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HIGH PERFORMANCE LIQUID CHROMATOGRAPHY, ppt

HIGH PERFORMANCE LIQUID CHROMATOGRAPHY, ppt

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  • 1. HIGH PERFORMANCE(PRESSURE) LIQUID CHROMATOGRAPHY
  • 2. HPLC
    • Presented By -
    • Mr. Shaise Jacob
    • Faculty
    • Nirmala College of Pharmacy
    • Muvattupuzha, Kerala
    • India
    • Email – jacobshaise@gmail.com
  • 3.
    • Liquid chromatography is a separation technique that involves:
    • the placement (injection) of a small volume of liquid sample into a tube packed with porous particles (stationary phase)
    • where individual components of the sample are transported along the packed tube (column) by a liquid moved by gravity.
  • 4.
    • The components of the sample are separated from one another by the column packing that involves various chemical and/or physical interactions between their molecules and the packing particles.
    • The separated components are collected at the exit of this column and identified by an external measurement technique, such as a spectrophotometer that measures the intensity of the color, or by another device that can measure their amount
    •  Note:The modern form of liquid chromatography is now referred to as “flash chromatography”
  • 5. Four types of high performance liquid chromatography (HPLC):
    • partition
    • adsorption (liquid-solid)
    • ion exchange
    • size exclusion or gel
  • 6. ◊ improved performance
    • ◊ high pressure
    • HPLC- Separation is accomplished by partitioning b/w a M.P & Stationary column material.
    • Packing material
    • small, uniform particle
    • gives high column efficiencies
    • High pressure
    • to achieved desired flow rates
  • 7. Types of HPLC techniques
    • Based on Modes of chromatography
    • 1. Normal phase mode:
    • S.P is polar
    • M.P is non polar
    • 2. Reverse phase mode:
    • S.P is non polar
    • M.P is polar
    • Different columns used: ODS,C18,C8,C4…
  • 8. Based on principle of separation
    • 1. Adsorption chromatography
    • 2. Ion exchange “
    • 3. Ion pair “
    • 4. Gel permeation / Size exclusion “
    • 5. Affinity “
    • 6. Chiral phase “
    • Based on elution technique
    • Isocratic separation
    • Gradient separation
  • 9. Based on scale of operation
    • Analytical HPLC
    • Preparative HPLC
    • Based on the type of Analysis
    • Qualitative analysis
    • Quantitative analysis
    • HPLC offers numerous advantages
    • ♠ Capable of handling “macromolecules”
    • ♠ Suitable for pharmaceutical compounds
    • ♠ Efficient analysis of liable natural products
    • ♠ Reliable handling of inorganic & ionic species
  • 10. ♠ Dependable analysis of biochemical's
    • PRINCIPLE
    • Adsorption
    • Particle size of the S.P material plays a crucial role in HPLC
    • Micro particulate column packing :
    • Silica particles -> uniform, porous, with spherical or irregular shape
    • Diameter -> 3.5 to 10 µm
  • 11. HPLC instrumentation comprises:
    • M.P reservoirs
    • Eluent degas module
    • Solvent delivery pumps
    • Manual / Auto injector
    • Analytical column
    • Detector
    • Data processor
  • 12.  
  • 13.  
  • 14.  
  • 15.  
  • 16. Mobile phase reservoir
    • stores M.P (HPLC grade solvents)
    • ♠ Resolution & Speed of analysis }
    • Flow rate, polarity & pH of M.P
    • Can't use metallic reservoir
    • Eluent degas module
    • Dissolved gases in M.P pose a number of problems
    • ∆ flow ∆ excessive detector noise
    • ∆ Rt fluctuation
    • ♠ Bubbling the pump & detector,
  • 17.
    • Degas module with reservoir of inert gases
    • He or N 2
    • Vacuum filtration
    • Helium purging
    • Ultrasonication (converts ultra high frequency to mechanical vibrations.)
