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15 October 2012
ContentsContentsINTRODUCTIONCLASSIFICATIONINSTRUMENTATIONDERIVATIZATIONREFERENCES
25 October 2012
High Pressure Liquid Chromatography
or
High Performance Liquid Chromatography
What is it?
Separation technique based on solid stationary phase + liquid mobile phase
How can achieve separation?
By partition
adsorption
ion exchange
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CLASSIFICATION OF HPLC:-1. NORMAL PHASE: Stationary phase: Polar
Mobile phase: Non polar
Eg. Assay of Pilocarpine, Tacopherol, Piperazine
2. REVERSE PHASE: Stationary phase: Non polar
Mobile phase: Polar
Eg. Assay of Nifedipine, Sulphamethoxazole
Partition chromatography is used for hydrocarbon soluble compound having molecular weight of lesser than 1000gm/mole.
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3. ION-EXCHANGE CHROMATOGRAPHY (IEX)
Based on the different affinities of the ions for the oppositely charged ions in the resin or adsorbed counterions in the hydrophobic stationary phase.
Consider the exchange of two ions A and B between the solution and exchange resin E :
A·E + B B·E + A
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4. SIZE-EXCLUSION CHROMATOGRAPHY (SEC)
SEC is the method for dynamic separation of molecules according to their size.
The separation is based on the exclusion of the molecules from the porous space of packing material due to their steric hindrance.
Hydrodynamic radius of the molecule is the main factor determining its retention.
In general, the higher the hydrodynamic radius, the shorter the retention.
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HPLC INTRUMENTATION CONSIST OF
DegasserSolvent Reservoir( HPLC solvent reservoir systems)Pumps Pre Guard Column Sample injection systemColumnsDetectorRecorder and integrators
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DEGASSERDegassing of mobile phase is required because bubble has
property to expand or compress.
Degasser is needed to remove dissolved air
1) By Subjecting the mobile phase under vacuum.
2) By Purging with fine spray of an inert gas at lower solubility such as Argon and Helium.
3) By heating and ultrasonic stirring.95 October 2012
HPLC SOLVENT RESERVOIR SYSTEMS
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HPLC SOLVENT RESERVOIR SYSTEMSThese are the glass bottles use to store the mobile phase.
The mobile phase is pumped under pressure from one or several reservoirs and flows through the column at a constant rate.
Desirable feature in the solvent delivery system is the capability for generating a solvent gradient.
Filtration is needed to eliminate suspended particles and organic impurities.
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PUMPS
Pass mobile phase through column at high pressure and at controlled flow rate.
Performance of pump directly affects the Rt, reproducibility, detector sensitivity.
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IDEAL CHARCETRISTIC OF A PUMPNon corrosive and compatible with solvent.
Provide High pressure to push mobile phase
Provide constant flow rate to mobile phase.
Easy to change for one mobile phase to another.
Should have reproducible flow rate and independent of column back pressure.
Should not leak & should be easy to dismantle and repair.
High pressure generated by pump should not lead to explosion.135 October 2012
TYPE OF PUMP USED IN HPLC
1) Reciprocating pump
2) Displacement pump
3) Pneumatic pump
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1) RECIPROCATING PUMP
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WORKINGContains reciprocating piston that moves back and forth in
hydraulic chamber.
By the movement of piston solvent flow into the column under high pressure.
When piston moves backward inlet valve open while exit valve closes. This result in mobile phase being drawn into the main chamber (cylinder).
The reduction in volume in main chamber due to forward motion of piston result in mobile phase moving out of the exit valve under high pressure.
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DISADVENTAGEPulsed flow which must be damped as they produce a base line
noise on the chromatogram
ADVANTAGES Generate high output pressure (upto10000 poise).Ready adaptability to gradient elusion. Provide constant flow rate.Pressure generated is so high that any back pressure generated in
the column due to higher viscosity of stationary phase can be easily overcome.
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2) DISPLACEMENT PUMP / SYRINGE PUMP
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WORKINGWorks on the principle of positive solvent pressure.
Consist of screw or plunger which revolves continuously driven by motor.
Rotatory motion provides continuous movement of the mobile phase which is propelled by the revolving screw at greater speed and pushes solvent through small needle like outlet.
Consist of large syringe like chamber of capacity 250 – 500 ml.
Double syringe pumps have also been developed in which one piston is delivering the solvent to the column while other one is refilled from the reservoir.
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ADVANTAGES
Flow is pulse free.Provide high pressure upto 200 – 475 atm. Independent of column back pressure and viscosity of solvent.Simple operation.
DISADVENTAGE
Limited solvent capacity Gradient elution is not easy.
