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HPLC

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HPLC

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HPLC

  1. 1. HPLCPresented byANU BALA
  2. 2. OUTLINE• INTRODUCTION• TYPES OF CHROMATOGRAPHY• RP LIQUID CHROMATOGRAPHY• INSTRUMENTATION• CHROMATOGRAM• METHOD DEVELPOMENT LAYOUT• ADVANTAGES OF HPLC
  3. 3. INTRODUCTION • High performance liquid chromatography • A form of liquid chromatography used to separate compounds that are dissolved in solution. • HPLC instruments consist of a reservoir of mobile phase, a pump, an injector, a column, and a detector • Compounds are separated by injecting a sample mixture onto the column • Separation is based on Partition of compounds towards stationary and mobile phase
  4. 4. TYPES OF CHROMATOGRAPHYA. Based on modes of chromatography 1. Normal phase mode 2.Reverse phase modeB. Based on principle of separation 1. Adsorption chromatography 2. Ion exchange chromatography 3. Partition chromatography 4. Size exclusionC. Based on elution technique 1. Isocratic separation 2. Gradient separation
  5. 5. D. Based on the scale of operation 1. Analytical HPLC 2. Preparative HPLCE. Based on the type of analysis 1. Qualitative analysis 2. Quantitative analysis
  6. 6. INSTRUMENTATION
  7. 7. HPLC Pump
  8. 8. Column
  9. 9. Bonded Phases• C-2 Ethyl Silyl -Si-CH2-CH3• C-8 Octyl Silyl -Si-(CH2)7-CH3• C-18 Octadecyl Silyl -Si-(CH2)17-CH3• CN Cyanopropyl Silyl -Si-(CH2)3-CN
  10. 10. Silica Stationery Phase supportThe longer the alkyl chains, the longer theretention time in a reversed phase column.
  11. 11. Injector
  12. 12. DetectorDetector is a device used in liquid and gaschromatography to visualize components of themixture being eluted off the chromatography columnThere are two general types of detectors1 Destructive detector2 Non destructive detector
  13. 13. UV Detector
  14. 14. Photodiode Array Detectors
  15. 15. CHROMATOGRAM
  16. 16. BAD CHROMATOGRAM
  17. 17. METHOD DEVELOPMENT PHYSICOCHEMICAL PROPERTIES (i.e. Structure, Solubility, Nature, components) SAMPLE PREPARATION (i.e. Concentration, Dilutions) SELECTION OF HPLC PARAMETERS1. Mobile phase : Selection of solvents and combinations Selection of Buffers Isocratic / Gradient Flow rate2. Column: NP/RP Temperature3. Detection: Selection of wavelength4. Chromatography: Parameters of Chromatogram
  18. 18. METHOD DEVELOPMENT PRE METHOD VALIDATION1. Selection of Upper and Lower Limit of Quantitation2. Impurity profiling3. Accuracy and Precision4. Linearity5. Selectivity (LOD, LOQ)6. Stability Validation
  19. 19. Advantages of HPLCHigh sensitivityHigh performanceRapid process and hence time savingCan be used for qualitative as well as quantitativeestimationCan be used for both analytical and preparativepurpose

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