Liquid Chromatography 1

4/30/2012 Liquid Chromatography 1 and Solid-Phase Extraction Lecture Date: April 9th, 2008 Reading Material ● Skoog, Holler and Crouch: Ch. 28 ● Cazes: Ch. 22, 26 ● For those using LC in their work, see: L. R. Snyder, J. J. Kirkland, and J. L. Glajch, “Practical HPLC Method Development”,2nd Ed., Wiley,1997. 1 4/30/2012 Basic LC Terminology ● Adsorption chromatography • The stationary phase is an adsorbent (like silica gel or any other silica-based packing) • The separation is based on repeated adsorption-desorption steps. ● Normal-phase chromatography • The stationary bed is strongly polar in nature (e.g., silica gel), and the mobile phase is nonpolar (such as n-hexane or tetrahydrofuran). • Polar samples are retained on the polar surface of the column packing longer than less polar materials. ● Reversed-phase chromatography • The stationary bed is nonpolar (hydrophobic) in nature, • The mobile phase is a polar liquid, such as mixturesof water and methanol or acetonitrile. • The more nonpolar the materialis, the longer it will be retained. Basic LC Terminology ● Size exclusion chromatography (SEC) • columnfilledwith material havingprecisely controlled pore sizes, and the sample is simply sieved or filteredaccording to itssolvated molecular size. • Larger moleculesare rapidly washed through the column; smaller moleculespenetrate inside the pores of the packing particlesand elute later. • Also called gel permeation chromatography(GCP) although the stationary phase is not restricted to a "gel" ● Ion-exchange chromatography (IC) • the stationary bed has a charged surface of opposite charge to the sample ions. • Used almost exclusively with ionic or ionizable samples. • The stronger the charge on the sample, the stronger it will be attractedto the ionic surface and thus, the longer it will take to elute • The mobile phase is an aqueous buffer,where both pH and ionic strength are used to control elution time 2 4/30/2012 Analytical Applications of LC  The “branches” of the LC family: Note– this means analyte polarity Basic Mechanisms used in LC Separations 3 4/30/2012 High Performance Liquid Chromatography (HPLC) ● HPLC utilizes a high-pressure liquid mobile phase (ca. 100-300 bar) to separate the components of a mixture ● These analytes are first dissolved in a solvent, and then forced to flow through a packed small-particle chromatographic column, where the mixture is resolved into its components ● HP = high pressure and high performance ● Resolution depends upon the extent of interaction between the solute components and the stationary phase Differences between HPLC and “Classical” LC  Small ID (2-5 mm), reusable stainless steel columns  Column packings with very small (3, 5 and 10 m) particles and the continual development of new substances to be used as stationary phases  Relatively high inletpressures and controlled flow of the mobile phase  Precise sample introduction without the need for large samples  Special continuous flow detectors capable of handling small flow rates and detecting very small amounts  Automated standardized instruments  Rapid analysis  High resolution  From now on, LC refers to HPLC 4 4/30/2012 Advantages and Disadvantages of LC Advantages: • Speed (minutes) • High resolution • Sensitivity • Reproducibility • Accuracy • Automation Disadvantages: • Cost • Complexity • Low sensitivity for some compounds • Irreversibly adsorbed compounds not detected • Co-elutiondifficultto detect More on Reversed-phase (RP) LC  RP is the most widely used mode of HPLC (75%?)  Separates molecules in solution on basis of their hydrophobicity – Non-polar stationary phase – Polar mobilephase  In practice: non polar functional group bonded to silica – Stationary phase  functionalgroup bonded to silica  thiscorresponds to a volume(Van deemter)  Alkyl groups ( C4, C8, C18)  retentionincreases exp. with chain length  Mobile Phases – Polar solvent (water) with addition of less polar solvent (acetonitrile or methanol) 5 ... - tailieumienphi.vn