Lecture 5: Liquid Chromatography A PDF

CHEM 2010: Chemical Analysis A Chromatography Lecture 5 of 8 Liquid Chromatography A Dr. Joyann Marks [email protected] Last time... The van Deemter Equation H = A +B/u + Cu A = Eddy Optimum value of the linear diffusion term...

CHEM 2010: Chemical Analysis A Chromatography Lecture 5 of 8 Liquid Chromatography A Dr. Joyann Marks [email protected] Last time... The van Deemter Equation H = A +B/u + Cu A = Eddy Optimum value of the linear diffusion term velocity of the mobile phase is that which gives a minimum value B/u = for H (maximum efficiency) Longitudinal diffusion term Cu = (resistance to) Mass transfer term Last time... Variation of Retention (elution) time and Resolution with Capacity (Retention) Factor: Rs improves greatly at small values of k′ But above k′ >5 (or 10), no improvement in Rs but longer tR Optimal k′ usually between 1 and 5 (or 10) k′ Lesson Objectives Lecture 5: Liquid Chromatography: Planar, column and high performance liquid chromatography (instrumentation, operating principles, columns, detectors, sample preparation, applications). Understand the operating principles of liquid chromatography Explore the components of liquid chromatography instrumentation Liquid Chromatography Liquid Chromatography Mobile phase - Liquid. Column or a plane. Very small packing particles and a relatively high pressure – High Performance Liquid Chromatography (HPLC).  Liquid Chromatography Classifications Liquid-Liquid Chromatography (Partition) vs Liquid-Solid Chromatography (Adsorption) Column Chromatography vs Planar Chromatography Thin Layer Chromatography (LSC) vs Paper Chromatography (LLC) HPLC  Liquid Chromatography ❑Column Chromatography- Stationary phase is held in a cylindrical tube and the mobile phase is forced through it by gravity or pressure. ❑Planar Chromatography- Stationary phase is supported on a flat plate or in the pores of paper and the mobile phase moves through the stationary phase by capillary action or gravity. Planar Chromatography Separation technique - Stationary phase is present as or on a plane. Paper – Paper Chromatography. Layer of solid particles spread on a support such as a glass plate - Thin layer Chromatography. Different compounds in the sample mixture travel different distances according to how strongly they interact with the stationary phase as compared to the mobile phase. Retention factor (Rf) for components compared. Thin Layer Chromatography Widely employed laboratory technique. Stationary phase - an adsorbent powder (SiO2 or Al2O3) on a solid plate (glass, aluminium or plastic). – Widely used in pharmaceutical & food stuff industry Advantages : ✓Simple, Rapid and Cheap ✓Faster runs. ✓Better separations. ✓Choice between different adsorbents. ✓Better resolution ✓Allows for quantification Thin Layer Chromatography Used to identify the unknown compounds and to determine the purity of mixture. Sample solution spotted on plate, evaporated TLC Plate - Aluminium or glass - coated by stationary phase. Coated material : 0.1-0.3mm in thickness. Fluorescent indicator that will make it fluoresce during the UV light exposure. STATIONARY PHASE: Silica gel, Alumina, or Cellulose on a flat, inert substrate. MOBILE PHASE: Volatile Organic solvents TLC Plate Development- The process by which a sample is carried through the stationary phase by the mobile phase in TLC Thin Layer Chromatography  Thin Layer Chromatography Dm dR dR dR = distance moved by compound Rf = Dm Dm = distance moved by solvent Examination by TLC of Fractions collected Compound 1 Compound 2 Compound 3 Compound 4 Column Chromatography Column chromatography involves the following: 1. Adsorption/retention of substance on stationary phase 2. Separation of adsorbed substance using mobile phase. 3. Recovery of individual components by continuous flow of mobile phase. 4. Quantitative and qualitative analysis of solute and the components which are recovered. Column Chromatography Column Chromatography Column Chromatography High Performance Liquid Chromatography Efficiency of a packed column increases as the particle size decreases However smaller particle size means greater resistance to the mobile phase flow New technology: 3mm diameter particles and pressures up to 6000psi  High Performance Liquid Chromatography High sensitivity Applicable to non-volatile and thermally unstable compounds Applicable to compounds of wide interest and importance Detection systems tend to be non-destructive Options for separation: Isocratic elution: constant mobile phase composition during run Improved selectivity by Gradient elution i.e. varying the mobile phase composition during the run  https://www.youtube.com/watch?v=kz_egMtdnL4 https://www.youtube.com/watch?v=9ZqL4MOWAi8&t=32s  HPLC Instrument Components Solvent Reservoir Solvent/ Solvent reservoir Column Pump (Gradient) Detectors Injection valve Recorder/data processing Solvent / Solvent Reservoir Reservoir must have adequate capacity Solvents must be degassed Why is this important? Solvent must be of high purity and particulate free. HPLC Pump Requirements Must be made from inert or corrosion resistant material Should be able to generate pressure of up to 6,000 psi PUMP PRESSURE: "Ultra High Performance Liquid Chromatography" systems 14,000 psi. Pulse free output (low noise) Should be able to deliver flow rates of 0.1 to 10 mL/min increments Flow reproducibility of 0.5% or better Gradient pumps can vary the proportion of a multi-component mobile phase https://www.waters.com/webassets/cms/category/media/overview_images/515HPLCPump_overview.jpg Injection Valve (Sample Introduction) Six port, rotary action Two positions (Load and Inject) Volume of sample determined by volume of loop attached Six Port Rotary Injection Valve for HPLC Load position: Inject Position: Sample loop is filled while Contents of the loop are transferred mobile phase flows through column to the column http://www.vici.com/support/app/app11j.php http://www.restek.com/images/animations/hplcsixport/6_Port_Injector_content.html  Injection Valve- Some Considerations Stainless steel construction to withstand high pressure Very reproducible in sample delivery Sample volume can be conveniently varied by changing the loop Relatively easy to operate compared to syringe type in GC End of Lecture #5