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HPLC Guide Chromatography Liquid High Performance
- 2. HPLC • HPLC instrument • Mobile phases • HPLC columns • Stationary phase • Sample introduction • Detectors for HPLC • Quantitative applications High Performance Liquid Chromatography (HPLC)
- 3. HPLC HPLC • The mobile phase is usually a liquid and the stationary phase is a solid or a liquid film coated on a solid surface. – Adsorption chromatography • Solutes separate based on their ability to adsorb to a solid stationary phase. – Partition chromatography • A thin liquid film coating a solid support serves as the stationary phase. • Separation is based on a difference in the equilibrium partitioning of solutes between the liquid stationary phase and the mobile phase.
- 4. HPLC HPLC – Ion exchange chromatography: • Stationary phases consisting of a solid support with covalently attached anionic (e.g., SO3 –) or cationic (e.g., –N(CH3)3 +) functional groups. Ionic solutes are attracted to the stationary phase by electrostatic forces. – Size-exclusion chromatography: • Porous gels are used as stationary phases. Separation is due to differences in the size of the solutes
- 5. HPLC Normal Phase and Reverse Phase • Normal Phase – Polar Stationary Phase eg. Silica – Non-polar Mobile Phase, eg. Hexane, pentane – Neutral compounds elute first; Polar compounds elute last • Reverse Phase – Non-Polar Stationary Phase, eg C18, C8 – Polar Mobile Phase, eg, H20, MeOH, CH3CH2CN – Polar Compounds elute first; Neutral Compounds elute last.
- 6. HPLC NP and RP • Reverse Phase is method of choice – Very broad scope, allows separation of wide variety of compounds with differing polarity – Inexpensive mobile phase (H20, methanol, acetonitrile) – Can be applied to ionic and ionisable compounds (ion pairing) – Generally experimentally easier with rapid equilibration, faster and more robust separations – Organic solvents can be more dangerous to use • Limitations – Columns are generally stable within the pH range of 3-8
- 7. HPLC HPLC instrument Schematic diagram of a high-performance liquid chromatograph
- 9. HPLC
- 10. HPLC HPLC column
- 11. HPLC HPLC Pumps • Isocratic – Pump delivers constant composition of mobile phase • Gradient – Pump delivers variable mobile phase composition or flow rate – Gradients are used for complex samples
- 12. HPLC Properties of HPLC mobile phases
- 13. HPLC Isocratic Elution • Performed with a single solvent • In a reverse phase separation, eluent strength decreases as the solvent becomes more polar.
- 14. HPLC Isocratic Elution - Peak Identity 1. benzyl alcohol 2. phenol 3. 3’,4’-dimethoxyacetophenone 4. benzoin 5. ethyl benzoate 6. toluene 7. 2,6-dimethoxytoluene 8. o-methoxybiphenyl
- 15. HPLC A - aqueous buffer; B - acetonitrile Isocratic Separation
- 19. HPLC HPLC stationary phases – In liquid–liquid chromatography the stationary phase is a liquid film coated on a packing material consisting of 3–10 m porous silica particles. – Bonded stationary phases are attached by reacting the silica particles with an organochlorosilane of the general form Si(CH3)2RCl, where R is an alkyl or substituted alkyl group
- 20. HPLC HPLC stationary phases – To prevent unwanted interactions between the solutes and any unreacted –SiOH groups, the silica frequently is “capped” by reacting it with Si(CH3)3Cl (end-capped columns)
- 21. HPLC HPLC Silica Bonded Phases Amino SAX Cyano Methyl Hexyl C18 C8 Phenyl SCX Butyl C8 Butyl C18
- 22. HPLC Surface of Reversed Phase Packing
- 23. HPLC Mobile Phase Considerations • Solid particles • Dissolved gases • Stabilisers / Antioxidants • U.V absorbing impurities • Viscosity • Toxicity
- 24. HPLC Solvent Filtration Removes particulate matter and dissolved gases Must always be done without exception!
- 25. HPLC UV spectrum of Acetonitrile 99.9%
- 26. HPLC UV spectrum of Methanol
- 27. HPLC HPLC Columns Silica Normal phase, gel permeation Alumina Normal phase Graphite Reversed phase Poly(styrene-divinyl benzene) Reversed phase Alkyl silane Reversed phase Phenyl Reversed phase Cyano Reversed phase Amino Normal phase, ion exchange Alkylated amino ion exchange Sulfonate ion exchange, ion exclusion Alkyl sulfonate ion exchange Diol Normal Phase Poly(methylmethacrylate) Ger permeation
- 28. HPLC Particle Size • Larger Particles – Lower backpressure – More economical – Higher Capacity • Smaller Particles – Higher efficiencies – Better sensitivity – Reduced run time
- 30. HPLC Sample Introduction – The sample is introduced using a loop injector Sampling loops are interchangeable, and available with different volumes.
- 32. HPLC Detectors • Ultra-violet (UV) detection • Photodiode array (PDA) • Conductivity • Electrochemical • Refractive index • Fluorescence • Evaporative Light Scattering • Radioactivity
- 33. HPLC Detector for HPLC UV/VIS detector
- 34. HPLC Variable UV Detector Flow Cell Lamp Slits Light Censor Grating
- 35. HPLC Photo Diode Array Detector
- 37. HPLC Derivatisation in HPLC • Why derivatise? – Increased sensitivity – Able to use different detectors – Eliminate interfering Compounds • Types – Pre-column derivatisation – Post-column derivatisation
- 38. HPLC Choice of HPLC Method
- 39. HPLC Choice of HPLC method
- 40. HPLC
- 41. HPLC
- 42. HPLC
- 43. HPLC Quantitative Calculations – Quantitative analyses are often easier to conduct with HPLC than GC because injections are made with a fixed-volume injection loop instead of a syringe – Quantitative measurements can be made using external standards
- 44. HPLC Quantitative Analyses - Example – A 2.013-g sample of dried soil is extracted with 20.00 mL of methylene chloride. After filtering to remove the soil, a 1-mL portion of the extract is removed and diluted to 10 mL with acetonitrile. Injecting 5 mL of the diluted extract into an HPLC gives a signal of 0.217 for the PAH fluoranthene. When 5 mL of a 20.0-ppm fluoranthene standard is analyzed using the same conditions, a signal of 0.258 is measured. Report the parts per million of fluoranthene in the soil. ppm1671 013.2 1000 201010 258.0 217.020 3