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3) PNEUMATIC PUMP
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WORKINGWORKINGThe driving air is applied, piston moves, inlet closes & outlet
open pushing mobile phase to the column.Pressure on solvent is proportional to the ratio of piston usually
50: 1.A lower pressure gas source of 1- 10 atm can be used to generate
high liquid pressure .( 1 – 400 atm )About 70 ml of the mobile phase is pumped from every stroke.
ADVENTAGES: Pulse free flow & Generates high pressure. DISADVANTAGES: 1) Limited volume capacity (70 ml ) 2) Pressure output and flow rate depends on the viscosity and
column back pressure. 3) Gradient elusion is not possible.
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SAMPLE INJECTION SYSTEM
Septum injectors
Stop flow Septumless injection.
Rheodyne injector / loop valve type.
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SEPTUM INJECTION PORT.
Syringe is used to inject the sample through an inert septum directly into the mobile phase.
Drawback: - leaching effect of the mobile phase in contact with septum, which may give rise to ghost peaks.
STOP FLOW SEPTUMLESS INJECTION.
Flow of mobile phase through the column is stopped while Syringe is used to inject the sample.
Drawback: formation of ghost peak.245 October 2012
RHEODYNE INJECTOR / LOOP VALVE TYPE. Sample is introduced in the column without causing interruption
to mobile phase flow.
Volume of sample ranges between 2 µl to over 100 µl.
Operation of sample loop.Sampling modeInjection mode.
Sample is loaded at atmospheric pressure into an external loop in the micro volume sampling valve, & subsequently injected into mobile phase by suitable rotation of the valve. 255 October 2012
COLUMN
Made up of stainless steel or heavy glass to withstand the pressure.
The columns are usually long (10 – 30 cm) narrow tubes. Contains stationary phase at particle diameters of 25 µm or less.
The interior of column should be smooth and uniform.
Column end fitting are designed to have a zero void volume.
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CLASSIFACTION OF CLOUMN
column
Main column Guard column
Analytical column Preparative column
Standard columnNarrow bore
Short fast column
Micro preparative Preparative columnMacro preparative
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A) BASED ON APPLICATION
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B) BASES OF COMPONENTS
Bonded phase column
Column where liquid is inpermagneted on solid inert support
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ANALYTICAL COLUMNSTANDARD COLUMN • Internal diameter 4 – 5 mm and length 10 – 30 cm. • Size of stationary phase is 3 – 5 µm in diameter.• Used for the estimation of drugs, metabolites, pharmaceutical
preparation and body fluids like plasma.
NARROW BORE COLUMN Internal diameter is 2 – 4 mm.Require high pressure to propel mobile phase.Used for the high resolution analytical work of compounds with
very high Rt.295 October 2012
SHORT FAST COLUMN Length of column is 3 – 6 cm.Used for the substances which have good affinity towards the
stationery phase. Analysis time is also less (1- 4 min for gradient elusion & 15 –
120 sec for isocratic elusion).
PREPARATIVE COLUMN Used for analytical separation i.e. to isolate or purify sample in
the range of 10-100 mg form complex mixture.Length – 25- 100 cmInternal diameter – 6 mm or more.
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TYPES OF PREPARATIVE COLUMN Micro preparative or semi preparative column Modified version of analytical columnUses same packaging and meant for purifying sample less
then 100 mg.
Preparative column Inner diameter – 25 mm .Stationary phase diameter – 15- 100 µm
Macro Preparative Column Column length – 20 – 30 cmInner diameter – 600 mm
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GUARD COLUMN
They are placed anterior to the separating column.
Serve as a protective factor that prolongs the life and usefulness of the column.
They are dependable column designed to filter or remove
Particles that clog the separation column.
Compounds and ions that could ultimately cause baseline drift, decrease resolution , decrease sensitivity and create false peaks.
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BONDED PHASE COLUMNHere the molecules, comprising the stationary phase i.e. the
surface of the silica particles, are covalently bonded to a silica based support particles.
The most popular bonded phase, siloxanes, are formed by heating the silica particles in dilute acid for the day so as to generate the reactive Silonal group.
- OH OH OH
ו ו ו
- Si – O – Si - O - Si -
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Silonal group is the treated with organochlorosilane.
These bonded phases are stable between the pH range 2 – 9 and upto temperature of 80º C.
Bonded phase is made with a linear C 18 hydrocarbon, also know as ODS (octadecyl silane) bonded phase. Used in pharmaceutical analysis or separation of less polar components.
An alkyl nitrile column or cyano column which has 12 carbon atoms with the last atom appearing as a nitrile group (CN), moderately polar column.
Amino alkyl bonded phase column which is normally C 8, last C atom bearing NH2 group, Polar column. Use full in separation of CHO, peptides, amino acids.
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Advantages
Can withstand high pressure exerted by mobile phase.
Life of column is more.
No bleeding effect
Disadvantages
Very expensive
Manually can not be fabricated
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COLUMN WHERE LIQUID IS INPERMAGNETED ON SOLID INERT SUPPORT.
