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Stationary phase Mobile phase

Stationary phase Mobile phase. Applications of HPLC I dentification Q uantification P urification 1- Gradient Elution 2- Isocratic elution. PAPER.Ch. THIN LAYER.Ch. R.Ph.Ch. N.Ph.Ch. Cation.Ex. Anion.Ex. G.F.Ch. G.P.Ch. Types of Liquid Chromatography

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Stationary phase Mobile phase

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  1. Stationary phase • Mobile phase Applications of HPLC Identification Quantification Purification 1- Gradient Elution 2- Isocratic elution

  2. PAPER.Ch THIN LAYER.Ch R.Ph.Ch N.Ph.Ch Cation.Ex Anion.Ex G.F.Ch G.P.Ch

  3. Types of Liquid Chromatography • Partition Chromatography • The stationary phase is a liquid adsorbed on a solid • Adsorption Chromatography • The stationary phase is a solid • Ion-Exchange Chromatography • The stationary phase is an ion-exchange resin • Size Exclusion Chromatography • The stationary phase is a liquid in a polymeric solid

  4. igh H erformance - Pressure ( price – patience) L iquid P C hromatography

  5. Advantages to HPLC • Efficiency increases as particle diameter decreases • smaller particles give better separation and more uniform flow • Higher resolution • Fast speed of analysis • Greater reproducibility and reused packing • Easy automation operation and data analysis • Preparative procedures • Possibility of using many detectors

  6. Basic Components of an HPLC System • Reservoirs (Solvents). • Pump System. • Mobil phase pressures up to 6000 psi .Typical flow rates are 0.1 to 10 mL/minute. • Injection System. • (0.1 to 500 µL) • 4. Chromatographic Column. • Typically 10-30 cm in length AND packing of 5-10 µm diameter. • 5. Detector. • UV, IR, refractive index, fluorescence, mass spectrometry, electrochemical,Diode array and etc.

  7. Injector

  8. Degassers Gases ( Oxygen,Nitrogen ) cause band broadening . ( Vacuum , Sparging w/inert gas,Sonication , thermal ) Mobile Phases 1- High purity required (to prevent extraneous peaks) 2- Low viscosity (to promote good mass transfer) 3- Immiscibility (avoids dissolving stationary phase)

  9. Pump System • Must generate pressures up to 6,000 psi • Flow-rates range from 0.1 to 10 mL/minute • Many HPLC systems have a dual pump system to minimize pulsing • Flow control and reproducibility < 0.5% • Corrosion resistance

  10. Reciprocating pumps • ~90% of HPLCs • Solvent in contact w/piston • Problem = pulsed flow • Advantage = Small volume, high pressure • Displacement pumps • Syringe-like chamber • Independent of viscosity • Pulse-free output • Limited solvent capacity

  11. Injection Valve Inject position Load position Sample loops available from 60 nL to 1000 L

  12. Columns • Stainless Steel (Pressures to 10,000 psi) Heavy Wall Glass (Pressures to 600 psi) Analytical Columns • 10 to 30 cm • Inside Diameters: 4 to 10 mm • Packing Diameters: 5 or 10 mm • Common: 25 cm long; 4.6 mm inside diameter; 5 mm particles; • Number of Plates: 40,000 to 60,000 • Guard Columns removes particulate matter and solvent contaminents • Column Thermostats for temperature control : 100° to 150° C • New columns (capillary) • 3-7 cm long • 1-4 mm i.d.

  13. UV (Single wavelength ,Variable wavelength, PDA), IR absorption,Fluorescence,Refractive Index ,Electrochemical Mass Spectroscopy,Light scattering ,NMR,FT-IR , ELSD ,…

  14. Detectors

  15. tr2 tr1 Detector Response time or volume tm tr1 Tm = Time for mobile phase to travel length of column (dead time) tr = Retention time tr= Adjusted retention time = tr - tm • Mobile phase flow rate: ml/min • Two ways to describe solute “retention” • 1- Retention Time, tr • 2- Retention Volume, Vr • Vr = F * tr = tr2/tr1= Relative retention

  16. Efficiency of Separation R = 0.75 RECOVERY = 50% R = 1 RECOVERY = 4% R = 1.5 RECOVERY = 0.3% R > 1.5 RECOVERY = 0

  17. R=0.50 R=0.75 t0 t0 time time 2s 3s R=1.00 R=1.50 t0 t0 time time 6s 4s • Resolution: higher R, better separation

  18. EXAMPLESubstances A and B were found to have retention times of 6.4 and 14.4 min, respectively, on a 22.6 cm column. An unretained sample of air passed through the column in 1.30 min. The widths of the peak bases were 0.45 and 1.07 min. Calculate the column resolution. 2((tR)y - (tR)x) 2(14.4 - 6.4) Rs = ----------------- = ---------------- = 10.5 Wx + Wy (0.45 + 1.07)

  19. Height Equivalent of a Theoretical Plate(HETP)Increase N by:Increasing column lengthDecreasing particle sizeDecreasing flow rate of mobile phase

