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High performance liquid chromatography is a convenient separation technique used for wide types of samples, with exceptional resolving power, speed and nano molecular detection levels. It is presently used in pharmaceutical research and development,
• To purify synthetic or natural products.
• To characterise metabolites.
• To assay active ingredients, impurities, degradation products and in dissolution assays.
• In pharmacodynamics and pharmacokinetic studies.2

High performance liquid chromatography is a convenient separation technique used for wide types of samples, with exceptional resolving power, speed and nano molecular detection levels. It is presently used in pharmaceutical research and development,
• To purify synthetic or natural products.
• To characterise metabolites.
• To assay active ingredients, impurities, degradation products and in dissolution assays.
• In pharmacodynamics and pharmacokinetic studies.2

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hplc.pptx

  1. 1.  HPLC stands for “High Performance Liquid Chromatography”.  High performance liquid chromatography is a powerful tool in analysis, it yields high performance and high speed compared to traditional column chromatography because of the forcibly pumped mobile phase Chromatography: • Separation of mixture of samples into individual samples. • It is a separation technique which separation take Between two phases 1.Stationary phase 2.Mobile phase 1.Stationary phase : The substance on which adsorption of analyte take place. 2.Mobile phase: Solvent which carries the analyte. 1
  2. 2. Liquid chromatography: 2  It is a chromatographic technique in which mobile phase is a liquid.  The main principle involved in liquid chromatography is adsorption. PRINCIPLE When a mixture of samples introduced into column various chemical and physical interactions takes place between column material and components present in the sample travel according to their relative affinities towards the stationary phase . the component which has more affinity towards the stationary phase ,travels slower. The components which having less affinity towards stationary phase travels faster. More over it work very efficiently compare toother chromatographic techniques. In this high pressure pump is used to transfer the sample to stationary phase. so it is also called as high pressure liquid chromatography.
  3. 3.  The principle of HPLC are based on Van Demeterequation which relates the efficiency of the chromatographic column to the particle size of the column, molecular diffusion and thickness of stationary phase.  The Van Deemter Equation is given as H or HETP = A + B/u + C υ Where, A= Represents eddy diffusion, B= Represents molecular diffusion, C =Represents rate of mass transfer, υ =Represents flow rate. 3
  4. 4. TYPES OF CHROMATOGRAPHY  Based on modes of chromatography 1. Normal phase mode 2.Reverse phase mode  Based on principle of separation 1. Adsorption chromatography 2. Ion exchange chromatography 3. Partition chromatography 4. Size exclusion  Based on elution technique 1. Isocratic separation 2. Gradient separation  Based on the scale of operation 1. Analytical HPLC 2. Preparative HPLC  Based on the type of analysis 1. Qualitative analysis 2. Quantitative analysis 3
  5. 5. 1.BASED ON MODE OF SEPARATION 5  Normal Phase HPLC: This method separates analytes on the basis of polarity. NP-HPLC uses polar stationary phase and non-polar mobile phase. Therefore, the stationary phase is usually Silica and typical mobile phases are hexane, methylene chloride, chloroform, diethyl ether, and mixtures of these.  Reverse Phase HPLC :The stationary phase is non polar (hydrophobic) in nature, such as C18, C8, Cyano columns used. While the mobile phase is a polar liquid, such as mixtures of water and methanol or acetonitrile.
  6. 6. 6
  7. 7. 2.BASED ON PRINCIPLE OF SEPRATION i)Adsorption chromatography: ⚫ In which stationary phase is an adsorbent. ⚫ The compounds separated based on their affinity towards stationa -ry phase. ⚫ More affinity-slow elution ⚫ Less affinity-fast elution ii)Partition chromatography: ⚫ A process of separation of solutes utilizing the partition of the solutes between two liquid phases ⚫ Namely the original solvent and the film of solvent on the adsorption column. 7
  8. 8. iii)Size-Exclusion HPLC: 8 ⚫ In which the stationary phase is a gel having a closely controlled pore size. ⚫ Molecules are separated based on molecular size, shape smaller molecules being temporarily retained in the pores and mainly Agarose , Dextran used as mobile phase. iv)Ion-Exchange HPLC: ⚫ Ion-exchange chromatography separates molecules based on their respective charged groups. ⚫ Ion-exchange chromatography retains analyte molecules on the column based on coulombic (ionic) interactions. ⚫ Essentially, molecules undergo electrostatic interactions with opposite charges on the stationary phase matrix.
  9. 9. 3.BASED ON ELUTION TECHNIQUE 9 During the chromatographic experiment, a pump can deliver a constant mobile phase composition(isocratic) or an increasing mobile phase composition (gradient). i) Isocratic Elution ⚫ Delivers constant mobile phase composition; ⚫ Solvent must be pre-mixed; ⚫ Lowest cost pump; ⚫ Best for simple preparation. ii) Gradient Elution ⚫ Delivers variable mobile phase composition; ⚫ In this mobile phase is programmed to change in composition during elution time; ⚫ Best for complex preparations.
