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HIGH PERFORMANCE LIQUID CHROMATOGRAPHY [ HPLC ]

HIGH PERFORMANCE LIQUID CHROMATOGRAPHY [ HPLC ]. Instructor Prof. Avinash P. Tupe. 1. Learning Outcomes. Define Chromatography State different types of Chromatographic Techniques. State Principle behind the Liquid chromatography Define HPLC. State Principle behind the HPLC technique.

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HIGH PERFORMANCE LIQUID CHROMATOGRAPHY [ HPLC ]

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  1. HIGH PERFORMANCELIQUID CHROMATOGRAPHY[HPLC] Instructor Prof. Avinash P. Tupe 1

  2. Learning Outcomes Define Chromatography State different types of Chromatographic Techniques. State Principle behind the Liquid chromatography Define HPLC. State Principle behind the HPLC technique.

  3. It is define as, it is analytical method in which separation of active constituent in complex mixture, and the mixture was distributed in two phases i.e. stationary phase and mobile phase is known aschromatography. • It isa techniqueused forseparation, purification, Identification and extraction ofcompound. • It is amethod it can consist of two phases stationary phase andmobilephase. • Stationary phase is constant phase or column packagingmaterial. • Mobile phase is moveablephase. • Thebasicprinciple of chromatographyisbased onAdsorptionand partitionchromatography. • Adsorption chromatography - The affinityof molecules towards stationaryphase is known as Adsorptionchromatography. • Partition chromatography - The molecule can moves in two phases of liquid is known as partitionchromatography. • It is important for qualitative and quantitativeanalysis. • 2 CHROMATOGRAPHY

  4. TYPES OFCHROMATOGRAPHY • Based on modes ofchromatography • Normal phasemode • Reverse phasemode • Based on principle ofseparation • Adsorptionchromatography • Ion exchangechromatography • Partitionchromatography • Sizeexclusion • Based on elutiontechnique • Isocraticseparation • Gradientseparation • Based on the scale ofoperation • AnalyticalHPLC • PreparativeHPLC • Based on the type ofanalysis • Qualitativeanalysis • Quantitativeanalysis 3

  5. PRINCIPLES OF LIQUIDCHROMATOGRAPHY 5

  6. TYPES OF LIQUIDCHROMATOGRAPHY 6

  7. HPLC • HPLC is a High Performance liquidChromatography. • High Pressure LiquidChromatography. • High Priced LiquidChromatography. • It is columnchromatography. • It is LiquidChromatography. • It is modified from of gas chromatography, it is applicable for both Volatile as well as Non volatilecompound. • It can mainly divided by two types 1. Normal phase HPLC 2. Reversed PhaseHPLC. • It is having a high resolution and separation capacity. • It is used as qualitative as well as quantitativeanalysis. • 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. 7

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  9. PRINCIPLE • High Performance Liquid Chromatography [HPLC] 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 volatilecompounds. • It is important for determination qualitative and quantitativeanalysis. • It is important for determination of Retention Time (the time is required , after sample injection maximum angle peak reaches todetector) 8

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  11. ADVANTAGES • It is simple, rapid ,reproducible. • High sensitivity. • Highperformance. • Rapid process and hence timesaving. • It is having a high resolution and separationcapacity. • Accuracy andPrecision. • Stationary phase was chemicallyinert. • Wide varities of stationaryphase. • Mobile phase was chemicallyinert. • Less requirement of mobile phase in developingchamber. • Early recovery of separatedcomponent. • Easy visualization of separatedcomponents. • It is having Good reproducibility andrepeatability. • It is ananalytical technique which is important for validation of product, quality control studies ofproduct. • It is important for qualitative and quantitativeanalysis. • It is used for both analytical and preparativepurpose. 10

  12. TYPES OF HPLCSEPARATIONS • Normal Phase: Separation of polar analytes by partitioning onto a polar,bonded • stationaryphase. • Reversed Phase: Separation of non-polar analytes by partitioning onto a non-polar, bonded stationaryphase. • Adsorption: In Between Normal and Reversed. Separation of moderately polar analytes using adsorption onto a pure stationary phase (e.g. alumina orsilica) • Ion Chromatography: Separation of organic and inorganic ions by their partitioning onto ionic stationary phases bonded to a solidsupport. • Size Exclusion Chromatography: Separation of large molecules based in the paths they take through a “maze” of tunnels in the stationaryphase.

  13. HPLCMETHODS

  14. HPLCCONTAIN • Stationary Phase - It is non polar and The stationary solid surface is coated with a 2nd liquid (the Stationary Phase) which is immiscible with mobilephase. • Mobile Phase - A polar mobile phase(ACN, MeOH, WATER +BUFFER). • Bonded Phases- • C-2 EthylSilyl • C-8 Octyl Silyl • C-18 OctadecylSilyl • CN CyanopropylSilyl -Si-CH2-CH3 -Si-(CH2)7-CH3 -Si-(CH2)17-CH3 -Si-(CH2)3-CN

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