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High performance liquid chromatography (HPLC)
High performance liquid chromatography (HPLC) is a separation technique used to separate mixtures. It uses columns packed with small particle sizes under high pressure, allowing better separation than traditional liquid chromatography. HPLC involves pumping a mobile phase through a column containing a stationary phase, separating components as they flow through at different rates based on interactions with the phases. Components are then detected and quantified as they exit the column. HPLC provides rapid, sensitive, and precise separation of mixtures and is widely used in fields like pharmaceuticals, chemistry, and environmental analysis.
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High performance liquid chromatography (HPLC)
- 1. High performance liquid chromatography(HPLC) Capt. Htet Wai Moe Resident Pharmacology
- 2. • A formof column chromatography to separate, identify, and quantify the compounds. • Developed in 1970s. • The most widely used analytical separation technique.
- 3. Chromatography • Chromatography isa technique which separates components in a mixture due to the differing time taken for each component to travel through a stationary phase when carried through it by a mobile phase.
- 4. Basically, all chromatographicsystems consists of two phases. •Mobile phase - liquid or gaseous and flows over or through the stationary phase •Stationary phase - solid, liquid or a solid/liquid mixture which is immobilized
- 5. Some chromatography terms Analyte •Substancethat is to be separated during chromatography Immobilized phase •Stationary phase which is immobilized on the support particles or on the inner wall of the column tubing
- 6. Mobile phase •Phase whichmoves in a definite direction. (liquid/gas/fluid). •Consists of the sample being separated/ analyzed and the solvent that moves the sample through the column. Effluent •Mobile phase leaving the column. Some chromatography terms
- 7. Different types ofchromatography methods • Paper chromatography • Liquid chromatography • Gas chromatography • High performance liquid chromatography
- 8. High performance liquidchromatography • HPLC is an extension of conventional liquid chromatography. • Powerful tool in analytical techniques • Columns are tightly packed, and the eluent is forced through the column under high pressure(up to 5,000 psi) by a pump.
- 9. • Allows touse a very smaller particle size for the column packing material which gives a much greater surface area for interactions between the stationary phase and the molecules flowing through it. • Allows a much better separation of the components of the mixture.
- 10. HPLC Technique • Utilizesliquid mobile phase to separate the mixture • Analytes are first dissolved in a solvent then through the column under high pressure of up to 400 atm • Mixture is resolved into its components in the column
- 11. • The totalseparation time is often 5 or 10 minutes rather than hours or even days required for some separations by gravity flow with the larger systems.
- 12. Components of HPLC •Pump • Injector • Column • Detector • Recorder or data system
- 13. A Flow Schemefor HPLC
- 15. Pump • A pumpforces the mobile phase through the column at a much greater velocity than gravity-flow columns. • The pump can be pneumatic, syringe- type, reciprocating, or hydraulic amplifier.
- 16. Pump (cont.) • Pneumaticpumps are used for preoperative purposes. • The most widely used pump today is the multihead pump with two or more reciprocating pistons.
- 17. • Pumps aredesigned in order to maintain a stable flow rate, avoiding pulsations even when the composition of the mobile phase varies • flow range – 0.01-10 ml/min
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- 19. Injectors • Inject theliquid sample within range of 0.1- 100 ml of volume under high pressure • Produce minimum band broadening • Produce possible flow disturbances • Volume must be small (0.1-500 uL)
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- 22. Columns • Smooth-bore stainlesssteel or heavy- walled glass tubing. • Hundreds of packed columns differing in size and packing are available from manufacture.
- 23. Columns • E.g. Columnpacking vary in size (3 to 20 um) with the smaller particles used mostly for analytical separations and the larger ones for preparative separation. • The most common material used for column packing is silica gel.
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- 26. Detector • HPLC detectorsmonitor the elute as it leaves the column • Produce an electronic signal proportional to the concentration of each separated component
- 27. Detector • Crucial intrace analysis • High sensitivity • Fast response • Simplifies quantitation • Insensitive to changes in type of solvent, flow rate and temp.