    • SOLVENT DELIVERY PUMPS
    • Reciprocating pumps:
    • » widely used
    • » maintain accurate flow rate
  • 18.  
  • 19.  
  • 20. Cross-sectional diagram of a simple single – piston reciprocating pump
  • 21. Solvent delivery systems
    • two types: 1. Isocratic system
    • 2. Low pressure gradient
    • 3. High pressure gradient
    • Injection system
    • a. Syringe system:- results best
    • b. Injection valve :- [Rheodyne injector]
    • » Loading through the sample loop (20-50µl)
  • 22. u
    • c. Automated injection device :-
    • commercially available, automatically inject 100samples
  • 23. Guard column
    • Pre-filter :- useful for industry
  • 24. Analytical column
    • Heart of any chromatographic system
    • » Actual separation of components takes place
    • Several S.P available
    • depending upon tech. or mode of separation
    • Column material
    • S.S, glass, polyethylene, PEEK
    • Column length Column diameter Particle size
    • 5-30 cm 2mm-50mm 1µ-20µ
  • 25. Particle nature:
    • Spherical, uniform sized porous material
    • Surface area
    • 1g S.P provides surface area 100-860 sq.m
    • Functional group
    • Depends on the type of chromatographic separations.
    • Normal phase mode: hydroxy group
    • Reverse phase mode: C 18 (octa decyl silane)
    • Bondapak ( waters)
    • C 8 octyl column, C 4 butyl column, CN Nitrile column
    • NH 2 Amino column
  • 26.  
  • 27. b
  • 28. Column packing
    • three forms
    • 1.Microporous support
    • 5-10 µm in d.m
    • 2. Pellicular
    • Porous & 40 µm in d.m
    • 3.Bonded phase
    • S.P bonded onto an inert support
  • 29. DETECTORS
    • 1. UV DETECTOR : Based on UV light ab.
    • > fixed WL detector (254nm)
    • > variable WL detector (190-600nm)
    • 2. REFRACTIVE INDEX DETECTOR :
    • Non specific / Universal detector
    • ↓ sensitivity & specificity
    • 3. PHOTODIODE ARRAY DETECTOR (PDA)
    • similar to UV detector, non destructive
    • 190-600 nm for quantization & identification
    • Spectra is 3D, Response vs time vs WL
  • 30. Photodiode Array Detector
  • 31. Flourimetric detector
    • Excitation & emission WL can be selected
    • ↑ sensitive than UV
    • Disadvantage: Some comp. are not fluorescent
    • Conductivity detector
    • based on electrical conductivity
    • Amperometric detector
    • Reduction / oxidation
  • 32. RECORDERS & INTEGRATORS
    • Recorders – to record the responses
    • Integrators - data processing capabilities
    • ◊ record individual peaks with Rt, height, width
    • of peaks, peak area, % of area..
    • Selection of solvent systems
    • Solvent compatibility with the detector
    • e.g.. Hexane, chloroform, ACN , Methanol…
    • Most widely used solvent in HPLC is water
    • Millipore Milli-Q apparatus produce water
  • 33. Selection of Column
    • Non polar & moderately polar comp. ->
    • ADSORPTION CHRO.
    • Highly polar molecules by -> R.P Chro.
    • Acids & Bases by -> Ion exchange Chro.
  • 34. APPICATIONS OF HPLC
    • ♥ Pharmaceutical field
    • ♥ Chemical & Petrochemical industry
    • ♥ Forensic
    • ♥ Biochemical separations
    • ♥ Food analysis
    • Qualitative analysis
    • Checking the purity of a compound
  • 35. Quantitative Analysis
    • Direct comparison method
    • injecting the sample & std. separately & comparing their peak areas.
    • Area of the peak = peak height x width of peak at half height
    • Calibration curve method
    • Multi component analysis
    • Isolation & identification of drugs
    • Stability studies
  • 36.  
  • 37.  
  • 38.  
  • 39.  
  • 40.