These are not use widely now days.
Stationary phase dose not have the strength to stay in the column on account of the physical forces exerted by the mobile phase at very high pressure.
Amount of loading on inner support is minimum
Stationary phase starts bleeding out of the column and can cause resistance to mass transfer.
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METHOD OF PACKINGDepends on the mechanical strength & Particle size of the
stationary phase.
Particle size greater then 20 µm – dry packingParticle size lesser then 20 µm – slurry packing / wet packing.
WET / SLURRY PACKING Particle size with diameter less then 20 µm can only be placed
wet as a suspension.
Suspension should be stable, it should not sediment, and agglomentation should be avoided.
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DRY PACKING
Particle size greater then 20 µm filled into vertical clamped column in small quantity.
Deposition is done by tapping or vibrating the column.
Column is unclamped and the tapped on the firm surface to obtain dense and reproducible packing.
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DETECTORS
Based on the application, the detectors can be classified into
Bulk property detectors
Solute property detectors.
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BULK PROPERTY DETECTORSCompare an over all change in physical property of mobile phase
with or without an eluting solute.These types of detectors tend to be relatively low sensitive and
require temperature control. e.g. Refractive index detector.
SOLUTE PROPERTY DETECTORS They respond to a physical property of the solute that is not
exhibited by the pure mobile phase. These detectors are more sensitive, detect the sample in
nanograms quantity. e.g. Uv visible detector , Electrochemical detector, Fluorescence
detector.415 October 2012
ULTRAVIOLET VISIBLE DETECTOR They measure the ability of a sample to absorb light. This can be
accomplished at one or several wavelengths.
A light source deliver a monochromatic parallel light beam which passes through a cell swept by the column effluent, and falls on photocell.
Selective in nature, detect only those solutes that absorb Uv/ visible radiation
E.g. alkenes, aromatic compounds and compound having multiple bonds between C and O, N or S.
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BASICALLY THREE TYPES OF ABSORBANCE DETECTORS ARE AVAILABLE
Fixed Wavelength Detector Variable Wavelength DetectorDiode Array Detector
FIXED WAVELENGTH DETECTOR
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Detectors which do not allow changing the wavelength of the radiation called fixed-wavelength detectors.
In this, most of the light may be emitted at a one wavelength, with most single wavelength UV lamps.
Low-pressure mercury lamp emits very intense light at 254 nm.By filtering out all other emitted wavelengths, utilize only 254
nm line to provide stable, highly sensitive detectors capable of measuring subnanogram quantities of any components which contains aromatic ring
The 254 nm was chosen since the most intense line of mercury lamp is 254 nm, and most of UV absorbing compounds have some absorbance at 254 nm.
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VARIABLE-WAVELENGTH DETECTORS
Detectors which allow the selection of the operating wavelength called variable wavelength detectors.
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Variable wavelength detector employs a lamp that emits light over a wide range of wavelengths and by using a monochromator, light of a particular wavelength can be selected for detection purposes.
Depending on the sophistication of the detector, wavelength change is done manually or programmed on a time basis into the memory of the system.
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DIODE ARRAY DETECTOR
It is also a multiwavelength UV detector, but functions on an entirely different principle.
The UV photo diode-array detector.5 October 2012 47
FLUORESCENCE DETECTORS Very sensitive, but very selective.
By definition, it will detect only those materials that will fluoresce or, by appropriate derivatization can be made to fluoresce.
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Fluorescence occurs when compounds having specific functional groups are excited by shorter wavelength energy and emit higher wavelength radiation.
Fluorescence is often collected at right angle to excitation beam.
With all sample cells, scattered radiation from the excitation source is selectively removed with cut off or band pass filters placed before photomultiplier tube.
Most important detectors for use in trace analysis both in environmental and forensic analysis.
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REFRACTIVE INDEX DETECTOR OR DIFFERENTIAL REFRACTOMETER
The detection principle involves measuring of the change in refractive index of the column effluent passing through the flow-cell.
It responds to any solute whose refractive index is significantly different from that of the mobile phase.
Principle: it is based on two principles.Deflection ( deflection type refractometer)Reflection (reflection type refractometer)
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DEFLECTION TYPE REFRACTOMETER. Measure the deflection of a beam of a monochromatic light by double
prism.Eluent passes through one half of prism & pure mobile phase to other
half known as reference compartment.Reference and sample compartment are separated by diagonal glass
divider.Auto zero is used to set, out put signal to zero when mobile phase is in
both the compartments.
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Tungsten lamp provides beam of light collimated through lens and passes through Eluent and reference compartment.
Reflected by the mirror through the same compartment againThe beam of light is focused on a beam splitter before passing
into the photo detector. Refractive index of the mobile phase is changed due to the
presence of solute, the beam from the sample compartment is deflected which produces the change signal that is proportional to the concentration of solute.
