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FOOD ANALYSIS: Principles of Chromatography

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FOOD ANALYSIS: Principles of Chromatography FOOD ANALYSIS: Principles of Chromatography B. Pam Ismail [email protected] FScN 146 612 625 0147 FOOD ANALYSIS: Principles of Chromatography The following intermolecular interactions are responsible for adsorption chromatography A. Electrostatic forces B. Hydrogen bonds C. Hydrophobic interactions D. A and B E. B and C F. A and C G. All of the above H. None of the above 1 FOOD ANALYSIS: Principles of Chromatography In reverse-phase chromatography: A. Polar compounds elute first B. Non-polar compounds elute first C. The mobile phase is polar liquid D. A and B E. B and C F. A and C G. All of the above H. None of the above FOOD ANALYSIS: Principles of Chromatography Historical Perspective David Day ---American Geologist o Crude petroleum---fuller earth Mikhail Tsvet ---- Russian Botanist o Leaf pigments --- column packed with chalk o Named the phenomenon chromatography o Credited for its discovery 1940’s: partition chromatography and paper chromatography 1960’s: Gas chromatography evolved because of petroleum industry followed by liquid chromatography and supercritical fluid chromatography (SFC) 2 FOOD ANALYSIS: Principles of Chromatography Uses? FOOD ANALYSIS: Principles of Chromatography Extraction: Transfer of a solute from one liquid phase to another Actual means of analysis Preliminary sample clean up Concentration of the component of interest 3 FOOD ANALYSIS: Principles of Chromatography Extraction: At equilibrium: - Batch extraction – Partition coefficient (K) http://carlwillis.files.wor dpress.com/2009/09/pur ex_4.jpg is constant – Continuous extraction K = Conc. of solute in phase 1 Conc. of solute in phase 2 – Countercurrent extraction Solute A is more soluble in (basis for partition phase 1 chromatography) Solute B is more soluble in http://195.134.76.37/applets/AppletCraig/Appl_Craig2. html phase 2 FOOD ANALYSIS: Principles of Chromatography Extraction: – Countercurrent extraction (basis for partition chromatography) http://195.134.76.37/applets/appletcraig/Images/Ani mation2.gif 4 FOOD ANALYSIS: Principles of Chromatography Chromatography Terminology Solute Stationary phase (liquid or solid) Mobile phase (eluting solvent/carrier gas/supercritical fluid) Series of equilibrations Eluent (noun), elute (verb) Chromatographic peak FOOD ANALYSIS: Principles of Chromatography Chromatography A separation technique used in food analysis Based on the partition or distribution of solute between a stationary phase and a mobile phase K = Conc. of solute in stationary phase Conc. of solute in mobile phase 5 FOOD ANALYSIS: Principles of Chromatography Chromatography is a separation technique used in food analysis Chromatography Gas Supercritical fluid Liquid Chromatography Chromatography Chromatography Paper Thin Layer Gas/Liquid Chromatography Chromatography (Liquid/Liquid) (Liquid/Liquid) Column Chromatography (Liquid/Liquid) (Liquid/Solid) FOOD ANALYSIS: Principles of Chromatography Chromatography is a separation technique used in food analysis Chromatography Column Liquid Chromatography Partition Adsorption Size-Exclusion (Liquid/Liquid) (Liquid/Solid) Reversed Phase Normal Phase Hydrophobic Ion-Exchange Affinity Interaction 6 FOOD ANALYSIS: Principles of Chromatography Characteristics of Different Chromatographic Methods Method Mobile/Stationary Phase Retention Varies with Gas-liquid chromatography Gas/liquid Molecular size/polarity Gas-solid chromatography Gas/solid Molecular size/polarity Supercritical fluid chromato- Supercritical fluid/solid Molecular size/polarity graphy Reversed-phase chromato- Polar liquid/nonpolar liquid Molecular size/polarity graphy or solid Normal-phase chromato- Less polar liquid/more polar Molecular size/polarity graphy liquid or solid Ion-exchange chromato- Polar liquid/ionic solid Molecular charge graphy Size-exclusion chromatography Liquid/solid Molecular size Hydrophobic-interaction Polar liquid/nonpolar liquid Molecular size/polarity chromatography or solid Table 12-1 Affinity chromatography Water/binding sites Specific structure FOOD ANALYSIS: Principles of Chromatography Chromatography is a separation technique used in food analysis Chromatography Gas Chromatography Gas/Liquid Separation of thermally stable volatiles Controlled temperature gradient Volatiles are separated based on molecular size, polarity and boiling point 7 FOOD ANALYSIS: Principles of Chromatography Recap Questions Define chromatography? What is the difference between stationary phase and mobile phase, what is the function of each? List three types of chromatography What is the form of mobile phase in GC and what is the form of the stationary phase? Same question for partition and adsorption