HPLC.pptx

High- performance Liquid Chromatography”/
(High- pressure Liquid Chromatography) is a powerful tool in analysis, it yields High Performance and high speed compared to traditional columns chromatography

1. Chromatography Dr. Suji V. S.

2. Chromatography Term derived from Greek word “ Chroma ” meaning ‘ Colour ’ “ Graphein ” meaning ‘to write’ collective term for a set of  laboratory techniques for the  separation of mixtures . The mixture is dissolved in a fluid called Mobile phase ,  which carries it through a structure holding another material called Stationary phase .

3. History 1 st employed by M. S. Tswett , a Botanist in 1903 for separation of plant pigments using a column of alumina

4. Types of Chromatography

5. Types of Chromatography Adsorption Partition Paper TLC Ion exchange Gel filtration Affinity HPLC

6. Adsorption chromatography

7. Adsorption chromatography Earliest form of chromatography Based on differences in adsorption at the surface of Solid Stationary Medium (Silica gel- Common adsorbing substance) Relative Lipophilic drugs readily analysed by Silica gel HPLC with UV detection

8. Partition chromatography Developed by Martin & Synge (1941) (Nobel Prize 1952) Use- Separation of mixtures of Amino Acids & Peptides

9. Partition chromatography Stationary phase- Solid/ Liquid over which Mobile phase (Liquid/Gas) moves. depends on Partition Co-efficient ( solubility ) of the particular substances in the mixture.

10. Partition chromatography Types Depending on phases of the components partitioned Solid-liquid, Liquid-liquid, Gas -liquid, etc.

11. Partition chromatography 2. Based On Mode Of Separation RPC- More Commonly used in TDM as Drugs are usually Hydrophilic Stationary phase Mobile phase Normal phase chromatography (NPC) Polar (hydrophilic) Non -polar (hydrophobic) Reverse phase chromatography (RPC) Non -polar (hydrophobic) Polar (hydrophilic)

12. 2A. Paper chromatography Stationary phase - water held on solid support of filter paper (cellulose) Mobile phase - mixture of immiscible solvents ( water+a non polar solvent+ acid/base) eg . Butanol - acetic acid- water

13. 2B. Thin Layer Chromatography (TLC) Stationary water phase- held on a silica gel ( Kieselguhr ) spread on a glass plate Mobile phase- Non Polar Solvent moves up Advantage of TLC over Paper Chromatography- Separation takes 2-4hrs

14. Visualisation of chromatography After chromatographic run is over, paper/ plate has dried  sprayed with a Location Reagent Location Reagent Ninhydrin Proteins Sulfuric acid Phospholipids Diphenylamine Sugars

15. Rf value Ratio of fronts ( Rf ) = Distance travelled by solute Distance travelled by solvent Is a constant for a particular solvent system at a given temperature.

16. Gas Chromatography Type of partition chromatography where Mobile phase is Gas . Stationary phase- liquid/solid supported by a column of inert material (silica) in a long narrow column Separation is based on: -Solute differences in vapor pressure -Interaction with stationary phase

17. GLC components Column Supply of carrier gas Flow control apparatus Injector Column oven Detectors Computer

18. GLC (Gas Liquid Chromatography) Mixture of substances to be separated is made volatile at one end of the column & the vapors are swept over the column by an inert carrier gas like argon/ nitrogen Fractions emerging from the column are detected & quantified by detecting devices Suitable for compounds ( eg Lipids ) which resists degradation & temperature.

19. GLC (Gas Liquid Chromatography) Advantage – Requires only a small sample; Specific. Disadv - Time consuming, High potential for Errors Volatile components Not suitable for TDM

20. Ion exchange chromatography Separation is based on Electrostatic Attraction between charged Biological molecules to oppositely charged groups on the ion exchange resins. Use- Separation of proteins & Amino Acids

21. Ion exchange chromatography

22. Gel Filtration Chromatography

23. Gel Filtration Chromatography ~ Gel Permeation /Molecular sieving/ Size Exclusion Chromatography Based on Molecular Size of solute Use- Separation not only of Macromolecules but also of Small Molecular Weight substances. Separation of proteins; Purification of Proteins Molecular weight determination

24. Affinity chromatography

25. Affinity chromatography Based on High Affinity of specific proteins for specific chemical groups Uses Co -enzymes can be used to purify enzymes Eg . NAD + used to purify Dehydrogenases Separating antibodies & antigens

26. HPLC

27. HPLC “High- performance Liquid Chromatography”/ (High- pressure Liquid Chromatography). is a powerful tool in analysis, it yields High Performance and high speed compared to traditional columns chromatography The liquid phase is passed through the column under High Pressure.

