HPLC-chiraglathiya

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HPLC BY CHIRAG LATHIYA

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HIGH PERFORMANCE LIQUID CHROMATOGRAPHY :

HIGH PERFORMANCE LIQUID CHROMATOGRAPHY PRESENTED BY: LATHIYA CHIRAG M.PHARM (1 ST SEM.) Department of pharmaceutical technology BHAGWAN MAHAVIR COLLEGE OF PHARMACY, SURAT. 1 5 October 2012

Contents:

Contents INTRODUCTION CLASSIFICATION INSTRUMENTATION DERIVATIZATION REFERENCES 2 5 October 2012

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H igh P ressure L iquid C hromatography or H igh P erformance L iquid C hromatography What is it? Separation technique based on solid stationary phase + liquid mobile phase How can achieve separation? By partition adsorption ion exchange 3 5 October 2012

CLASSIFICATION OF HPLC:-:

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. 4 5 October 2012

3. ION-EXCHANGE CHROMATOGRAPHY (IEX):

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 5 5 October 2012

4. SIZE-EXCLUSION CHROMATOGRAPHY (SEC):

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. 6 5 October 2012

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HPLC INTRUMENTATION CONSIST OF Degasser Solvent Reservoir( HPLC solvent reservoir systems) Pumps Pre Guard Column Sample injection system Columns Detector Recorder and integrators 7 5 October 2012

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8 5 October 2012

DEGASSER:

DEGASSER Degassing 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. 9 5 October 2012

HPLC SOLVENT RESERVOIR SYSTEMS :

HPLC SOLVENT RESERVOIR SYSTEMS 10 5 October 2012

HPLC SOLVENT RESERVOIR SYSTEMS:

HPLC SOLVENT RESERVOIR SYSTEMS These 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. 11 5 October 2012

PUMPS:

PUMPS Pass mobile phase through column at high pressure and at controlled flow rate. Performance of pump directly affects the Rt, reproducibility, detector sensitivity. 12 5 October 2012

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IDEAL CHARCETRISTIC OF A PUMP Non 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. 13 5 October 2012

TYPE OF PUMP USED IN HPLC :

TYPE OF PUMP USED IN HPLC 1) Reciprocating pump 2) Displacement pump 3) Pneumatic pump 14 5 October 2012

1) RECIPROCATING PUMP :

1) RECIPROCATING PUMP 15 5 October 2012

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WORKING Contains 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. 16 5 October 2012

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DISADVENTAGE Pulsed 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. 17 5 October 2012

2) DISPLACEMENT PUMP / SYRINGE PUMP :

2) DISPLACEMENT PUMP / SYRINGE PUMP 18 5 October 2012

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WORKING Works 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. 19 5 October 2012

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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. 20 5 October 2012

3) PNEUMATIC PUMP :

3) PNEUMATIC PUMP 21 5 October 2012

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WORKING The 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. 22 5 October 2012

SAMPLE INJECTION SYSTEM :

SAMPLE INJECTION SYSTEM Septum injectors Stop flow Septumless injection. Rheodyne injector / loop valve type. 23 5 October 2012

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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. 24 5 October 2012

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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 mode Injection 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. 25 5 October 2012

COLUMN:

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. 26 5 October 2012

CLASSIFACTION OF CLOUMN:

CLASSIFACTION OF CLOUMN 27 A) BASED ON APPLICATION 5 October 2012

B) BASES OF COMPONENTS:

B) BASES OF COMPONENTS Bonded phase column Column where liquid is inpermagneted on solid inert support 28 5 October 2012

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ANALYTICAL COLUMN STANDARD 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. 29 5 October 2012

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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 cm Internal diameter – 6 mm or more. 30 5 October 2012

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TYPES OF PREPARATIVE COLUMN Micro preparative or semi preparative column Modified version of analytical column Uses 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 cm Inner diameter – 600 mm 31 5 October 2012

GUARD COLUMN :

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. 32 5 October 2012

BONDED PHASE COLUMN:

BONDED PHASE COLUMN Here 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 - ו ו ו ו 33 5 October 2012

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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. 34 5 October 2012

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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 35 5 October 2012

COLUMN WHERE LIQUID IS INPERMAGNETED ON SOLID INERT SUPPORT. :

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. 36 5 October 2012

METHOD OF PACKING:

METHOD OF PACKING Depends on the mechanical strength & Particle size of the stationary phase. Particle size greater then 20 µm – dry packing Particle 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. 37 5 October 2012

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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. 38 5 October 2012

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39 5 October 2012

DETECTORS :

DETECTORS Based on the application, the detectors can be classified into Bulk property detectors Solute property detectors. 40 5 October 2012

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BULK PROPERTY DETECTORS Compare 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. 41 5 October 2012

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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. 42 5 October 2012

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BASICALLY THREE TYPES OF ABSORBANCE DETECTORS ARE AVAILABLE Fixed Wavelength Detector Variable Wavelength Detector Diode Array Detector FIXED WAVELENGTH DETECTOR 43 5 October 2012

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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. 44 5 October 2012

VARIABLE-WAVELENGTH DETECTORS:

VARIABLE-WAVELENGTH DETECTORS Detectors which allow the selection of the operating wavelength called variable wavelength detectors. 45 5 October 2012

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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. 46 5 October 2012

DIODE ARRAY DETECTOR:

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 :

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. 48 5 October 2012

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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. 49 5 October 2012

REFRACTIVE INDEX DETECTOR OR DIFFERENTIAL REFRACTOMETER :

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) 50 5 October 2012

DEFLECTION TYPE REFRACTOMETER. :

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. 51 5 October 2012

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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 again The 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. 52 5 October 2012

REFLECTION TYPE REFRACTOMETER:

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” 53 5 October 2012

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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. 54 5 October 2012

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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 55 5 October 2012

ELECTROCHEMICAL DETECTOR OR AMPEROMETRIC DETECTOR :

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. 56 5 October 2012

RECORDER AND INTEGRATORS :

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. 57 5 October 2012

DERIVATIZATION:

DERIVATIZATION The 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. 58 5 October 2012

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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

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IDEAL CHARACTERISTICS The 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:

1. CARBOXYLIC ACIDS 5 October 2012 61 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:

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. 5 October 2012 62

3. AMINES:

3. AMINES The same acylating reagents used for alcohol can also be used for amines. R-NH 2 + R’COCl R’CONHR + HCl This reaction has been used for the analysis of tobramycin in serum. 1 0 & 2 0 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. 5 October 2012 63

4. ALDEHYDES & KETONES:

4. ALDEHYDES & KETONES Nucleophilic 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. 5 October 2012 64

REFERENCES :

REFERENCES B.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. 76-80 http//hplc.chem.shu.edu/new/ hplcbook /detector 5 October 2012 65

THANK YOU :

THANK YOU 66 5 October 2012

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