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PRINCIPLE
High Performance Liquid Chromatography [HPLC] is principle is based on
adsorption as well as partition chromatography is depending on the
nature of stationary phase, if stationary phase is solid principle is based
on adsorption chromatography and if stationary phase is liquid principle is
based on partition chromatography.](https://image.slidesharecdn.com/hplc-221230113334-bc85ef9a/85/HPLC-pptx-4-320.jpg)
HPLC.pptx
- 2. CONTENTS 2 Introduction. Modes of separation. Instrumentation. HPLC method development,optimization and validation. References.
- 3. INTRODUCTION 3 HPLC is a High Performance (Pressure or Priced)liquid Chromatography. It is column chromatographic technique.It is applicable for both Volatile as well as Non volatile compound. Most of the drugs in multicomponent dosage forms can be analyzed by HPLC method because of the several advantages like: • Improved resolution of the separated substances • Faster separation times • The improved accuracy, precision, & sensitivity with which the separated substances may be quantified. • It can be used for both qualitative and quantitative analysis.
- 4. 4 PRINCIPLE High Performance Liquid Chromatography [HPLC] is principle is based on adsorption as well as partition chromatography is depending on the nature of stationary phase, if stationary phase is solid principle is based on adsorption chromatography and if stationary phase is liquid principle is based on partition chromatography.
- 5. MODESOFSEPARATION IN HPLC 5 There are different modes of separation in HPLC: 1.Normal phase mode. 2.Reversed phase mode. 3.Reversed phase ion pair chromatography. 4.Affinity chromatography. 5.Size exclusion chromatography
- 6. NORMALPHASE MODE 6 Stationary phase-polar in nature. Mobile phase-non polar in nature. Generally used for separation of non polar compounds.
- 7. REvERSEDPHASE MODE 7 Stationary phase-non polar . Mobile phase-polar in nature. Generally used for separation of polar compounds.
- 8. ION EXCHANGE CHROMATOGRAPHY : 8 The stationary phase contains ionic groups like NR⁺з, SO⁻з which interact with the ionic groups of the sample molecules. This method is suitable for the separation of charged molecules only. ION PAIR CHROMATOGRAPHY: This may be used for the separation of ionic compounds. Strong acids & basic compounds may be separated by reversed phase mode by forming ion pairs with suitable counter ions.
- 9. AFFINITY CHROMATOGRAPHY: • It uses highly specific biochemical interactions for separations. • The stationary phase contains specific groups of molecules which can absorb the sample if certain steric & charge related conditions are satisfied. • This technique can be used to isolate proteins, enzymes, as wel as antibodies from complex mixture. SIZE EXCLUSION CHROMATOGRAPHY: • Separates molecules according to their molecular mass. • Largest molecules are eluted first and smaller molecules last. 9
- 10. MODES OF HIGH PERFORMANCE LIQUID CHROMATOGRAPHY Types of Compounds Mode Stationary Phase Mobile Phase Neutrals Weak Acids Weak Bases Reversed Phase C18, C8, C4 cyano, amino Water/Organic Modifiers Ionics, Bases, Acids Ion Pair C-18, C-8 Water/Organic Ion-Pair Reagent Compounds not soluble in water Normal Phase Silica,Amino, Cyano, Diol Organics Ionics Inorganic Ions Ion Exchange Anion or Cation Exchange Resin Aqueous/Buffer Counter Ion High Molecular Weight Compounds Polymers Size Exclusion Polystyrene Silica Gel Filtration- Aqueous Gel Permeation- Organic
- 11. BLOCK DIAGRAM OF HPLC Reservoir Pump Gradient controller Mixing chamber Pump Solvent Conditioning column Reservoir Injector Precolumn Analytical column Detector Recorder Fraction collector 11
- 14. VARIOUS COMPONENTSOFHPLC 14 Solvent storage bottle Gradient controller and mixing unit De-gassing of solvents Pump Pressure gauge Pre-column Sample introduction system Column Detector Recorder
- 15. SOLVENTSTORAGE BOTTLE / MOBILE PHASE RESERVOIR 15 Glass or stainless-steel containers capable of holding up to 1 liter mobile phase (pure organic solvents or aqueous solutions of salts and buffers). Inert to a variety of aqueous and non aqueous mobile phases. Stainless steel should be avoided for use with solvents containing halide ions. For normal phase separation eluting power increases with increasing polarity of the solvent but for reversed phase separation, eluting power decreases with increasing polarity.
