This document provides an overview of high performance liquid chromatography (HPLC). It discusses the principle, types, instrumentation, parameters, advantages, disadvantages and applications of HPLC. The key points are: - HPLC was developed in the 1970s to improve separation efficiency by using high pressure to force the mobile phase through the column faster. - There are various types of HPLC based on the mode of chromatography (normal vs reverse phase), principle of separation, elution technique, scale of operation, and type of analysis. - The basic instrumentation includes solvent reservoirs, pumps, an injection system, columns, detectors and recorders. Common pumps are syringe pumps, reciprocating pumps and pneumatic pumps.

1. HPLC
HIGH PERFORMANCE LIQUID
CHROMATOGRAPHY
Tenzin palmo
Md Fayazuddin
(M.pharm)
Al-ameen college of pharmacy

2. CONTENTS
 INTRODUCTION
PRINCIPLE
TYPES OF HPLC TECHNIQUES
INSTRUMENTATION
PARAMETERS USED IN HPLC
ADVANTAGES OF HPLC
DISAVANTAGES OF HPLC
APLLICATIONS

3. INTRODUCTION
HPLC was developed in early 1970’s from a knowledge of already established
chromatographic techniques.
Originally , HPLC was referred to as High Pressure Liquid Chromatography
because , high pressure were used to increase the efficiency of separation .
But now-a-days , the term High Performance Liquid Chromatography is preferred
as it describes the characteristic of the chromatography .
Earlier , the separation of component by liquid chromatography was dependent on
gravity and it took lot of time to separate components . This problem was overcome
by HPLC in which the separation is assisted by means of increasing the force of
mobile phase at high rate .

4. PRINCIPLE
The principle of high performance liquid chromatography is based on adsorption
as well as partition chromatography depending on the nature of stationary
phase .
If the stationary phase is solid , principle is based on adsorption chromatography
.
If the stationary phase is liquid , principle is based on partition chromatography
.

5. TYPES OF HPLC TECHNIQUES :
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

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 on analysis
1. Qualitative analysis
2. Quantitative analysis

7. TYPES OF HPLC TECHNIQUES
A. Based on modes of Chromatography: This depends on the polarity of stationary and mobile phase.
1. Normal phase mode: In this mode, the stationary phase is polar in nature and the mobile phase is non-
polar. In this technique, the non-polar compounds travel faster and are eluted first, which is due to its
less affinity between solute and stationary phase . The polar compounds are retained for a longer time
in the column because of more affinity towards stationary phase and takes more time to be eluted from
the column.
This technique is not advantageous in pharmaceutical applications, since most of the drug substance
are polar in nature
and takes longer time t be eluted and detected, and hence not widely used.
2. Reverse phase mode: In this mode, the stationary phase is non-polar and mobile phase is polar in
nature. In this technique, the polar compounds get eluted first and the non-polar compounds re
retained for a longer time,
and hence this technique is widely used.
B. Based on Elution technique:
1. Isocratic separation: In this technique the same mobile phase (single solvent or combination of solvents)
is used throughout the process of separation i.e. the same polarity or elution strength is maintained
throughout the process.
2. Gradient separation: In this technique, a mobile phase combination of lower polarity or elution strength
is used followed by gradually increasing the polarity or elution strength. The separation efficiency is
greatly enhanced by gradient elution, here the proportion of two or more solvents is greatly varied in a
programmed way, sometimes continuously or in a series of steps.

