instrumentation of hplc

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INSTRUMENTATION OF HPLC Prepared by M.D.Dwivedi B.Pharm ɪv th Yr.

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Gives basic idea about instrumentation of high performance liquid chromatography

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Page 1: Instrumentation Of HPLC

INSTRUMENTATION OF HPLC

Prepared by

M.D.Dwivedi

B.Pharm ɪvth Yr.

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Instrumentation

Modern HPLC essentially consist of following main components:

Solvent delivery systems Pumping systems Sample Injector systems HPLC Column(s) Detector Data System

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HPLC Instrumentation Overview

Principle Pattern An Example

Solvent ReservoirsController

Solvent Cabinet

Vacuum Degasser

Binary Pump

Auto sampler

Column Compartment

Detector

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Solvent delivery systems

Continuously provide eluent (solvent).

Provide accurate mobile phase compositions.

Includes solvent reservoirs, inlet filter, and degassing facilities which works in conjugation.

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Solvent Reservoirs

A good HPLC unit should have 3-4 solvent reservoirs to release eluent into a mixing chamber at varying rate.

Inert container for holding the solvent (mobile phase).

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Inlet Filters

Type of filter. Stainless Steel or glass with 10

micron porosity.

Removes particulates from solvent.

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Degassing System

Removed dissolved gases (such as oxygen and nitrogen).

May consist of vacuum pump system, a distillation system, a heating and stirring device, or a system for spearing.

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Pumping System

Constant, reproducible, and pulse free supply of eluent to the HPLC column.

Flow rate in between 0.1-10 cm3 min-1 .

Operating pressures from 3000 psi to 6000 psi.

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Types of Pumping System

Mainly three types1. Constant flow reciprocating

pump2. Syringe (or displacement) type

pump.3. Pneumatic (or constant

pressure) pump.

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Constant flow reciprocating pump

The term "reciprocating" describes any continuously repeated backwards and forwards motion.

Widely used (~90% in HPLC system) type of pump.

It gives a pulsating delivery of the eluent. Pulse damper is used to make the flow pulse free. Deliver solvent(s) through reciprocating

motion of a piston in a hydraulic chamber.

Solvent is sucked during back stroke and gets deliver to the column in forward stroke.

Flow rates of eluent can be set by adjusting piston displacement in each stroke.

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Reciprocating pump

Working:

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Reciprocating Pump Continuous…

Advantages: Small internal volume (35-400 µL) High output pressures up to 10,000

psi. Smart adaptability to gradient

elution. Constant flow rates independent of

column back pressure, solvent viscosity and temperature.

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Syringe (or displacement) type pump

Consist of large syringe like chamber (capacity up to 500 Cm2).

Plunger activated by screw-driven and hydraulic amplifier machine.

Suitable for small bore column.

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Displacement Pump Continuous…

Working:

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Displacement Pump Continuous…

Advantages: Flow is independent of viscosity, back

pressure. Deliver pulseless flow. Provide pressure up to 78,000 psi.Disadvantages: Costly Low flow rate (1 to 100 mL/min). Limited solvent capacity. Inconvenience in frequent refilling i.e. in

changing solvent

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Pneumatic Pump

Gas is used to pressurize the mobile phase present in a collapsible solvent container.

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Pneumatic Pump Continuous…

Working:

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Pneumatic Pump Continuous…

Advantages: Not very costly. Provide pulse free flow.Disadvantages: Produce pressure only up to 2000 psi. Not suitable for gradient elution. Flow rate depends upon column back

pressure, and viscosity. Small capacity for filing of solvent.

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Sample Injection system

Introduce required sample volume accurately into the HPLC system.

Introduction of sample without depressurizing the system.

Volume of sample must be very small (2 µL to 500 µL).

Types of injection system:(a) Manual injection(Rheodyne/Valco

injectors)(b) Automatic injection

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Manual Injector

Also know as Rheodyne / Valco injectors. User manually loads sample into the

injector using a syringe. Overloading of column causes band

broadening hence volume used must be very small (2 µL to 500 µL).