- 28. The most widelyused detection methods • Spectrophotometers • Fluorometers • Electrochemical detectors • Mass spectrometer • Refractive index detector
- 29. Detectors used inHPLC Type Principle Detection limit Comments Spectro- photometer Measure absorbance of light <1 ng Analyte must absorb UV or visible light Fluorometers Measures fluorescence pg to ng Analyte must fluoresce Electro- chemical detectors Electrochemically measures oxidized/ reduce analyte pg to ng Useful for catecholamine s
- 30. Detectors used inHPLC Type Principle Detection limit Comments Mass spectrometer Detects ions after separation by mass-to-charge ration fg to ng Analyte must be converted to ionized form Refractometer Measure change in refractive index 1 µg Detection of most compounds but relatively low sensitivity
- 31. Depending on therelative polarity of the solvent and stationary phase, there are two variants in use in HPLC 1.Normal phase HPLC • Utilize polar adsorbent surface and non- polar eluent • Polar substance in the mixture sticks to polar adsorbent than non-polar • Non-polar ones will pass more quickly through the column
- 32. 2. Reversed phaseHPLC • Utilize non-polar adsorbent surface and polar eluent • Attraction between non-polar compound in the mixture and non-polar adsorbent
- 33. 2. Reversed phaseHPLC (cont.) • Polar molecules will travel through the column more quickly because there is strong attraction between polar solvent and polar molecules when pass through the column • Reversed phase HPLC is the most commonly used form of HPLC
- 34. Solvents used inmobile phase • hexane, heptane, cyclohexane, carbon tetrachloride, benzene, toluene, diethyl ether, chloroform etc. Adsorbents used in stationary phase • silica gel, alumina, celite, cellulose powder, ion-exchange, cellulose, starch
- 35. Retention time • Thetime taken for a particular compound to travel through the column to the detector • From the time at which the sample is injected to the point at which the display shows a maximum peak height for that compound.
- 37. Types of chromaticseparation • Adsorption Chromatography • Ion- exchange Chromatography • Size Exclusion Chromatography
- 38. Adsorption Chromatography • Competitionfor adsorption sites occurs between the molecules of the mixture to be separated and the molecules of the mobile phase • Mobile phase can be either a single solvent or two or more solvents depend on the analytes to be desorbed • Speed of migration of the component along the column depend on adsorptive affinity
- 39. Ion- exchange Chromatography •Molecules can be separated by their ionic charges in a process known as Ion- exchange Chromatography. • Ion-exchange resins are used as the column packing materials. • This method is used for separation of ionic species, such as amino acids.
- 41. • Known asgel permeation chromatography or gel filtration chromatography. • Packing material with very small pore is used. • Precisely controlled pore size materials in the column • Large molecules, such as polymers are physically prevented from passing through the column Size Exclusion Chromatography
- 42. Applications HPLC is usedfor • Chemistry and biochemistry research analyzing complex mixtures, • Purifying chemical compounds • Quality control to ensure the purity of raw materials • Analyzing air and water pollutants,
- 43. Applications (cont.) • Monitoringmaterials that may jeopardize occupational safety or health • Monitoring pesticide levels in the environment. • To survey food and drug products, • To identify confiscated narcotics • To determine the amount of such chemical compounds found in new drugs in pharmaceutics
- 44. HPLC as comparedwith the classical technique • Small diameter, reusable stainless steel columns • Column packing with very small particles • Control flow of mobile phase • Precise sample introduction
- 45. HPLC as comparedwith the classical technique (cont.) • Good pumping system • Special continuous flow detectors- can handle small flow rates and detect very small amounts • Rapid analysis • High resolution
- 46. Disadvantages of HPLC •Cost • Complexity • Low sensitivity for some compounds • Irreversibly adsorbed compounds not detected • Co-elution difficult to detect
- 47. Summary • The modernday technique is greatly enhanced in terms of selectivity, resolution, through miniaturization and the use of very elaborate stationary phases. • Therefore HPLC is widely used for separation of molecules in biological, pharmaceutical, food, environmental and industrial process
- 48. References: • Harold Varleypractical clinical chemistry • http://scimedia.com • http://www.forumsci.co.il/HPLC
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Editor's Notes
- #36 Different compounds have different retention times. For a particular compound, the retention time will vary depending on: Pressure used -(affects flow rate of the solvent) Nature of stationary phase (material, particle size) Exact composition of the solvent Temperature of the column