8. C. Based on scale of Oparation:
1. Analytical HPLC: In this technique only analysis of sample is done, recovery of samples for reusing is
normally not done, since the sample used is very low.
2. Preparative HPLC: In this technique, the individual fractions of pure compounds can be collected using
fraction collector. The collected samples can be reused or analysed.
D. Based on Type of Analysis:
1. Qualitative analysis: It is used to identify the compounds, detect the presence of impurities i.e. by using
the retention time.
2. Quantitative analysis: It is done to determine the quantity of individual or several components in a
mixture i.e. by comparing the peak area of standard and sample.
E. Based on principle of separation:
1. Adsorption Chromatography
2. Ion Exchange Chromatography
3. Size Exclusion or Gel Permeation Chromatography
4. Affinity Chromatography
5. Ion pair Chromatography
6. Chiral Phase Chromatography

9. INSTRUMENTATION OF HPLC
In HPLC, the mobile phase from the solvent reservoir is filtered, pressurized and
pumped through the chromatographic column. A mixture of solute injected at the top
of the column is separated into components on travelling down the column and the
individual solutes are monitored by the detector and recorded as peaks on the chart
recorder.

10. Thebasiccomponentsof HPLCChromatographare:
 Solvent delivery system or Solvent reservoirs-----Mobile phase
 Pumping system or pumps
 Sample injection systems
 Column-Guard and Analytical columns----Stationary phase
 Detectors
 Recorders
Solvent delivery system or Solvent Reservoir:
• Solvent delivery system consist of solvent reservoirs, inlet filters, degassing facilities and mixing
units which work in conjugation with vacuum pumps and flow controls.
• Solvent reservoirs: HPLC system is equipped with one or more glass or stainless steel reservoirs,
each of which can hold 500ml or more of the solvent. The reservoirs are often equipped with a
means of removing dissolved gases usually oxygen and nitrogen, which interferes by forming
bubbles in the columns and detector systems by causing band spreading.

11. Degassers---- which are used to remove the dissolved gases in the mobile solvents several gases are soluble in organic
solvents, and when solvents are pumped under high pressure, gas bubbles are formed which interferes with the separation
process , steady baseline and shape of the peak.
• Degassing can be carried out by any one technique:
I. Vacuum filtration– It is the filteration of solvent using a vacuum pump.
II. Helium purging—It is by passing the helium gas through the solvent in which the dissolved gases are swept out of the
solution by fine bubbles of inert gas of low solubility.
III. Distillation system—In this the solvent is distilled out and used.
IV. Ultrasonication—by using sonicator , it converts ultra high frequency to mechanical vibrations, which causes the
removal of air bubbles.
V. Heating and Stirring the solvents
Filters---They are used for removing dust and particulate matter from the solvent . The solvents are usually filtered
through a Millipore filter (2μm-0.45μm) under vacuum before introduction into the solvent reservoir . this treatment
removes gases as well as suspended matter.
Mixing unit—is used to mix the solvents and pass through the column. There are two types of mixing units, low pressure
mixing unit(using inert gas helium) and high pressure mixing unit(by using static or dynamic mixers or stirrers and
operates under high pressure).

12. PUMPING SYSTEMS OR PUMPS
• Pumps are used to pass the mobile phase through the column at high pressure and at controlled flow
rate.
Properties of pumps---
• It should
 Generate pressure upto 6000psi
 Have flow rates ranging from 0.1ml-5ml per minute.
 Have flow control and flow rate reproducibility of ±0.5%
 Should be composition resistant and give a pulse free output.
 Easy to change from one mobile phase to another.
 Easy to dismantle and repair.
 It must be resistant to corrosion by a variety of solvents
 The high generated pressure by the pumps should not led to any kind of explosion hazards.
• Check valves----are used to control the flow rate of the solvent and back pressure.

13. There are three kinds of pumps:
1.Constant displacement pumps or Syringe pumps
2.Reciprocating pumps
3.Constant pressure pumps or Pneumatic pumps
1.Constant displacement pumps or Syringe pumps
• It consist of a large syringe like chamber equipped with a plunger that is activated by a screw
driven mechanism powered by a stepping motor. This works on the principle of positive solvent
displacement by a piston mechanically driven at constant rate in a piston chamber with the
generation of pulse less flow with high pressure capability. Double syringe pumps have also been
developed in which one is delivering solvent to the column while the other is refilled from the
reservoir.
Advantages:
 It is pulse free and flow rate is controlled.
 Flow rate is independent of viscosity and column back pressure.
Disadvantages:
 Lack of solvent capacity(limited to 200-500ml)
 Solvent changing is inconvenient.