Sample should be introduce without depressing the system.

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Manual Injector

Working:

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Automatic Injector System

Also know as Autosampler. Programmed based sample delivery system. User loads vials filled with sample solution into the

autosampler tray (100 samples). Autosampler automatically 1. Measures the appropriate sample volume, 2. Injects the sample, 3. Flushes the injector to be ready for the next sample, etc., until all sample vials are processed. Also controls the sequence of samples for injection from vials.

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HPLC Columns

Material: Stainless steel (highly polished surface).

External diameter: 6.35 mm Internal diameter: 4-5 mm (usual 4.6

mm) Length: 10-30 cm (usual 25 cm) Packing particles size (3 µm, 5µm, 10 µm) Stainless steel frits or mess discs (porosity< 2µm) retain packing material.

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HPLC Columns Continuous Efficiency or performance of a column may be measured by fallowing

expression : N = 16(VR/WB)2 …(a)

H = L/N …(b)

VR = Retention volume of the solute

WB = Volume occupied by a solute

( For efficient column WB < VR )

N = Plate number of the column (dimensionless) H = Height of the column (mm × µm) L = Length of the column (cm)

For more efficient column ‘N’ should be larger and correspondingly ‘H’ gets smaller.

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HPLC Columns Continuous

Factors affecting efficiency of column: Particle size Flow rate Thickness of stationary phase Mobile phase viscosity Diffusion of solute in mobile and

stationary phases How well a column is packed

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HPLC Columns Continuous…

For prolonged life of HPLC columns Guard column Scavenger column Column thermostats Guard column: Also know as pre-column. Placed in between injector and analytical

column. Having same material as in column but with larger size particles ~ 30-40 µm.

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HPLC Columns Continuous…

Scavenger column: Place between the pump and injection valve. Saturate the aqueous eluent (specially high or low pH buffers)with silica.

Column thermostats: HPLC is performed at ambient temperature in number of cases. Controls temperature of the column for better resolutions (chromatograms). HPLC is performed at ambient temperature in many cases.

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HPLC Columns Continuous…

On the basis of chromatographic objective HPLC column can be categorized as follows:

Scale Chromatographic Objectives

Analytical Information ( compound identification and concentration)

Semi-preparative Data and small amount of purified compound[<0.5 g]

Preparative Large amount of purified compound [>0.5 g]

Process (industrial) Manufacturing quantities ( g to kg)

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Stationary Phase (column packing)

The stationary phase is the substance fixed in place for the chromatography procedure.

The stationary phase can be a solid, a liquid, or a bonded phase.

Bonded phase is a stationary phase that is covalently bonded to the support particles or to the inside wall of the column tubing.

Chemically-modified silicas, unmodified silica or cross-linked co-polymers of styrene and divinyl benzene, commonly used as stationary phase.

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Stationary Phase Continuous…

Silica particles as the basis of the support.

Sizes 3 µm, 5 µm, and 10 µm (spherical and regular in shape).

Pore size normally are in the 60 – 100 Å range.

Pore size of 300 Å or larger being used for larger biomolecules.

Columns are packed using high-pressure to ensure that they are stable during use.

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Stationary Phase Continuous…

Several types of particles are used in HPLC column packing.

1. Micro porous (or diffusive) particle/Porous microsphere

2. Perfusion particles3. Nonporous (or micropellicular)4. Chiral (bounded) stationary phase

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Stationary Phase Continuous…

Thorough pore

Diffusive Pore

Perfusion Particle

5 µm

Microporus Particle

Micropellicularparticles

Solid core

Liquid or ion exchange film

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Stationary Phase Continuous…

Microporus (or diffusive) Particles: Main surface area is within the pores to

interact with the stationary phase. Small particles reduces the diffusion path

length and thereby band broadening. Zorbax Rx (Sil) (Silica sol) is a porous

microsphere silica particle with 50% porosity and a pore size of 100 Å.

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Stationary Phase Continuous…

Perfusion Particles: The particle consist of both small

(diffusive) and large (through) pores in them.