chromatograph What is partition coefficient? What are some applications? FOOD ANALYSIS: Principles of Chromatography Liquid Chromatography Paper chromatography Paper (cellulose) serves as a support for the liquid stationary phase The stationary phase is usually water Mobile phase is a solvent immiscible in water 8 FOOD ANALYSIS: Principles of Chromatography Liquid Chromatography Paper chromatography If sample mixture is colorless (amino acid mixture) Visualized by autoradiography FOOD ANALYSIS: Principles of Chromatography Liquid Chromatography Paper chromatography 2-D techniques for complex mixtures 9 FOOD ANALYSIS: Principles of Chromatography Liquid Chromatography Paper chromatography Components of a mixture is characterized by their relative mobility o Rf = Distance moved by component Distance moved by solvent o Rf values are supposedly constant Can be affected by stationary phase thickness, humidity, developing distance, and temperature FOOD ANALYSIS: Principles of Chromatography Liquid Chromatography Paper chromatography Can also be reversed-phase o Support impregnated with a non-polar solvent o Developed with polar solvents or water The stationary phase can be ion exchange paper o OH groups on the cellulose derivatized with acid or base moieties 10 FOOD ANALYSIS: Principles of Chromatography In reversed-phase paper chromatography A. The most polar travels the most B. The least polar travels the most C. Rf value of the polar compound will be higher than the less polar one D. A and C E. B and C FOOD ANALYSIS: Principles of Chromatography Liquid Chromatography Thin layer chromatography (TLC) Similar principle as paper chromatography Developed to replace paper chromatography o Better resolution Smaller more uniform particle size o Faster, o Better reproducibility 11 FOOD ANALYSIS: Principles of Chromatography Liquid Chromatography Thin layer chromatography (TLC) Thin layer (250 μm thick) of sorbent or stationary phase bound to inert support (glass, plastic, or aluminum foil) Sorbent used: silica, alumina, or cellulose Can be applied as adsorptive, normal phase or reversed- phase partition, ion exchange chromatography FOOD ANALYSIS: Principles of Chromatography Liquid Chromatography Thin layer chromatography (TLC) Different techniques can be applied to detect or visualize the components o Colorimetrically (ninhydrin, sulfuric acid, iodine vapor, dichloroflurescein) o Measure of absorbed or emitted radiation (fluorescence) o Measure of radioactivity of radioactive labeled compounds Quantitative evaluation can be carried out by means of o Densitometer o Scraping off the zone, eluting the compound and then analyze the resulting solution 12 FOOD ANALYSIS: Principles of Chromatography Column liquid chromatography General procedure Stationary vs. mobile phase Column dimensions Packed dry or wet (making a slurry) Isocratic vs. gradient mobile phase FOOD ANALYSIS: Principles of Chromatography Chromatography is a separation technique used in food analysis Chromatography Supercritical fluid Chromatography Mobile phase commonly used liquid CO2, can sometimes be combined with methanol High diffusivity and low viscosity – Stationary phase similar to that of better resolution and less time HPLC Used for separation of non-polar Equipment similar to HPLC, and molecules, and non-volatile detectors used are similar to those compounds that are thermally used for GC labile (instead of GC) 13 Physicochemical principle of chromatography separation FOOD ANALYSIS: Principles of Chromatography Adsorption (solid/liquid) chromatography: Stationary phase: (adsorbent) finely divided solid; permit differential interaction with components to be separated Intermolecular forces: Van der Waals forces Electrostatic forces Hydrogen bonding Hydrophobic interactions Adsorption coefficient is not a constant 14 FOOD ANALYSIS: Principles of Chromatography Adsorption (solid/liquid) chromatography: Stationary phase: o Silica: (slightly acidic) (Silica bounded to C8 or C18) o Alumina: (slightly basic) o Charcoal: (non-polar) The elution order of solutes depend on their relative polarities Solute and solvent (mobile phase) compete for active sites on the stationary phase Changing the strength of the mobile phase will alter solute – stationary phase interactions FOOD ANALYSIS: Principles of Chromatography Recap Questions What is supercritical fluid chromatography? What is reverse phase chromatography? In reverse phase paper chromatography what type of compound travels the most with the mobile phase? What does TLC stand for? How does TLC differ from paper chromatography? What is the difference between isocratic and gradient elution? What does increasing the strength of the mobile phase mean? 15 FOOD ANALYSIS: Principles of Chromatography Partition (liquid/liquid) chromatography: Solutes
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