14. Constand displacement pump:

15. 2. Reciprocating pumps:
• A reciprocating pump is driven electrically, the driven piston pressurizes the eluent directly or hydraulic
oil, which is acting by via a diaphragm, pressurizes the liquid from the solvent reservoir. The movement
of a plunger piston causes suction of the eluent during the backward stroke and the delivery of the
pressurized solvent during the forward stroke. The pump is equipped with ball check valves to ensure the
positive direction of flow.
• Pulse dampers are used in this type of pumps as the back and forth motion of the piston causes pulses
that will affect the detection. A simple pulse damping method involves a compressible gas is capped
upright portion of the tea tube to take up some of the portion of the pulsation energy. When the pump
refills, this energy is resealed to help smooth the pressure pulsation.
Advantages:
 Small internal volume(35-400μL)
 High output pressure(up to 10000psi)
 Readily used for gradient elution.
 Constant flow rates that are independent of column back pressure and solvent viscosity.
Disadvantages:
• It produces pulses, so pulse dampers are used, otherwise they produce a baseline noise on the
chromatogram.
• Pulse dampers---are used to dampen the pulses observed from the wavy baseline caused by the pumps.

16. Reciprocating pump:

17. 3. Pnematic pumps or Constant pressure pumps
• They use a source of pressurized gas(or pressurized by a compressed gas) to drive the
liquid mobile phase to the column either directly in holding coil or a stainless steel
cylinder or by means of flexible or moving separator in order to prevent substantial
dissolution of the gas in upper part of the liquid.
Advantages:
 Simple, inexpensive and pulse free.
Disadvantages:
 Limited capacity and pressure output only upto 2000psi.
 Gradient elution not possible.
 Flow rate depends on solvent viscosity column back pressure.

18. Pneumatic pump:

19. Sample injection system
• It is a device that is used to introduce the sample into the HPLC setup. The volume to be injected is
determined by the concentration of the components present in the sample. The overloading of sample
in the column causes band broadening. The sample is usually injected at the head of the column with
minimum disturbance of the column material.
• A good injection device should have:
 The smallest possible contribution to peak broadening.
 Should be convenient to use.
 Able to operate at high pressure.
 Chemically inert with the eluent and the sample.
 reproducible.
Two important types of sample injectors used are:
1. Syringe injection
2. Variable volume valve injection or Stop flow injection

20. 1. Syringe injection
• This is the earliest and simplest technique, here the syringes are used to inject the sample
and are designed to withstand high pressure. Fixed volume is introduced by making use of
the fixed volume injector. The sample devices are six port rotary valve. The sample is loaded
by means of a loading syringe. Excess sample is used in order to ensure that the storage line
is completely filled. This line may either be an external loop with adequate volume is to be
changed or an external cavity or fixed volume grooved at the surface of the rotor or drilled
into the rotor.

21. 2. Variable volume valve injection or Stopflow injection:
• A variable volume Is introduced by making use of an injection valve. Variable volume
injectors contain a needle port, which can be closed or sealed at high pressure to insert the
syringe. This too is a syringe injection but here the solvent flow is stopped momentarily. A
fitting at the column head is removed and sample is injected directly into the head pf the
column packing at atmospheric pressure, then the fitting is replaced and the system is again
pressurized. Then solvent line switching on causes the sample to be injected into the column.