- 16. DEGASSING & FILTRATION OF MOBILE PHASE In many cases, aqueous solvents & some organic solvents are degassed prior to use Degassing is done to prevent formation of gas bubbles in the pump or detector ( Mobile phases are degassed by stirring of the mobile phase under vacuum, sonication or sparing with helium gas) The mobile phase are filtered to remove particulate matter that may clog the system Tubing Should be inert, have ability to withstand pressure able to carry sufficient volume 26
- 17. PUMPS 17 The pump is one of the most important component of HPLC, since its performance directly affects retention time, reproducibility and detector sensitivity. The solvents or mobile phase must be passed through a column at high pressures at up to 6000 psi(lb/in²) or 414 bar. As the particle size of stationary phase is smaller (5 to 10µ) the resistance to the flow of solvent will be high. That is, smaller the particle size of the stationary phase the greater is the resistance to the flow of solvents. Hence high pressure is recommended.
- 18. REQUIREMENTS FOR PUMPS Generation of pressure of about 6000 psi. Pulse free output & all materials in the pump should be chemically resistant to solvents. Flow rates ranging from 0.1 to 10 mL/min Pumps should be capable of taking the solvent from a single reservoir or more than one reservoir containing different solvents simultaneously.
- 19. Types of pumps used in HPLC DISPLACEMENT PUMPS RECIPROCATING PUMPS PNEUMATIC PUMPS
- 20. DISPLACEMENT PUMPS ⚫ It consists of large, syringe like chambers equipped with a plunger activated by a screw driven mechanism powered by a stepping motor. ⚫ So it is also called as Screw Driven Syringe Type Pump. ⚫ Advantages:- It produces a flow that tends to be independent of viscosity & back pressure. ⚫ Disadvantages:-It has a limited solvent capacity(250 ml) & considerably inconvenient when solvents must be changed.
- 21. RECIPROCATING PUMPS This pump transmits alternative pressure to the solvent via a flexible diaphragm ,which in turn is hydraulically pumped by a reciprocating pump. Disadvantages Produces a pulsed flow which is damped because pulses appear as baseline noise on the chromatograph. This can be overcome by use of dual pump heads or elliptical cams to minimize such pulsations. 33
- 22. Advantages: Solvent is pumped back and forth by a motor driven piston Two ball check valves which open & close which controls the flow The piston is in direct contact with the solvent Small internal volume 35-400μL High output pressure up to 10,000 psi Ready adaptability to gradient elution and constant flow rate
- 23. PNEUMATIC PUMPS In this pumps, the mobile phase is driven through the column with the use of pressure produced from a gas cylinder. Disadvantages: It has limited capacity of solvent Due to solvent viscosity back pressure may develop.
- 24. SAMPLE INJECTOR SYSTEM Several injector devices are available either for manual or auto injection of the sample. Septum Injector or on column injection: These are used for injecting the sample through a rubber septum. They deliver the fixed amount of volume by making use of fixed volume loop injector. This kind of injectors cannot be commonly used , since the septum has to withstand high pressures. Stop Flow Injector or valve injection: In this type the flow of mobile phase is stopped for a while & the sample is injected through a valve. Rheodyne Injector or loop injection: It is the most popular injector and is widely used.This has a fixed volume of loop, for holding sample until its injected into the column, like 20µL, 50µL or more.
- 25. COLUMN 1. Precolumn 1. It contains a packing chemically identical to that in analytical column. 2.Mainly used to remove the impurities from the solvent and thus prevents contamination of the analytical column, it can protect analytical column. 3. It is also called as guard column or protective column. 4. it is having large particle size. 5. It is having short length of 2 to 10 cm, so does not affect separation. 2.Analytical column 1. The success or failure of analysis depends upon choice of column. 2. Actual separation is carried out here. 3. Stainless –steel tube 4. size – length -25 to 100 cm 5. Internal diameter – 2 to 4.6 mm 6. Column is filled with small particles 5 – 10 micron. The solid support can be silica gel, alumina. 7. The separation is result of different components adhering to or diffusion into the packing particles when the mobile phase is forced through column. 45
- 26. 8.C8 and C18 columns are considered as examples of reversed phase liquid chromatography (RP). 9.The stationary phase here is seen as a thin film of non-polar liquid phase that has been designed to be chemically similar to an inert material (Silica gel particles). 10.The non-polar layer is chemically linked to the silica particles surface by reaction with the polar silanol groups on the stationary phase surface and so rendering them less polar or non-polar. 11. The difference between the two columns will be in the length of the carbon chain attached to the silica surface. 12.Accordingly C8 hplc columns have packing material composed of silica particles attached to C8 carbon units. 13. C18 will, of course, have packing materials coated with C18 hydrophobic units. 14.Categorically both are reversed phase but C18 columns will definitely be more "hydrophobic rather than the C8 columns.