Diffusive pore provide sorption power. Through pore permits the mobile phase

to pass directly through the packing. Slightly larger than microporous particles (~ 12 µm).

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Stationary Phase Continuous…

Nonporous particles: Made from either silica or resin. Smaller in size (1.5 - 2.5 µm) with thin

porous layer.

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Mobile Phase

Also know as eluent. Solvent used in HPLC must be of HPLC grade

i.e. Filtered using 0.2 μm filter. Eluting power of the mobile phase is

determined by its overall polarity, stationary phase polarity and the nature of the sample components.

For 'normal-phase‘ separations eluting power increases with increasing polarity of the solvent, while for 'reverse-phase' separations eluting power decreases with increasing solvent polarity.

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Solvent Solvent strength e° parameter, (adsorption)

Solvent strength parameter, p’(partition)

UV cut-off (nm)

n-Hexane 0.01 0.1 195

Cyclohexane 0.04 -0.2 200

Tetrachloromethane 0.18 1.6 265

Methylbenzene 0.29 2.4 285

Trichloromethane 0.40 4.1 245

Dichloromethane 0.42 3.1 230

Tetrahydrofuran 0.56 4.0 212

Propanone 0.56 3.9 330

Acetonitrile 0.65 5.8 190

iso-Propanol 0.82 3.9 205

Ethanol 0.88 4.3 205

Methanol 0.95 5.1 205

Ethanoic acid >1 4.4 255

Water >1 10.2 170

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Detectors

The detector refers to the instrument used for qualitative and quantitative detection of analytes after separation.

Monitors the eluent as it emerges from column. Establishing both the identity and concentration of

eluting components in the mobile phase stream.Characteristics of detectors: Adequate sensitivity (10-8 to 10-15 g solute sec-1). Desired stability and reproducibility. Sort response time Minimal internal volume (minimize zone

broadening).

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Detectors Continuous

Sensitivity: Expressed as the noise equivalent concentration, i.e.

the solute concentration, Cn, which produces a signal equal to the detector noise level.

The lower the value of Cn for a particular solute, the more sensitive is the detector for that solute.

A linear response: The linear range of a detector is the concentration

range over which its response is directly proportional to the concentration of solute.

Type of response: Detector is either universal or selective. Universal (sense all the constituents of the sample). Selective (respond to certain components).

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Detectors continuous…

Types of Detectors: 1. Bulk property detectors2. Solute property detectorsBulk property detectors: Measure the difference in some physical

property of the solute present in the mobile-phase in comparison to the individual mobile-phase.

Not suitable for gradient elution.(a) Refractive - index detector(b) Conductivity detector

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Detectors continuous…

Solute property detectors: Respond to a particular physical or

chemical characteristic of the solute which should be ideally and absolutely independent of the mobile-phase being used.

(a) UV - detectors(b) Fluorescence Detectors(c) Electrochemical detectors

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Detectors continuous…

Detectors Used in HPLC:Type Limit of

detection (µg/cm3)

Response

Flow rate sensitivity

Temperature sensitivity

Gradient elution

UV/Visible absorption

10-4 Selective No Low Yes

Fluorescence 10-5 Selective No Low Yes

IR absorption 10-3 Selective No Low Yes

Refractive index 10-2 Universal No ± 10-4 oC No

Conductometric 10-2 Selective Yes ± 1 oC No

Amperometric 10-5 Selective Yes ± 1 oC _

Mass spectroscopy 10-5 Universal No None Yes

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Refractive - index detector

Also know as ‘RI-Detector’ and ‘Refract meter’. Based on refractive index measurement. Determine change of refractive index of the

eluant from the column with respect to pure mobile phase.

Types:(a) Deflection refractometer(b) Fresnel refractometer Referactive index (n) = c = speed of light in vacuumv = speed of light in medium

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Refractive - index detector continuous…

The RI of a few commonly used mobile-phase is stated below :

Mobile-Phase Refractive-Index

Benzene 1.501

Decane 1.410

Hexane 1.375

Octane 1.397

Tetrayhydrofuran 1.405

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Refractive - index detector continuous…

Working:

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Refractive - index detector continuous…

Advantages: Universal response Independent of flow rate.Disadvantages: Less sensitivity Temperature dependent, strict

temperature control (±0.001 °C). Not suitable for gradient elution.