22. Guard columns or precolumns:
• A guard column is placed between injector and press. Pump to protect the HPLC column from damage
and loss of efficiency caused by particulate matter and strong adsorbant in solvent. In HPLC, guard
columns serve to saturate the mobile phase with the stationary phase so that loses of stationary phase
from analytical column is minimised. The composition of the guard column should be similar to that of
the analytical column. The particle size is usually large, to minimise the pressure drop. It is mainly used
to remove the impurities from the solvent and thus prevent contamination of the analytical column.
Analytical columns:
• Column chromatography columns are constructed from smooth bore stainless steel tubing, or heavily
walled glass tubing. Column length ranges from 10-100cm and internal diameter of 2-6mm for analytical
column and ID of 6mm and more for preparative columns. Column packaging have a particle size of 3-
10μm. Recently micro columns are available with ID of 1-4.6mm, particle size of 3-5μm and length of 3-
7.5cm which works with high speed and gives high resolution and minimal solvent consumption.
Types of columns:
1. Standard columns
2. Radial compression column
3. Narrow-bore columns
4. Short and fast columns

23. 1. Standard columns:
• HPLC are done on columns with an internal diameter of usually 4-5mm. They provide a good
compromise between efficiency, sample capacity and the amount of packing and solvent required.
Column packing materials are uniformly sized and mechanically stable
2. Radial compression columns:
• In the radial column the packing takes the form of a cylinder and the flow of mobile phase passes
from the outside of the cylinder, through the packing to the outside of the cylinder, the separation
taking place on the way through. The packing is supported between two cylindrical frits and the
gap between represents the bed height or column length. The outer frit is the column inlet and
consequently the sample initially has a large area of stationary phase with which to interact. The
sample is injected into a radial column that disperses the sample over the total peripheral area of
the external frit. This type of column has a low resolution but high loading capacity and is not
suitable for separating complex mixtures. It can be used effectively with gradient elution
development techniques. Radial columns are slurry packed.
3. Narrow-bore columns:
• Bore size or the internal diameter of the column is decreased, then there is an increase in the
signal of the sample component. When bore size is decreased the volume of solvent requires is
decreased. Mobile phase of high purity is used. In these columns there is better homogeneity in
packing density and smaller temperature gradient across the column.

24. 4. Short and fast columns:
• A short, conventional bore column packed with small particles can save solvent cost, increase sample
throughput, and deliver higher sensitivity than conventional length columns. Time required is 15-
120secomds for isocratic elution and 1-4minutes for gradient elution. These columns are used where
analytical speed is essential i.e. in QC dept.
Types of column packing material in HPLC:
1. Pellicular or Superficially porous material: it consist of a core hat, surrounded by a crust or a layer of
material that is chromatographically active. The core is usually a non-porous spherical glass beads or
polymer beads, and the outer layer consist of porous particles. The particle size ranges from 30-40μm.
Limitations of this is that there is reduction or decrease in the surface area available in the column. E.g.
: perisorb A- contains silica, perisorbRP- contains silinised silica.
2. Microparticulate packing or small porous particles: this packing material is totally porous packing
material, and the particle size ranges from 3-10μm. Usually silica, alumina or an ion exchange resins
are used. Mainly silica particles are used with small particle size and spherical shapes. The limitation is
that silica is not chemically stable, it reacts with many compounds and also dissolves away in the mobile
phase. Silica also requires to use buffer solutions to maintain the Ph and prevent the stationary phase
from degrading. Eg: adsorbosphere-silica, IBM-silica.
3. Bonded phase packing: the bonded phase HPLC packings consist of a silica support with an organic
moiety bonded to it through a silicon carbon a covalent bonding system. This bonding is obtained by
chemical bonding reactions, and it is used in HPLC-partition chromatography.

25. Requirements of Stationary phase
1. The pore size of the stationary phase should be small. The optimum pore size should be ranged
between 100 to 1000 angstrom for small molecules. The most frequently used pore size is 300
angstroms for proteins and peptides and 100 angstrom for small molecules.
2. Particle size of the stationary phase is also an important factor as it effects the pressure
development. Larger particle size generates less pressure but small particle size gives more
pressure. The particle size range used is from 3-5μm and most frequently used is 5μm.
3. The stationary phase is required to be mechanically stable (especially to high pressure and liquid
shear forces) and it should be chemically inert under chromatography conditions.
4. In HPLC using partition as principle bonded phase supports should be used as stationary phases,
here the stationary phase is covalently bonded to the support particles or the inside wall of the
column tubing. This is done to prevent bleaching of stationary phase.

