1. INSTRUMENTATION AND APPLICATION OF HPLC
PREPARED BY: PARMAR PRATIK
GUIDED BY: MS.JENEE CHRISTIAN
ASSISTANT PROFESSOR
FACULTY OF PHARMACY
DHARMSINH DESAI UNIVERSITY, NADIAD
2. HPLC
High Performance Liquid chromatography.
Hplc is the fastest growing analytical technique for analysis of drugs.
The analytical technique in which component of mixture are separated
by ushing high pressure is called hplc or high pressure liquid
chromatography.
It is widely used in industry, research and development center, quality
control laboratories and educational institutions.
4. Solvent Reservior
• Also known as mobile phase.
• Mobile phase is usually organic or aqueous or mixture of both.
• Mobile phase is placed in bottle glass.
Characterstic of mobile phase
• Pure
• Law viscocity
• Law price
• Solubility of the sample
• Chemically inert
5. • The volume capacity of reservoir should be 500ml.
• The reservoir should be placed in plastic container.
6. Tubing
• The nature of tubing used to connect all parts of the system deserve
some attention.
• The inside diameter of diameter of tubing prior to injection device is
not critical but the tubing should be inert, have ability to withstand
pressure and be able to carry sufficient volume.
7. PUMP
• Pump is used for forcing the mobile phase through the column.
• Pump can be constructed from material that are inert to all mobile
phases.
• Material commonly used are glass,stainless steel,Teflon.
• The pump is capable for delivering high pressure upto 500psi at flow
rates of upto 3ml/min for analytical solution.
TYPES OF PUMP
1. Constant-Flow-Rate-Pump
2. Constant -Pressure Pump
8. 1.Constant-Flow-Rate-Pump
• The two principle type of pump in this category are the reciprocating
piston and syringe drive pump.
• The reciprocating piston pump is presently the most widely used pump
in hplc.
• This pump is characterized by a filling and pumping cycle.
• During the filling cycle a piston is with drawn from a syringe type
chamber.
• Two check valve are connected to this chamber such that during the
piston withdrawal solvent flow from the solvent reservoir but not into
pump outlet.
• When the piston direction is reversed the check valve operateto allow
solvent to flow from the outlet valve only.
9. • A principle advantage of reciprocating piston pump is the essentially
unlimited volume of the solvent reservoir since it external to the pump.
• A major disadvantage lies in the potential for producing pulses in the
flow rate.
• A more sophisticated ‘’single-head’’ reciprocating piston pump
(e.g.Altex Model 110A) provides for a very rapid filling cycle thus
minimizing the dead time between the cycle.it produce flow.
• Dual piston pump, operated 180 out of phase, further reduce the
pilsation since one head filling is while the other is discharging giving
flow.
10. 2.Constant-Pressure pump
• Constant pressure pump can deliver a constant-flow-rate if the pump
operates against a constant column back pressure and viscosity of
mobile phase remain constant.
• Consequently the temperature should be controlled.
• The simplest form of pump is reservoir such as coil of tubing to which
pressure is applied from a gas cylinder.
• While this system is simple,is suffer from very limited solvent
capacity.
• Since the mobile phase is direct contact with the pressurized gas a
significant amount of gas may be dissolved.
• Thus a portion of mobile phasemay contain sufficient dissolve gasesto
produce bubbles in the detector.
11. Sample injection system
• The injection of sample onto the column presents some unique
problem because of some unique problem present on hplc .
• Direct sample introduce via syringe in a gas chromatography is
simplest form of injection.
• The syringe must be specifically constructed to withstand pressure
upto1000-1500psi without leakage.
• Syringe injection technique can be used at much high pressure if stop
flow technique are used.
• While mobile phase is flowing the sample can be introduce into the
one chamber of piston .
• The piston is move rapidly to align .
• The most widely used sample injection system is loop injection valve.
12. • Sample is fliused through the sample loop with the excess going to
drain.
• For sample injection the valve is rotated so that mobile phase flows the
sample loop fluishing the sample to the column.
• The rotation of valve may be controlled by air –actuated device.
• A major drawback of this device is that the sample loop must be
changed.
13. Columns and Fittings
• Is the hardware used to connect the various components of lc system.
• The diameter of tubing prior of the sample injection device is
generally not critical.
• Lines connecting the injector to the column and column to detector
must be very small inner diameter in order to minimize extra column
broadening.
• It is good practise to place a 2µm in line filter between the sample
injection device and column.
• This traps particle introduce through the mobile phase or sample.
• Fitting is used to connect column to tubing and tubing to tubing must
be law volume in order to minimize extra-column brand broadening.
14. Detector
• Detector of hplc fall into three general categories.
• Diffential detector or bulk property detector provide a differtial
measurement of a bulk property that is possessed by both solute(analyte)
and mobile phase.
• The refractive index detector falls into this category.
• The solute property or selective detector measure a property of sample
which is not possessed by the mobile phase. Ultraviolet and fluorescence
detector falls into this category.
• The ideal hplc detector would possess several properties.it would have high
sensitivity,produce reoroducuble and predictable response and give a
response to any analyte.
• The detection limit or detecter sensitivity is generally consider to be the
concentration or mass of analyte entering the detector that will be produce a
signal-to-noise ratio of 2.
• A rule of thumb, the detector time constant is not greator than 0.5sec for
high efficiency saparation.
15. Uv-visible detector
• Measure the ability of solute to absorb light at a purticular
wavelength in the ultraviolet (UV) or visible (VIS) wavelength range.
• When light of certain wavelength is directed at flow cell, the
substance inside flow cell absorb the light. As a result, the intensity of
light that leaves the flow cell is less than of the light that enters.
• An absorbance detector measures the extents to which the light
intensity decreases
• All uv visible detector operates on principle of beer’s law.
• Uv-visible detector claasified based as affixed or variable wavelenth
detector.
• The most common wavelenth detector are based on the 254nm line
generated by a law pressure mercury vapour lamp.
16. • By ushing appropriate phosphor, the 254nm cause emission of line at
280nm
• Schematic representation of uv visible hplc detector.
17. Refractive Index Detector
• Refractive index detector or differential refractometers are universal
detector in that they respond to virtually any solute provided that the
refractive index of solute is significantly different from that of the
mobile phase.
• A major disadvantage of refractive index detector is its severe sensitivity
to temperature.
• Temparature of mobile phase, column and detector must be rigorously
controlled if accurate measurement of high sensitivities of 10-5
RIUFS(Refractive index units full scale ±1% noise) are common with
commercial instruments.
• The most common RI detector is deflection refractometer.
• In this detector the beam of light is collimated and focus on detector cell.
18. • The Fresnel refractometer is based on Fresnel’s law of reflection .
• This law states that the percentage of light reflected at a glass-liquid
interface is proportional to the angle of incidence and the refractive
indices of the two substances.
• The interferometric refectometer is based on the shearing
interferometric refractometer.
• In this detector the light beam is spilt by a beam spilter, then
recombined by a collimating lens and beam spilter, and focussed on a
photodetector.
19. Recorders
• Recorders are integraters used in hplc are essentially the same as used
in gc.
20. Application of hplc
• Quality control testing of drugs.
• In qualitative and quantitative analysis.
• Saparation and control of impurities.
• In analysis of biological fluids.
• Stability studies.
• Therapeutic monitoring of drug metabolism studies.
• Industrial application.
• Determination of synthetic intermediates ex: Atenolol.
• Stability testing ex:Acyclovir