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Conductivity detectors

Conductivity measurement of effluent. Mainly measure inorganic ions and small

organic substances, including organic acids and amines.

Conductivity detector measures electronic resistance and measured value is directly proportional to the concentration of ions present in the solution.

Employed as a detector in an ion chromatography.

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Conductivity detectors continuous…

Working:

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Ultraviolet Detector

Based on the principle of absorption of UV visible light as the effluent from the column is passed through a small flow cell placed in the radiation beam.

High sensitivity (detection limit of about 1x10-9 g mL-1 for highly absorbing compounds).

Detector cells are generally 1 mm diameter tubes with a 10 mm optical path length.

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Ultraviolet Detector Continuous…

Ultraviolet detector are of fallowing types:1. Fixed-wavelength detector2. Variable-wavelength detector3. Photodiode-array detectorsFixed-wavelength detector: Simplest UV absorption detector. Mercury lamp source, optical filters to

select a limited number of wavelengths 220, 250, 254, 280, 313, 334, 365, 436, and 546 nm.

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Ultraviolet Detector Continuous…

Variable-wavelength detector: Deuterium lamp (for UV region) or

Tungsten filament light source (for visible region) a diffraction grating monochromator for wavelength selection and a photomultiplier detector.

Allow monitoring at any wavelength within the working range of the detector.

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Ultraviolet Detector Continuous…

Photodiode-array detectors: A photodiode array (PDA) is a linear array

of discrete photodiodes on an integrated circuit (IC) chip.

A photodiode is a type of photodetector capable of converting light into either  current or voltage, depending upon the mode of operation.

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Ultraviolet Detector Continuous…

Photodiode-array detectors:

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Fluorescence Detectors

Based on filter-fluorimeters or spectrofluorimeters.

Flow cell has a capacity 10-25µL with a narrow depth (1.07 mm) and large surface area for excitation-emission collection. The fluorescent radiation emitted by the sample

is usually measured at 90° to the incident beam. Simplest detector: mercury excitation source,

and filters (one/more). Advanced detector: xenon source and a grating

monochromator to isolate emitted fluorescent radiation.

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Fluorescence Detector Continuous…

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Fluorescence Detector Continuous…

Emission Monochromatorsignal & spectra mode

PMT detector

Reference Diode

8 µl Flow Cell, auto-recognition

Trigger pack

Exitation Monochromator,signal & spectra mode

Mirror

Lens(condensor EX)

Lens (condensor EM)

Slit EM Slit PMTSlit EX

Diffuser

Xenonflash Lamp,15 W

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Electrochemical Detector

The term 'electrochemical detector' in HPLC normally refers to amperometric or coulometric detectors.

Measure the current associated with the oxidation or reduction of solutes.

Complete removal of oxygen is almost difficult, therefore, electrochemical detection is normally based upon the oxidation of the solute.

Amperometric detector is presently considered to be the best electrochemical detector.

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Electrochemical Detector Continuous…

Working electrode: Commonly made of glassy carbon, is the

electrode at which the electro active solute species is monitored.

Reference electrode: Usually a Ag-AgCl electrode, gives a stable,

reproducible voltage to which the potential of the working electrode is referred.

Auxiliary electrode: Current-carrying electrode and usually made

of stainless steel.

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Electrochemical Detector Continuous…

Potentiosta

t

From Column To waste

Auxiliary Electrode

Reference

Electrode

Working Electrode

Electro Chemical Detector

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Electrochemical Detector Continuous…

Advantages: Very small internal cell-volume, High degree of sensitivity, More limited range of applications, and Excellent for trace analyses as UV-

detector lacks adequate sensitivity.

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Applications of HPLC

Used for both qualitative and quantitative analyses of environmental, pharmaceutical, industrial, forensic, clinical, and consumer product samples.