  20. As HETP , resolution increases (N )

  21. EXAMPLESubstances A and B were found to have retention times of 6.4 and 14.4 min, respectively, on a 22.6 cm column. An unretained sample of air passed through the column in 1.30 min. The widths of the peak bases were 0.45 and 1.07 min. Calculate the av. no. of plates in the column and then calculate the plate height for component B. N = 16 * (tR/W)2 for component A NA = 16 * (6.4/0.45)2= 3.2 x 103plates for component B NB = 16 * (14.4/1.07)2 = 2.9 x 103plates AVG=3.05*103 PLATES H = L/N H = L/NB = (22.6 cm)/(2.9 x 103 plates) = 7.8 x 10-3cm/plate

  22. GENERAL FACTORS INCREASING RESOLUTION • 1. Increase column length • 2. Decrease column diameter • 3. Decrease flow-rate • 4. Use uniform stationary phase (packing material) • 5. Decrease sample size • 6. Select proper stationary phase • 7. Select proper mobile phase • 8.Use gradient elution • Change temperature (affects diffusion)

  23. PARTITION CHROMATOGRAPHY (gas or liquid) MOBILE PHASE Sample out Sample in STATIONARY PHASE • Separation is based on the analyte’s relative solubility between two liquid phases

  24. Partition Chromatography

  25. Typical Applications of Partition Chromatography

  26. Reverse Phase

  27. Reverse Phase Stationary phase with different chain lengths

  28. General Rule Polarity of analytes ≈ polarity of stationary phase ≠ polarity of mobile phase. To achieve good separation, the analytes should interact with the stationary phase, but not too strongly or the retention time will become very long. • Normal Phased Chromatography • Highly polar stationary phase • Silica or alumina oxides • Relatively non-polar solvent • hexane,i-propylether,Decane,Pentane,Cyclohexane • Less polar solutes elute first • Increasing mobile phase polarity decreases elution times • Reversed Phased Chromatography • Non-polar stationary phase • a hydrocarbon • Relatively polar mobile phase • water, methanol,acetonitrile,propanol,tetrahydrofuran • More polar solutes elute first • Increasing the mobil phase polarity increases elution time

  29. Increased organic → decreased retention Polar Solvents: Water > Methanol > Acetonitrile > Ethanol >Oxydipropionitrile Non-polar Solvents : Decane >Hexane > Pentane >Cyclohexane

  30. Not only is solvent Selection important… But also the proper mixture of the solvents (And flow rate….!)

  31. Chromatography Decision Chart Water soluble Water soluble Water insoluble Ionic Nonionic Size exclusion ch. Adsorption.ch Partition. ch Anion Exch Ch. Cation Exch ch. R.P. partition ch. Size exclusion ch. • M.W >2000: Are they water soluble? Yes or No • If Yes: Use size exclusion chromatography with water as the mobile phase. • If No: Use size exclusion chromatography with a non-polar mobile phase.

  32. Critical parameters in reversed phased chromatography 1- COLUMN LENGTH 2-gradient elution is significant rather than column length 3-FLOW RATE 3-1 An important factor for resolution of small molecules, Small peptides,… 3-2 Low flow rates,typically used with long columns,may actually decrease resolution due to increased longitudinal diffusion 4- TEMPERATURE An important factor for resolution of small molecules, Small peptides,… Temperature above decrease viscosity of the mobile higher mass transfer between st.phase and m.phase better resolution 5- MOBILE PHASE General Rule: Start with high polar and the end with low polar eluting

  33. Adsorption or Liquid-Solid Chromatography • Two common stationary phases • Silica (most common) • Alumina • Highly polar stationary phase, less polar mobile phase • Suitable for non-polar compounds of low molecular weight • Compounds That Can be Separated • Olefins,Aromatic hydrocarbons,Halides, sulfides, • Nitro- compounds,Esters, aldehydes, ketones, • Amines,Sulfones,Sulfoxides,Amides,Carboxylic acids

  34. Intermolecular Attractive Forces (IMAF) • Ion-Dipole • Dipole-Dipole • Hydrogen bonding • Dipole-Induced dipole • London dispersion forces (Induced dipole-Induced dipole)

  35. Ion-Dipole Between an ion and a polar molecule: Na+ Cl–

  36. + + – – Dipole-Dipole Between neutral polar molecules Cl – H + – + + –

  37. Hydrogen Bonding Special kind of dipole-dipole H covalently bonded to N, O, or F O=C –N–H Hydrogen bond

  38. + + – –  – + – + – + + – Dipole-Induced Dipole Dipole in one polar molecule induces temporary dipole in another Nonpolar molecule

  39. London Dispersion ForcesInduced dipole-induced dipole Electrons are moving charged particles can concentrate in one area of molecule to produce temporary charged region + + + + + – – – – – + + + + – – – –

  40. Induced dipole-Induced dipole attractive forces two nonpolar molecules center of positive charge and center of negative charge coincide in each + – + –

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