- 27. DETECTORS Absorbance (UV/Vis and PDA) Refractive index (detects the change in turbidity) Fluorescence (if the analyte is fluorescent) Electrochemical (measures current flowing through a pair of electrodes, on which a potential difference is imposed, due to oxidation or reduction of solute) Conductivity (for ions) Light scattering Mass spectrometry (HPLC-MS)
- 28. SELECTION OF DETECTORS Detectors Type of compounds can be detected UV-Vis & PDA RF CDD • Compounds with chromophores, such as aromatic rings or multiple alternating double bonds. • Fluorescent compounds, usually with fused rings or highly conjugated planar system. • Charged compounds, such as inorganic ions and organic acid. ECD For easily oxidized compounds like quinones or amines. RID & ELSD For compounds that do not show characteristics usable by the other detectors, e.g. polymers, saccharides.
- 29. TYPES OF HPLC DETECTORS UV-Vis Works w/all molecules Non-specific; complex samples; absorption wavelength DAD Works for all wavelengths High LOD Fluorescence Very specific; low LOD Not everything fluoresces IR Works w/all molecules Many solvents IR active Refractive Index Works w/nearly all molecules Temperature sensitive; high LOD Scattering Uniform response; 5ng/25mL LOD Non-specific; interference from solvent Electrochemical Commercially available Non-specific; high LOD Mass Spec Low LOD; analyte identification Ability to ionize analyte 49 Name Advantage Disadvantage
- 30. IDEAL DETECTOR PROPERTIES High Sensitivity Universality or predictable specificity Large linear response range Low dead volume Non-Destructive Insensitive to temperature & mobile phase Continuous operation Reliable and easy to use No single detector fits all these criteria
- 31. DETECTORS 31 The function of detector in HPLC is to monitor the mobile phase as it merges from the column. Detectors are usually of two types: 1.Bulk property detectors: It compares overall changes in a physical property of the mobile phase with and without an eluting solute e.g. refractive index ,dielectric constant or density. 2.Solute property detectors: It responds to a physical property of the solute which is not exbited by the pure mobile phase.e.g.UV absorbance,fluoroscence or diffusion current.
- 32. TYPESOF DETECTORS 32 There are mainly 4 types of detectors are used in HPLC: 1. Photometric detectors. Single wavelength detectors. Multiwavelength detectors. Variable wavelength detectors. Programmable detectors. Diode array detectors . 2. Fluorescence detectors. 3. Refractive index detectors. 4. Electrochemical detectors.
- 33. PHOTOMETRIC DETECTORS 33 • These normally operate in the ultra violet region of the spectrum . • Most extensively used in pharmaceutical analysis.
- 34. SINGLE WAVELENGTH DETECTORS Equipped with a low pressure mercury discharge lamp. Fixed wavelength detectors employ filter as a source to provide appropriate wavelength.The absorbance is measured at the wavelength of mercury at 254nm. MULTI-WAVELENGTH DETECTORS Employ mercury and other discharge sources. When used in combination with interference filters,allow a no of monochromatic wavelengths to be selected e.g. 206, 226, 280 , 313, 340 or 365 nm. 34
- 35. VARIABLE WAVELENGTH DETECTORS 35 Use a deuterium light source. A grating monochromator to allow selection of any wavelength in deuterium continuum (190-360nm). Variable wavelength detectors employ a spectrophotometer to produce a dispersion of light and selection of wavelength in the UV visible region. PROGRAMMABLE DETECTORS This are programmed detectors which alters their wavelength automatically for the required detection.
- 36. PHOTODIODE A R R AY 36 DETECTORS They are microprocessor – controlled photodiode array spectrophotometers in which light from an UV source passes through the flow cell into a polychromator which disperses the beam so that the full spectrum falls on the array of diodes.