A few typical examples: Isolation of natural pharmaceutically active

compounds Control of microbiological processes Assay of cephalosporins Assay of frusemide Assay of theophylline Assay of corticosteroids Assay of dichlorphenamide Assay of human insulin

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Applications of HPLC Continuous…

Isolation of natural pharmaceutically active compounds

Chromatographic Conditions :Column : Size-25 cm × 4.6 mm ID Adsorbent : Lichrosorb RP-8 Mobile-phase : Water/Acetonitrile-Gradient Elution Detector : UV 254 nm

Category of Natural Products

Constituents Used as

Alkaloids Morphine; Codeine Analgesic, Antitussive

Glycoside Digitalis glycosides Sennosides

Cardiovascular diseases, Laxatives

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Applications of HPLC Continuous…

Control of microbiological processes: Determine kinetics of the microbiological

process Monitoring of the on-going process Isolation and purification of active

ingredients Purity control of active constituents Monitoring derivatization reactions

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Applications of HPLC Continuous…

Controlled analysis of a microbiological process during Penicillin Production

Chromatographic conditions:Column : Size-25 cm × 4.6 mm ID Adsorbent : Lichrosorb-NH2 (10 μm)

Mobile-phase : 0.005 M H2SO4

buffer (pH4.4)/acetonitrile (50:50)Flow rate : 3 ml min-1

Detector : UV-220 nm

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Applications of HPLC Continuous…

Assay of Cephalosporins: Several commercially available

cephalosporin antibiotics have been adequately separated by HPLC methods under the following experimental parameters

Column : ODS-SIL-X-IIMobile-phase : 0.95 M Ammonium Carbonate/Methanol (95 : 5) Detector : UV-220 nm

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Applications of HPLC Continuous…

Assay of Theophylline: Theophylline contains other related substances as

impurities, namely : theobromine, caffeine and β-hydroxypropyltheophylline

Chromatographic conditions:Sample size : 10 μL Column : size – 250 × 4.6 mm ID Adsorbent : Lichrosorb (R) RP-8, 10 μmMobile-phase : 0.02 M KH2PO4 Buffer

(pH 3.5)/Acetonitrile (95 : 5) Detector : UV-254 nm

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Applications of HPLC Continuous…

Field Typical Mixture

Pharmaceuticals Antibiotics, Sedatives, Steroids

Biochemicals Amino acids, Proteins, Carbohydrates, Lipids

Food Products Additives, Artificial Sweeteners, Anti - oxidents

Polluants Pesticides, Herbicides, PCBs

Forensic Chemistry Drugs, Poisons,

Some other applications:

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References

1. Kar Ashutosh “Pharmaceutical Drug Analysis”, Revised Second Edition, New Age Internal (P) Limited Publishers, page no. 452-474.

2. Jeffery G.H., Bassett J., Mendham J. , Denney R. C., “Vogel's Textbook Of Quantitative Chemical Analysis”, Fifth edition 1989, Longman Scientific & Technical, Page no. 220-229.

3. Patnaik Pradyot, ”Dean’s Analytical Chemistry Hand Book”, Second Edition, McGRAW-HILL, Page no. 5.60-5.91.

4. Kealey D. , Haines P. J. , “Instant Notes Analytical Chemistry” , Frist edition 2002, BIOS Scientific Publishers Limited, Page no. 155-173.

5. Harvey David, “Modern Analytical Chemistry”, McGraw-Hill Higher Education, Page no. 578-589.

6. Lee David C. and Webb Michael L. , “Pharmaceutical Analysis” , Frist Published 2003, Blackwell Publishing Ltd, Page no. 44-49.

7. Kamboj P.C. , “Pharmaceutical Analysis volume ɪɪ Instrumental Methods” First Edition 2010, Vallabh Publication , Delhi, Page no. 239-280.

8. WWW.Google.co.in

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“Optimism is the faith that leads to achievement, nothing can be done without hope and

confidence” —-

Helen Keller