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  1. 1. Introduction Definition:  High performance liquid chromatography (HPLC) is a chromatographic technique used to separate a mixture of compounds in analytical chemistry and biochemistry with the purpose of identifying, quantifying or purifying the individual components of the mixture.  Also called High Pressure Liquid Chromatography.  It is column chromatography.  It is modified from of gas chromatography, it is applicable for both Volatile as well as Non volatile compound.  It is having a high resolution and separation capacity  It is used as qualitative as well as quantitative analysis.
  2. 2. Principal  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.  It is important for determination of volatile and non volatile compounds.  It is important for determination qualitative and quantitative analysis.  It is important for determination of Retention Time (the time is required , after sample injection maximum angle peak reaches to detector)
  3. 3. Advantages  It is simple, rapid , reproducible.  High sensitivity.  High performance.  Rapid process and hence time saving.  It is having a high resolution and separation capacity.  Accuracy and Precision.  Stationary phase was chemically inert.  Wide verities of stationary phase.  Mobile phase was chemically inert.
  4. 4. Advantages  Less requirement of mobile phase in developing chamber.  Early recovery of separated component.  Easy visualization of separated components.  It is having Good reproducibility and repeatability.  It is analytical technique is important for validation of product, quality control studies of product.  It is important for qualitative and quantitative analysis.  It is used for both analytical and preparative purpose.
  5. 5. Types of HPLC 1. Normal Phase: Separation of polar analyses by partitioning onto a polar, bonded stationary phase. 2. Reversed Phase: Separation of non-polar analyses by partitioning onto a non-polar, bonded stationary phase. 3. Adsorption: In Between Normal and Reversed. Separation of moderately polar analyses using adsorption onto a pure stationary phase (e.g. alumina or silica). 4. Ion Chromatography: Separation of organic and inorganic ions by their partitioning onto ionic stationary phases bonded to a solid support. 5. Size Exclusion Chromatography: Separation of large molecules based in the paths they take through a “maze” of tunnels in the stationary phase
  6. 6. Selection of Separation System
  7. 7.  Table: A List of Stationary Phases used in Various Modes of HPLC
  8. 8. Six major components needed to perform HPLC I. A solvent reservoir to store the mobile phase. II. High pressure pump to push the mobile phase through the column. III. A device to inject the sample into the mobile phase. IV. A column in which the separation will take place. V. A detector used in detecting the concentration of the sample components as they come out of the column. VI. A potentiometric recorder to produce a chromatogram.
  9. 9. Figure : Presents a schematic diagram of the instrumentation required for HPLC
  10. 10. Solvent Reservoir and the Solvents  The solvent reservoir should meet the following criteria: I. It must contain volume enough for repetitive analysis II. It must have a provision for degassing the solvents; III. It must be inert to the solvent.
  11. 11. Solvent/ mobile phase reservoirs  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. Pooja Gupta
  12. 12. 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)  Generally. glass and steel containers of 0.5-2.0 liter capacity are suitable as solvent reservoirs.
  13. 13. Degassing & filtration of mobile phase  Glass bottles in which the HPLC solvents are sold also make for a very good solvent reservoir.  The solvent container should preferably be insulated against contamination through laboratory atmosphere.  All solvents to be used in HPLC must be extra pure since even the smallest impurity interferes with the detection system.  This is more so if the detection system is measuring the absorbance below 200nm.  Thus. even the extra pure HPLC solvents are passed through a 1- 5 mm filter placed before the pump.
  14. 14. Pump  Pumping system can be said to be the heart of HPLC.  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
  15. 15. Qualities of pump: 1. A pulseless stable flow. 2. Absence of pulsations minimizes detector noise. 3. suitable pump should provide solvent flow-rates of 0.5-10 ml/min, which is compatible with most HPLC modes. 4. A constant volume delivery. 5. Amenability to high pressures of up to 6000 psi. 6. The pump should be adaptable to gradient operation.
  16. 16. Types of pumps 1. Liquid displacement by compressed gases (holding coil). 2. Pneumatic amplifier 3. Piston/diaphragm driven by a moving fluid. 4. Reciprocating piston. 5. Syringe pumps.
  17. 17. Types of pumps 1. Holding coil. I. This unit is usually available with less expensive HPLC systems. II. A large holding coil made up of stainless steel tubing is filled up with the solvent. III. Compressed gas from a cylinder forces the liquid at constant pressure from the holding coil into the chromatographic column. IV. Flow rates are dependent upon column permeability and the gas pressure applied.