26. Requirement of Mobile phase:
1. Polarity – it is an important criterion for mobile phase selection. The solvent system chose should
have neither high polarity nor should it have low polarity, the polarity of the solvent should be
moderate, in normal phase chromatography the solvent system used is non-polar while in reverse
phase it is polar solvent. one other criterion is that the polarity of the solvent is also decided on
the functional group of the analyte to be separated, in better words solvent can be selected by
matching the relative polarity of the solvent to that of the sample component or a solvent is
chosen to match the most polar functional group in the sample. Eg: alcohols for hydroxyl group
and ketones, acetates for carbonyl group etc.
2. The solvent system should be able to carry loosely held substances or weakly soluble substances
and carry out the process of separation efficiently.
3. Mobile phase used must be compatible to the detector that is used for the specific HPLC process.
Viscosity is the major consideration and requirement for the development of high column
efficiencies.
4. Grade of solvent should be selected for HPLC which is recently constant physical and chemical
characteristics. High grade solvents or analytical grade solvents are used.
5. Solvent system containing buffers are also used in HPLC, where the pH should be maintained.
Mainly the buffering solutions that are used in the range pf 4-8 Ph.

27. • Detectors in HPLC:
• Detectors are devices that will identify a particular substance and show a specific response that
indicates this substance.
Criteria for HPLC detectors:
1. Good sensitivity to all eluting compounds.
2. Should not degrade the separation obtained in the column.
3. Reliable and easy to operate.
4. Good reproducibility.
There are mainly two types of detectors
1. Bulk property detectors- which compare an overall change in physical property of the mobile
phase with and without an eluting solute.
• eg: refractive index detector, conductivity detector
2. Solute property detector-respond to physical property of the solute that is not exhibited by the
pure mobile phase. It gives a good sensitivity and detects even the smallest quantity of sample.
• Eg: ultra violet, visible adsorption , fluorescent and electrochemical detectors.

28. Refractive index detector
• The detection is based on the fact that the refractive index of the mobile phase may be different
from that of mobile phase containing a solute and the differences is directly proportional to the
concentration.
• Detection occurs when light is bent due to sample eluting from the column, and this is read as a
disparity between the two channels.
Types of refractive index detectors
1. Deflection refractometer: it measures the deflection of a beam of monochromatic light by a double
prism. Eluent passes through half of the prism, pure mobile phase passes through or fills the
other half. The two compartment are separated by a glass plate mounted at an angle such that
bending of the incident beam occurs if the two solutions differ in refractive index. The resulting
displacement of the beam with respect to the photosensitive surface of detector causes variation in
the output signal, which when amplified and recorded, provides the chromatogram.
Advantages:
 They respond nearly to all solutes.
 They are reliable.
 They are unaffected by flow rate.

29. Disadvantages
 They lack in high sensitivity.
 They are not much suitable for gradient elution.
 They need strict temperature control to be operated at their higher sensitivity.

30. Reflection type refractometer / Frensel refractometer
• This type of detector measure the change in percentage of reflected light at a glass liquid
interface as the refractive index of the liquid changes. In this detector the column, the column
mobile phase as well as the reference flow of solvents are passed through small cells on the
back surface of the prism. When the two are liquids are identical there is no difference
between the two beams reaching the photocell. A change in the amount of light transmitted to
the photocell, if the mobile phase containing solute passes through the cell, and as a result a
signal is produced.

