS
Food and Flavour analysis :-
• Rapid screening and analysis of components in
non - alcoholic drinks.
• Measurement of quality of soft drugs and water.
• Sugar analysis in fruit juices.
• Analysis of polycyclic compounds in vegetables.
• preservative analysis.
• Multi residue analysis of lots of pesticides in
food samples by LC triple quadrupole MS.
Environmental applications :-
• Detection of phenol compounds in drinking
water.
• Identification of diphenhydramine in
sedimented samples.
• Bio-monitoring of pollutant.
• Rapid separation and identification of carbonyl
compounds by HPLC.
• LC/MS/MS solution for pharmaceuticals and
personal care products in water, sediment, soil
and bio - solids by HPLC/MS/MS.
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17. THANK YOU
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