28. Principle of HPLC Chromatography principle Resolving power (Resolution) of HPLC increases with Column length and Smaller particle size of stationary phase. High Pressure (500-6000 psi) Because of small particle size of stationary phase <10µ  high resistance to the flow of solvent

29. Factors determining HPLC Mechanical separation power ( Efficiency ) Column length Particle size Packed bed uniformity Chemical separation power ( Selectivity ) Interaction between Packing materials & Mobile phase

30. HPLC setup sketch

31. HPLC system

32. UPLC (Ultra Performance HPLC) If resolution increased by Decreasing particle size ( 1-1.7 μ ); Very high pressure, 15,000-1,00,000 psi is used to deliver mobile phase.

33. A . Solvent delivery system (Mobile Phase) acts as a carrier to the sample solution

34. B. Pumps Force the solvent (mobile phase) through the liquid chromatograph at a specific flow rate (1-2mL/min). Typical pumps can reach pressures of 6000-9000 psi.

35. C. Injector introduces liquid sample into the flow stream of the mobile phase for analysis Typical sample volumes : 5-20 μL

36. D. Column (Stationary phase) “ Heart of the Chromatograph ” Column Inner Diameter & Packing play imp. role in Efficiency of HPLC separates the sample components using various physical and chemical parameters. “ Packing ” Small particles (< 10µ ) inside the column ( Silica gel) Resp. for the High Back Pressure at normal flow rates.

37. Column Made of Stainless Steel to withstand high pressure HPLC Column Dimensions

38. E . Detector Detect & measure the amount of individual molecules that elute from the column convert the data into an electrical signal  provides an output to a Recorder (Computer)  Liquid Chromatogram

39. Types of Detector Detector is selected based on the analyte or the sample under detection Ultraviolet detector Fluorescence detector Refractory index detector Mass spectrometry detector coupled with HPLC called LC-MS

40. F. Recorder (Computer) controls all the modules of the HPLC instrument takes the signal from the detector and uses it for analysis of sample components ( qualitative analysis) and the amount of sample ( quantitative analysis).

41. HPLC chromatogram Conc. of each detected component is calculated from the area or height of the corresponding peak

42. TYPES OF HPLC Based On Mode Of Separation RPC- More Commonly used in TDM as Drugs are usually Hydrophilic Stationary phase Mobile phase Normal phase chromatography (NPC) Polar (hydrophilic) Non -polar (hydrophobic) Reverse phase chromatography (RPC) Non -polar (hydrophobic) Polar (hydrophilic)

43. Types of HPLC II. Based On Principle of Separation Adsorption (Liquid- solid) Partition Ion exchange Gel permeation Selection of proper HPLC mode is necessary for each drug

44. Types of HPLC Stattionary phase Mobile phase Type of interaction Feature NPC Silica gel (polar) Organic solvent (n-Hexane/ IPE) 100% organic (Non polar) Adsorption Fat soluble RPC Silica – ODS (Silica-C18) Non polar MeOH / water (Polar) Partition / Hydrophobic MC used SEC Porous polymer Aqueous porous polymer Organic solvent (THF) Buffer solution Gel permeation Molecular Wt distribution, protein separation IEC Ion exchange gel Buffer solution Ion exchange Separation of ionic substance

45. Advantages of HPLC Separations fast and efficient (High Resolution Power) Continuous monitoring of the column effluent Separation and analysis of very complex mixtures Accurate quantitative measurements. Repetitive and reproducible analysis using the same column. Adsorption, partition, ion exchange and exclusion column separations are excellently made.

46. Advantages of HPLC HPLC is more versatile than GLC because Not being restricted to volatile & thermally stable solute Choice of mobile and stationary phases is much wider. Both aqueous and non aqueous samples can be analyzed with little or no sample pre treatment High degree of Selectivity for specific analyses. As variety of solvents and column packing are available Determins multiple components in a single analysis.

47. Applications Widely used in Biotechnology, Biomedical, Biochemical research MC used analytical technique for TDM In other fields & industries Cosmetics Food & Flavour Forensic Environmental studies

48. Applications of HPLC Clinical used for assaying or monitoring many substance like Amino acids, peptides, proteins, carbohydrates, lipids, vitamins, nucleic acids, hormones, metabolites, Drugs- Antiarrhythmics , Antibiotics, Antiepileptics , Analgesics, Tricyclic Antidepressents , etc.

49. Advantages of HPLC Versatality Efficacy Precision Sensitivity Speed Most popular, widely accepted, powerful form of chromatography for analysis.

50. Thank you