31. Ultraviolet detectors
• It works on the principle of adsorption of UV-visible light as the effluent from the column is
passed through small flow cell held in the radiation beam. The UV detector measure the
change in the UV adsorption as the solute passes through a flow cell (usually 10μl in volume)
In a UV transparent solvent. A light source delivers a monochromatic parallel light beam
which passes through a cell swept by a column effluent, and fall on the photo cell. A signal
proportional to the amount of light received is measured and recorded. The source of light of
the sample UV photometer is generally a mercury lamp providing work at 200 to 400nm.

32. UV detectors are of three types:
1. Fixed wavelength detectors- uses a light source that emits maximum light intensity at one or several
wavelength that are isolated by appropriate filters. HPLC detectors which do not allow changing the
wavelength of the radiation that is at fixed wavelength is called Fixed wavelength detectors.
• A fixed wavelength detectors uses a light source that emits maximum light intensity at one or several
discrete wavelength that are isolated by appropriated filters.
• Such a detector offers a minimum of noise but no free choice of wavelength.
• Using a medium pressure mercury lamp, wavelength of 254, 280, 313, 334 and 365nm can be selected by
the use of narrow band pass interference filters.

33. 2. Variable wavelength detectors-
• It offers a wide selection of UV and visible wavelengths, but at an increased cost. To
obtain a complete spectrum, the eluent flow must be stopped to trap the component
of interest in the detector cell while the UV- visible spectrum region is scanned.

34. 3. Scanning wavelength detectors-
• To obtain a real time spectrum for each solute as it elutes , solid state arrays are
required like PHOTODIODE ARRAY DETECTORS: light from continuous source
example: deuterium lamp passes through a lens system which focuses polychromatic
light onto the flow cell (containing sample). The transmitted light then falls on a
holographic gratings, where it is dispersed into a photodiode array(PDA).
ADVANTAGES OF PDA DETECTORS
 It provides spectra of each peak and can be used for peak purity analysis.
 A very selective detector which will detect only such solutes that specifically absorb
UV/visible radiation e.g. alkenes, aromatic and compounds having multiple bonds
between C, O, N , S.
DISADVANTAGES OF PDA DETECTOR
 It is less sensitive and more expensive than single wavelength detector

35. Fluorescence detector
• The fluorescence detectors are based on the principle that a UV beam of light is focused in a
detector cell, swept by the column eluent. Light emitted in perpendicular direction is collected on
the photocell and its intensity measured a s a function of concentration elute in the effluent
stream. Its easy to combine a fluorescence detector to a UV photometer. It is a specific detector that
sense only those substances that fluoresce. A flow cell is used as a sensor through which the
excitation light passes axially. A photocell is situated at the side of the cell to receive a radially
emitted light. The cell wall is made of Pyrex glass to prevent the excitation light from reaching the
photocell. When the solute that fluoresces in the excitation light is situated in the cell, the
fluorescent light passes through the walls of the cell onto the photocell, the output of which is
electronically processed and passed to a computer.
• The excitation light may be UV at 254nm produced by the mercury lamp or it may be light of any
wavelength selected from the light produced by the deuterium lamp using a monochromator.
Advantages
 It has higher detection sensitivity than UV detection.
 Lower detection limits.
 Less sensitive to fluctuations

36. Disadvantages
 Careful choice of mobile phase, Ph and mobile phase composition
• eg: aniline is cation at acidic Ph and do not fluorescent but in pH range of 7-12 it exist as a neutral
species and fluorescent.
Recorders
• The signals from the detector are recorded as deviations from the baseline. The peak position along the
curve(retention time) denotes a particular compound and a peak area or height is a measure of amount of
component in the sample.
•

37. PARAMETERS USED IN HPLC:
1. Retention time:
Retention time is the difference in time between the point of injection and
appearance of peak maxima . It is defined as the time required for 50% of the
components to be eluted from a column . Its is measured in minutes and seconds .
2. Retention volume :
Retention volume is the volume of carrier gas required to elute 50% of the
components from the column . It is the product of retention time and flow rate .
Retention volume = Retention time x flow rate

38. 3. Resolution :
Resolution is the measure of extent of separation of 2 components and the
base line separation achieved .
Rs = 2(Rt1-Rt2)/w1+w2
4. Separation factor :
Separation factor is the ability of the chromatography system to chemically
distinguish between sample components . It is usually measured as the ratio of the
retention factor of the two peaks in the compositions.