Advantages Wide range of linearity.Covers entire refractive index range.
525 October 2012
REFLECTION TYPE REFRACTOMETER
Measure change in % of reflected light at glass liquid interface as the reflective index of liquid changes.
Based on the Fresnel's law of reflection which states
“The amount of liquid reflected at a glass- liquid interface varies with the angle of incidence and the refractive index of the liquid”
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working:Two collimated beams from the projector (light source & lens)
illuminate the reference and sample cell.Cells are formed of Teflon gasket, which is clamped between the
cell prism and a stainless steel reflecting back plate.
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As the light of beam is transmitted through the cell interfaces, it passes through the liquid film and imposes on the surface of the reflecting back plate.
Diffused, reflected light appears as two spots and passes through the lens and detected by photo detector.
The ratio of the reflected light to transmitted light is function of refractive index of the two liquid, the illumination of the cell back plate is direct measure of the refractive index of the liquid in each chamber
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ELECTROCHEMICAL DETECTOR OR AMPEROMETRIC DETECTOR
It is based on the measurements of the current resulting from an oxidation/reduction reaction of the analyte at a suitable electrode.
The level of the current is directly proportional to the analyte concentration
Also called coulometric detector.
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RECORDER AND INTEGRATORS
Recorders are used to record the response obtained from the detector after amplification. They record the baseline and all the peaks obtained, with respect to time. Retention time for all the peaks can be calculated.
Integrators are improved versions of recorder with data processing capabilities. They can record the individual peaks with retention time, height and width of peak, peak area, etc.
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DERIVATIZATIONThe most commonly used detector in HPLC is 254 nm UV
detector, many methods have been developed to introduce or enhance chromophores that will absorb light at this wavelength.
Also, reactions have been developed to produced a fluorophore for the purposes of fluorimetric detection.
While it is common to derivatize analytes in order to improve chromatographic properties, the emphasis in this section will be on derivatization for the benefit of detectability.
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Derivatization may be either pre-column or post-column.
PRE-COLUMN: Derivatization-Injection-Separation-Detection.
Ex. Treatment of ketosteroids with 2, 4, DNP,
Benzoylation of hydroxysteroids,
Esterification of fatty acids.
POST-COLUMN: Injection-Separation-Derivatization-Detection.
Ex. Reaction of amino acid with ninhydrin,
Reaction of fatty acids with o-nitrophenol,
Thermal or acid/phenol treatment of carbohydrates.5 October 2012 59
IDEAL CHARACTERISTICSThe ideal derivatization reaction is rapid, goes to completion,
produces a stable product.
Product has suitable chromatographic & spectral properties.
The unreacted derivatizing reagent should not interfere with the chromatographic separation.
The derivatization reactions are characteristics of
functional group, their description will be classified according to functional group.5 October 2012 60
1. CARBOXYLIC ACIDS
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Ex.
PDBI (O-p-nitrobenzyl-N,N’-diisopropylisourea) &
1-(p-Nitro)benzyl-3-p-tolytriazine also reacts with carboxylic acids to produce esters.
4-Bromomethyl-7-methoxycoumarin (BMC) reacts with carboxylic acids to form a fluorigenic product.
2. ALCOHOL
Activated carboxylic acid derivatives such as acyl chlorides are the most common reagents.
This reaction gives a product that has a molar absorptivity at 254 nm too low to be analytically useful.
P-nitrobenzoyl chloride, 3,5-dinitrobenzoyl chloride & anisyl chloride form esters that have much higher molar absorptivity.
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3. AMINESThe same acylating reagents used for alcohol can also be used
for amines.
R-NH2 + R’COCl R’CONHR + HCl
This reaction has been used for the analysis of tobramycin in serum.
10 & 20 amines react with 7-chloro-4-nitrobenzyl-2-oxa-1,3-diazole(NBD chloride) to produce a fluorescent derivative by displacement of 7-chloro group.
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4. ALDEHYDES & KETONESNucleophilic addition to a carbon-heteroatom double bond are
most frequently employed for derivatization of carbonyl compounds.
A prototype reaction is the condensation of a ketone with 2,4-dinitrophenylhydrazine(2,4-DNPH) to form the hydrazone.
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REFERENCESB.K.Sharma, Instrumental method of chemical
analysis, GOLE Publishing House, Page no 292-304.Ashutosh kar, Pharmaceutical drug analysis -2nd
edition, page no 453 456,459,466 Dr.A.V.kasture, Pharmaceutical analysis vol-2, page
no 52, 53.Elena katz, Roy Eksteen, Peter Schoenmarkers, Neil
Miller, Handbook of HPLC, volume 78, Special Indian Edition, page no. 536-550.
Munson, Pharmaceutical Analysis, Page no. 76-80http//hplc.chem.shu.edu/new/hplcbook/detector
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THANK YOU
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