  10. 10. 4.BASED ON SCALE OF OPERATION i)Analytical HPLC: No recovery of individual components of substance. ii)Preparative HPLC: Individual components of substance can be recovered. 5.BASED ON TYPE OF ANALYSIS i)Qualitative Analysis: Determine the quality of sample. ii)Quantitative Analysis: Determine the quantity(concentration) of sample. 10
  11. 11. INSTRUMENTATION OF HPLC 11  Solvent storage bottle  Gradient controller and mixing unit  De-gassing of solvents  Pump  Pressure gauge  Pre-column  Sample introduction system  Column  Detector  Recorder
  12. 12. HPLC INSTRUMENTATION OVER-VIEW 12
  13. 13. 13
  14. 14. I. Solvent reservoir & Mixing unit & degassing system 14 ⚫ The appropriate solvents [mobile phase] from the reservoirs are allowed to enter the mixing chamber where a homogenous mixture is obtained. ⚫ Several gases are soluble in organic solvents. ⚫ When solvents are pumped under high pressure, gas bubbles are formed which will interfere with the separation process, steady base line and the shape of the peak. ⚫ Hence degassing of solvent is important. This can be done by -Vacuum filtration, -Helium purging, -Ultrasonication.
  15. 15. II. High pressure pump ⚫ The role of the pump is to force a liquid (called the mobile phase)through the liquid chromatograph at a specific flow rate, expressedin milliliters per min (mL /min). ⚫ Normal flow rates in HPLC are in the 1-to 2-mL/min range. ⚫ Typical pumps can reach pressures in the range of 6000- 9000psi (400-to 600bar). ⚫ There are several types of pumps used for HPLC most commonly used are:- 1.Reciprocating Piston Pump, 2.Syringe Pump, 3.Constant Pressure Pump. 15
  16. 16. HPLC Pump Constant flow rate (Mechanical) Pump Constant Pressure Pump (Pneumatic) Manner in which they operate syringe (Displace ment pump) Reciprocating pump Single piston reciprocati ng pump Double piston reciprocating pump Reciprocating diaphragm pump Direct pressure pump Amplifier pump 16
  17. 17. 1.Reciprocating pump: -The piston is moved in and out of a solvent chamber by an - eccentric cam or gear. -The forward-stroke closes the inlet-check value while the outlet valve opens and the respective mobile phase is duly pumped into the column. -Consequently, the return-stroke-closes the outlet valve and it - refills the chamber. Advantages: *The internal-volume can be made very small from 10-100 μl, *The flow-rate can be monitored either by changing the length of the piston or by varying the speed of the motor. *It has an easy access to the valves and seals. 17
  18. 18. DISPLACEMENT PUMPS 18 ⚫ 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) & considerably inconvenient when solvents must be changed.
  19. 19. 19
  20. 20. 20 3.Constant Pressure Pump : -A constant-pressure pump acts by applying a constant pressure to the mobile-phase. -The flow rate through the column is determined by the flow resistance of the column.
  21. 21. PNEUMATIC PUMPS  In this pumps, the mobile phase is driven through the column with the use of pressure produced from a gas cylinder.  It has limited capacity of solvent  Due to solvent viscosity back pressure may develop. 21
  22. 22. III. Injector:  The injector serves to introduce the liquid sample into the flow stream of the mobile phase.  Typical sample volumes are 5 to 20μL.  The injector must also be able to withstand the high pressures of the liquid system. Types of injectors : A.Manual injectors: User manually loads sample into the injector using a syringe and then turns the handle to inject sample into the flowing mobile B. Auto sampler injector: User loads vials filled with sample solution into the auto sampler tray(100 samples) -Measures the appropriate sample volume, -Injects the sample, 18
  23. 23. SAMPLE INJECTOR SYSTEM 23  Several injector devices are available either for manual or auto injection of the sample. (i) Septum Injector (ii) Stop Flow Injector (iii) Rheodyne Injector
  24. 24. i. SEPTUM INJECTOR 24 ⚫ 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.
  25. 25. 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. 25
  26. 26. 26
  27. 27. SELECTION VALVE BY USING THE SELECTION VALVE WE CAN SELECT WHETHER THE PURPOSE IS FOR ANALYTICAL PURPOSE OR PREPARATIVE PURPOSE. INJECT POSITION In this position the loaded sample is injected into the column by the forceful flow of the solvent into the sample loop by which the sample is introduced into the column. LOAD POSITION In this position the sample is loaded into the sample loop . 43 43
  28. 28. 44 44 HPLC AUTO INJECTORS Inside of SIL-20AC
  29. 29. 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
  30. 30. 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. 30
  31. 31. DETECTORS 31  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)
  32. 32. SELECTION OF DETECTORS Detectors 32 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.