39. 5. Height equivalent to a theoretical plate (HETP) :
The theoretical plate is a imaginary or hypothetical unit of a column where
distribution of solute between stationary phase and mobile phase has attained
equilibrium . It can also be called as a functional unit of the column .
6. Asymmetry factor :
The asymmetry factor is a measure of peak tailing . It is defined as the distance
from the centre line of the peak to the backslope divided by the distance from the centre
line of the peak to the front slope , with all measurements made at 10% of the maximum
peak height.

40. ADVANTAGES OF HPLC
1. Separation fast and efficient (high resolution power).
2. Continues monitoring of the column effluent .
3. It can be applied to the separation and analysis of very complex mixture .
4. Accurate quantitative measurements .
5. Repetitive and reproducible analysis using the same column
6. Adsorption , partition , ion exchange and exclusion column separations are
excellently made .
7. High separation capacity , enabling the batch analysis of multiple components .

41. 8. Superior quantitative capability and reproducibility .
9. Moderate analytical condition .
10. Generally high sensitivity
11. Low sample consumption .
12. Easy preparative separation and purification of samples .
13. HPLC has no restriction to volatile and thermally stable solute and the choice of
mobile and stationary phase is much wider .
14. Both aqueous and non aqueous sample can be analysed with little or no sample
pre treatment .
15. A variety of solvents and column packings are available , providing a high degree
of selectivity for specific analyses .
16. It provides means for determination of multiple components in single analysis .

42. DISADVANTAGES OF HPLC
1. Complexity , need a skill person to run the instrument .
2. Solvent consumption is more .
3. High cost .
4. Coelution
5. Adsorbed compounds .

43. APPLICATION OF HPLC
• The main applications of HPLC in pharmacy are:
 Natural products: HPLC is an ideal method for the separation of various components in plant
extracts which resemble in structure like analysis of digitalis, cinchona, liquorice and ergot extracts.
 Stability studies: HPLC is used for ascertaining the stability of various pharmaceutical substance
and products. I.e in impurity profiling and analysis of various degradation products.
 HPLC can be used to complement bioassays, which are very costly, gives poor precision, requires
more time and necessitates replication. HPLC is for analysis of penicillin, chloramphenicol, sulpha
drugs, peptide hormones, clotrimoxazole etc.
 HPLC has been used in the design of pharmaceutical dosage form by studying the pharmacokinetics
of the drugs.
 HPLC has been used for analysis of various categories of compounds like alprazolam, captopril,
diclofenac, haloperidol, folic acid, norfloxacin, omeprazole etc.
• The other applications are:
 Qualitative analysis: identification of compounds. This is done by comparing the retention time of
the sample as well as the standard.
 Checking the purity of a compound: by comparing the chromatogram of the standard and the
sample, the purity of the compound can be found.

44. • Continued…..
 Presence of impurities- this can be found out by the additional peaks in the
chromatogram when compared with a reference material.
 Quantitative analysis
• Direct comparison method: by injecting a sample and standard separately and
comparing their peak areas, the quantity of the sample can be determined.
• Calibration curve method- a calibration curve of peak area Vs concentration of the drug
is plotted. From the peak area of unknown sample, by interpolation, the concentration of
the sample can be determined.
• Internal standard method: a known concentration of the internal standard is added to
sample solution whose conc. Is not known. A compound with similar retention
characteristics is used.
 Isolation and identification of drugs or metabolites in biological fluids like urine,
plasma, serum etc can be carried out.
 Isolation and identification of mixture of components of natural or synthetic origin.
 Purification of some compounds of natural or synthetic origin on preparative scale.

45. THANK YOU