- 37. FLUORESCENCE DETECTORS 37 • Fluorescence is a type of luminescence in which the light energy is released in the form of a photon in nanoseconds to microseconds.These are essentially filter fluorimeter or spectro -fluorimeters equipped with grating monochromators,and micro flow cell. • Their sensitivity depends on the fluorescence properties of the components in the eluate. More sensitive and specific. The disadvantage is that most compounds are not fluorescent in nature.
- 38. REFRACTIVE INDEX DETECTORS 38 • Detection occurs when the light is bent due to samples eluting from the columns, and this is read as a disparity b/w the two channels.It is not much used for analytical applications because of low sensitivity & specificity. When a solute is in the sample compartment, refractive index changes will shift the light beam from the detector. • The sensitivity of the refractive index detector is much less than that of specific solute property detectors, they are useful for the detection of substances(e.g,carbohydrates& alcohols) which do not exhibit other properties that can be used as the basis for specific detection. 12/30/2022
- 39. ELECTROCHEMICAL DETECTORS 39 • These are based on standard electrochemical principles involving amperometry,voltametryand polarography. • These detectors are very sensitive for substances that are electroactive ,i.e. those that undergo oxidation or reduction . • They have found particular application in the assay of low levels of endogenous catecholamines in biological tissues,pesticides,tryptophan derivatives and many drugs.
- 40. RECORDERSAND INTEGRATORS 40 Recorders are used to record responses obtained from the detectors after amplification, if necessary. They record the baseline & all the peaks obtained with respect tot time. Retention time can be found out from this recordings, but area under curve cannot be determined. The Integratorsare improved versions of recorderswith some data processing capabilities. They can record the individual peaks with retention time, height, width of peaks, peak area, percentage area, etc. Integrators provides more information on peaks than recorders. In recent days computers and printers are used for recording and processing the obtained data & for controlling several operations.
- 41. USES OF HPLC Simultaneous analysis High resolution High sensitivity Good repeatability Moderate analysis condition Easy to fractionate and purify Not destructive
- 42. APPLICATION Drug Discovery ClinicalAnalysis Proteomics Forensic Chemistry Drug Metabolism study Environmental chemistry Diagnostic studies Cosmetic analysis Determination of Green Florescent Protein Structural Determination PharmaceuticalApplications Identification of BileAcid Metabolite ClinicalApplications Biochemical Genetics qualitative and quantitative analysis Therapeutic Drug Monitoring 63
- 43. COMPARISION BETWEEN HPTLCAND HPLC Sr.no. HPTLC HPLC 1 High Performance Thin Layer High Chromatography Performance Liquid Chromatography 2 Column Chromatography 3 Adsorption Planar Chromatography Principle based on Chromatography Principle is based on Adsorption and Partition Chromatography 4 Simultaneous method for test as well as reference material Not simultaneous method for test as well as reference material 5 It is simple, rapid, reproducible method It is Tedious method 6 Sample preparation is simple Sample preparation is complex 7 Limited Flexibility Extreme Flexibility 8 Semiautomatic Technique Automatic (Instrumental) Technique 59 9 Determination of SurfaceArea Determination of Retention Time
- 44. COMPARISION BETWEEN GCAND HPLC 60 SR.NO. GC HPLC 1 Gas Chromatography High Performance Liquid Chromatography 2 Less resolution High resolution 3 Limited Flexibility Extreme Flexibility 4 Determination of Volatile compounds Determination of Volatile and Non Volatile Compounds
- 45. COMPARISION BETWEEN HPLC AND UPLC Parameters HPLC Assay UPLC Assay Column XTerra,C18,50 × 4.6mm AQUITY UPLC BEH C18,50 ×2.1mm Particle size 4µm particles 1.7µm particles Flow rate 3.0 ml per min 0.6 ml per min Injection volume 20 µl 3 µl partial loop fill or 5 µl full loop fill Total run time 10 min 1.5 min Theoretical Plate 2000 7500 count
- 46. 62 Parameters HPLC Assay UPLC Assay Lower limit of quantization 0.2 µg/ml 0.054µl/ml Total solvent consumption Acetonitrile:10.5ml, water:21ml Acetonitrile:0.53ml, water:0.66ml Delay volume 720 µl 110 µl Column temperature 30 °C 65 °C Maximum back pressure 35-40 Mpa less 103.5 Mpa more Resolution Less High Method development cost High Low
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