  18. 18. Types of pumps Disadvantage: I. These pumps can at best provide pressures up to 1500 pSi .,and cannot be used for gradient elution separations. II. These pumps is that many times the driving gas can inadvertently get dissolved in the mobile phase and cause problems in resolutions. III. These pumps are consequently not very popular.
  19. 19. Types of pumps 2. Pneumatic amplifier. • In this pumps, the mobile phase is driven through the column with the use of pressure produced from a gas cylinder. • The pump has a piston driven by the compressed gas. • The pump uses gas at comparatively low pressure of about 200 p.s.i. • The gas is in contact with a large surface area of the piston. • A smaller surface area of the piston is in contact with the solvent.
  20. 20. Types of pumps  The pressure of the gas is thus increased 10-20 fold when it is applied to the solvent.  This pump gives a pulseless flow and is ideal for quantitative purposes.
  21. 21. Pneumatic amplifier
  22. 22. Types of pumps 3. Moving fluid type.  These pumps use either a piston or a diaphragm driven by moving liquid.  These pumps give a pulseless flow and they are adaptable to gradient elution.
  23. 23. Types of pumps 4. Reciprocating piston.  These pumps use a piston that is in direct contact with the solvent.  The piston may be driven with motors and gears or by solid-state pulsing circuits.  A piston moves rapidly back and forth in a hydraulic chamber.  On the backward move the piston sucks in solvent from the reservoir.  which it pushes into the column on the forward move.  The outlet to the columns close during the backward move to maintain the pressure in the column.
  24. 24. Types of pumps  The pump. however. fails to produce a pulseless flow. In order to suppress the pulses a pulse dampening system has to be employed.  The pump is not very popular.
  25. 25.  A. Single-piston pump with slow filling cycle  B. Single-piston pump with a rapid filling cycle  C. A dual-piston pump with rapid filling cycles and operate 1800 out of phase.
  26. 26. Advantages  Have small internal volume of 35-400µL  Higher output pressures up to 10,000 psi.  Adaptability to gradient elution.  Large solvent capacities & constant flow rates.  Largely independent of column back pressure & solvent viscosity. Pooja Gupta
  27. 27. Types of pumps 5. Syringe pump.  These pumps operate by a screw gear displacing a plunger through the solvent reservoir.  These pumps provide a stable flow rate high pressure. The pump is well suited to gradient operations.
  28. 28. Sample Injection  Sample introduction on to the HPLC column is, as for all conventional types of chromatography. An important factor in achieving a satisfactory resolution.  Two methods are available to introduce the sample as a narrow band.  The first method employs a micro syringe designed to withstand high pressures.  With the help of this micro syringe.  The sample is introduced either directly onto the column or onto an inert material directly above the column.  Preferably-the sample is injected when the pressure has dropped to almost one atmosphere after switching the pump off.
  29. 29. Sample Injection  This technique is known as stop low.  Alternatively, the sample can be injected while the system is under high pressure. 2. The second method employs a small volume metal loop which can be filled with the sample. • An appropriate valve then channels the eluant from the pump through the loop directly onto the column. • The sample is thus carried spontaneously with the eluant to the column. Pooja Gupta
  30. 30. Types Sample Injection  Several injector devices are available either for manual or auto injection of the sample. I) Septum injector  These are used for injecting the sample through a rubber septum.  This kind of injectors cannot be commonly used , since the septum has to withstand high pressures. II) Stop Flow  In this type the flow of mobile phase is stopped for a while & the sample is injected through a valve.
  31. 31. Types Sample Injection III) Rheodyne injector  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.  Through an injector the sample is introduced into the column.  The injector is positioned just before the inlet of the column.
  32. 32. Column  The columns for HPLC are usually made up of stainless steel, glass, aluminum, copper, or PTFE.  Stainless steel columns are preferred Since they can withstand pressures up to 8000 psi. relatively easily. Straight columns of between 20-50 cm in length a!"e generally used.  Short columns are required for liquid adsorbent and liquid- liquid chromatography.  Whereas, for other modes longer columns are necessary. The internal diameter of the columns is usually 1-4 mm.  The columns usually possess an internal mirror finish, which allows efficient packing.  The packing material is supported by a porous stainless steel or on plug/disc at the end of the column.
  33. 33. Column Packing :  Maximal separation without or with minimal band broadening is a function of the stationary and mobile phases chosen.  Coarser particles induce increased band broadening.  To minimize this unwelcome phenomenon.  stationary supports for HPLC have been so designed that the individual particles are as small and as uniform as possible.