  33. 33. 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
  34. 34. IDEAL DETECTOR PROPERTIES 34  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
  35. 35. 19 Manual injectors Auto sampler injector
  36. 36. IV. Column  The column [stationary phase] separates the sample components of interest using various physical and chemical parameters.  The columns used for HPLC are generally made up of stainless steel . so they can withstand up to high pressure [8000psi].  Straight columns of 20-50 cm in length & 1-4mm in diameter are used .  Particle size should be for porous particles 20-40μm. For porous micro particles it should be 3-10 μm.  porous plugs of stainless steel are used in ends of column to retain the packing material. Stationary phases used in column: -Alumina -Silica -Polystyrene -Polyvinyl acetate beads 20
  37. 37. TYPES OF COLUMNS IN HPLC: A. Guard Column B. Fast Column C. Preparative(i.d. > 4.6 mm; lengths50-250mm) D. Capillary (i.d. 0.1 -1.0 mm; various lengths) A. Guard column: Guard columns, set between the injector and an analytical column, are used to protect analytical columns from chemical impurities in samples. We have two types of guard columns. Cartridge type which can be changed easily by hand, and Packed type, which are packed in stainless cartridge like analytical columns. 21
  38. 38. . B. Fast column: 38 One of the primary reasons for using these column is to obtain improved sample output ( amount of compound per unit time). Fast column are designed to decrease the time of chromatographic analysis. C. Capillary column: They allow the user to work with nano liter sample volume , decreased flow rate and decreased solvent usage volume , led to cost effectiveness. D. Preparative column: It Used when objective is to prepare bulk ( milligrams) of sample for laboratory preparatory application. It has usually a large column diameter , which is designed to facilitate large volume injections into the HPLC system.
  39. 39. COLUMN EFFICIENCY IN SEPARATION PROCESS A. Theoretical plates: ⚫ Chromatographic column contains a large number of separate layers, called ‘Theoretical Plates’. ⚫ Separate equilibrations of the sample between the stationary and mobile phase occur in these "plates". ⚫ The analyte moves down the column by transfer of equilibrated mobile phase from one plate to the next. ⚫ It is important to remember that the plates do not really exist ; they are a figment of the imagination that helps us understand the processes at work in the column. B. HETP [Height Equivalent to a Theoretical plate]: ⚫ A theoretical plate is an imaginary unit of a column, where distribution of solute between , stationary phase and mobile phase, has attained equilibrium. 39
  40. 40. ⚫ It also called as functional unit of the column. ⚫ It can beof any height ,which describes the efficiency of separation HETP = L /N Where HETP = LENTH OF COLUMN /NUMBER OF THEORETICAL PLATES ⚫ If HETP is less , the column is more efficient . ⚫ If HETP is more , the column is les s efficient . C. RESOLUTION: ⚫The most important thing in HPLC is to obtain the optimum resolution in the minimum time ⚫ Resolution is the ability to separate two signals i.e., separation of two constituents. 40
  41. 41. D. PEAK ASYMMETRY ⚫ In the ideal world all chromatographic peaks would be symmetrical (or Gaussian) . ⚫ However, due to the effects of instrument ,adsorptive effects of the stationary phase and the quality of the column packing peaks may often show tailing & fronting behavior. Fronting : Deformation at the beginning of the peak. It is due to saturation of stationary phase with higher quantity of components. Tailing: Deformation at the end of the peak. It is due to similarity of polarity for a component towards stationary phase. 41
  42. 42. FACTORS AFFECTING EFFICIENCY OF COLUMN 42
  43. 43. V.DETECTORS: 43 1. UV- Visible Dectectors. 2. Photo Diode Array Detectors. 3. Refractive Index Detectors. 4. Fluorescence Detectors. 5. Conductivity Detectors. 6. Mass Spectrometer. 7. Evaporative Light Scattering Detectors.
  44. 44. Refractive index detector Fluorescence detectors 44
  45. 45. 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 innert.  Wide varities of stationary phase.  Mobile phase was chemically innert.  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. 10
  46. 46. 46 HPLC DISADVANTAGES :  Co-elution.  Cost.  Complexity.
  47. 47. APPLICATIONS 47  Purification Of Samples.  Identification Of Compounds.  Determination Of Impurities.  Determine the Concentration Of Drug.  Biopharmaceutical & Pharmacokinetic Studies.  Drug Stability Studies.  Qualitative & Quantitative Analysis.  Environmental Applications[ analysing air & water pollutants.  Separation Of Mixture Of Samples. Ex: Carbohydrates
  48. 48. QUANTITATIVE ANAYSIS  To measure the concentration of each compound in a sample.  There are 2 main ways to interpret a chromatogram.  Determination of the peak height of a chromatographic peak as measured from the baseline.  Detection of the peak area. 48
  49. 49. QUALITATIVE ANALYSIS  The identification of individual compounds in the sample.  The most common parameter for compound identification is its Retention Time [Is the amount of time a compound spends on the column after injection]  Depending on the detector used, compound. identification is also based on the chemical structure, molecular weight or some other molecular parameter. 49
  50. 50. REFERENCES 50  Principles of instrumental analysis- Doglas A skoog  Instrumental methods of chemical analysis- Gurudeep R.chatwal, sham K. anand  Textbook of chemical analysis- Francis Rouessac and Annick Rouessac 2nd edition john wiley & sons ltd  Text book of modern analytical chemistry- David harvey

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