  34. 34. Three forms of column packing material are available based on a rigid solid rather than a gel structure: I) Microporous supports where micropores ramify through the particles.  These particles are generally 5-10 mm in diameter. II) Pellicular supports consist of a solid inert core onto which are coated several porous particles (e.g. a glass bead of about 40 mm diameter). • These supports are therefore superficially porous III) Bonded phases where the stationary phase is chemically bonded to an inert support. Pooja Gupta
  35. 35. Pooja Gupta
  36. 36. Pooja Gupta
  37. 37. The Guard Column  The resolution power of HPLC is so high that an elaborate sample preparation before chromatography is not necessary.  Thus. sera. or other biological materials can be applied to the column without any pretreatment.  This. however. clogs the column after a few applications as the column dwing separation retains many undesirable components of the biological samples.  To circumvent this problem. a short column (2-10 cm) precedes the main column.  This short column is known guard column and its function is to retain those biological components .  The guard column has the same diameter an the same packing as the main column.  The packing of the guard column can be replaced at regular intervals.
  38. 38. Types of Detectors 1. Absorbance (UV/Vis and PDA) 2. Refractive index (detects the change in turbidity) 3. Fluorescence (if the analyte is fluorescent) 4. Photodiode array (PDA). 5. Electrochemical (measures current flowing through a pair of electrodes, on which a potential difference is imposed, due to oxidation or reduction of solute) 6. Conductivity (for ions) 7. Light scattering 8. Mass spectrometry (HPLC-MS) Pooja Gupta
  39. 39. Types of Detectors 1. UV-Visible Detector  UV visible detector is widely used as it detects large number of compounds because most drugs have appropriate structural characteristics for light absorption.  These are useful for aromatic compounds and other type of unsaturated systems.  These are classified as (Two modes)  1. Fixed detectors  2. variable wavelength detectors.  Fixed wavelength detectors employ filter as a source to provide appropriate wavelength.  Most common fixed wavelength detectors are based on 254 nm. Pooja Gupta
  40. 40. Types of Detectors 2. variable wavelength detectors:  UV/VIS spectrophotometers with wavelength selection range of 200-800 nm are very popular HPLC detectors.  These can be either recording spectrophotometers or manual wavelength selection spectrophotometers.  The advantage with these is that a range of biological substances can be detected by selection of appropriate wavelengths.
  41. 41. Types of Detectors 2. Refractive index (RF) detector:  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 (non specific).  When a solute is in the sample compartment, refractive index changes will shift the light beam from the detector. Pooja Gupta
  42. 42. Types of Detectors 3. Photodiode array (PDA).  A photodiode array (PDA) is a linear array of discrete photodiodes on an integrated circuit (IC) chip.  Allows a range of wavelengths to be detected simultaneously.  In this regard it can be thought of as an electronic version of photographic film.  Array detectors are especially useful for recording the full Uv- vis is a absorption spectra of samples that are rapidly passing through a sample flow cell, such as in an HPLC detector.  PDAs work on the same principle as simple.  Photovoltaic detector similar to UV detector, non destructive 190-600 nm for quantization & identification Spectra is 3D, Response vs time vs W.
  43. 43. Types of Detectors 4. Fluorimetric Detectors:  It is based on the fluorescent radiation emitted by some compounds.  The excitation source passes through the flow cell to a photo detector while a monochromatic measures the emission wavelengths.  More sensitive and specific.  Disadvantage is that most compounds are not fluorescent in nature.  Fluorescence is a type of luminescence in which the light energy is released in the form of a photon in nanoseconds to microseconds.
  44. 44. Pooja Gupta
  45. 45. Recorders and Integrators  Recorders are used to record responses obtained from the detectors after amplification, if necessary.  They record the baseline & all the peaks obtained with respect to time.  Retention time can be found out from this recordings, but area under curve cannot be determined.  The Integrators are improved versions of recorders with 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. Pooja Gupta
  46. 46. Application  Drug Discovery  Clinical Analysis  Proteomics  Forensic Chemistry  Drug Metabolism study  Environmental chemistry  Diagnostic studies  Cosmetic analysis
  47. 47.  Determination of Green Florescent Protein  Structural Determination  Pharmaceutical Applications  Identification of Bile Acid Metabolite  Clinical Applications  Biochemical Genetics  qualitative and quantitative analysis  Therapeutic Drug Monitoring
  48. 48. Thank you