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Chromatographic Separation

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Adam Hasham, Sam Heavenrich, Jayanth Prakhya, Enoch Tieu. Chromatographic Separation. Chromatography. Separates mixtures (physical method) Involves: Stationary Phase; Surface Mobile Phase; Solvent Quantifiable: Retention Factor / Rate of Flow (Rf) Can be: - PowerPoint PPT Presentation
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  • Adam Hasham, Sam Heavenrich, Jayanth Prakhya, Enoch Tieu

  • Separates mixtures (physical method)Involves:Stationary Phase; SurfaceMobile Phase; Solvent Quantifiable:Retention Factor / Rate of Flow (Rf)Can be:Preparative: separates mixture for further useAnalytical: measures relative proportions of componentsPreparative more easily studied

    Rf = Distance traveled by solute/Distance traveled by solvent

  • Mikhail Semenovich TsvettSeparate Tints / Types of ChromatographyTrickled mixture through glass tube with Calcium Carbonate powderPigments stuck to powderDifferent degrees of strength Coloured bands

  • PartitionBased on the thin film formed on the surface of a solid support by a liquid stationary phaseSolute equilibrates between the mobile phase and the stationary liquid.Ion ExchangeResin is used to covalently attach anions or cations by electrostatic forcesSolute Mobile Liquid Phase have opposite charge

  • Molecular ExclusionNo attraction between stationary phase and soluteLiquid or Gaseous Mobile Phase separates molecules according to sizeAffinityMost selectiveInteraction between only 1 component of solute and stationary phase moleculeOnly the specific component is attracted to the stationary phase

  • Adsorption: When a fluid solute accumulates on the surface of a solid or liquidDifferent amounts of the components of a mixture are adsorbed to the 2 phasesAs mobile phase moves through stationery phase, the substances that are easily adsorbed will lagSeparation created

  • Archer JP Martin and Richard LM Synge in 1941Strip of porous filter paper substituted for powderUsed to separate proteinsLater used to Separate and Identify components of various mixturesIn industrial settings, Paper Chromatography has been replaced by more sophisticated methods

  • Place a drop of a mixture on the paperDip one edge of the paper into the mobile phaseThrough capillary action, adsorbent will move up the paperOnce adsorption occurs and the paper dries, spray-on reagentReagent will reveal change in color

  • Purpose:To predict the composition of various mixtures using Paper Chromatography

  • Pigment applied to Strip of Chromatography PaperStrips hung on rack at equal lengthsAdsorbent added (70% Isopropyl Alcohol) Rack removed once solvent travelled to the top of each stripThe final position of solvent markedThe final position of pigments markedDistance travelled by the mobile phase and each colour measured

  • View Demo using the Play button after each step. Do not proceed after the Gas Chromatography Demo appears. Use arrow keys for next slide.

  • The Mixture involving Red, Yellow, and Blue Food Coloring PigmentsApproximate elapsed time 1 minute

  • The Mixture involving Red, Yellow, and Blue Food Coloring PigmentsApproximate elapsed time 6 minutes

  • The Mixture involving Red, Yellow, and Blue Food Coloring PigmentsApproximate elapsed time 18 minutes

  • The Spinach, Pomegranate and Blue PigmentsApproximate elapsed time 0 minutes

  • The Spinach, Pomegranate and Blue PigmentsApproximate elapsed time 5 minutes

  • The Spinach, Pomegranate and Blue PigmentsApproximate elapsed time 13 minutes

  • Defined as Rate of Flow or Retention FactorCalculated by dividing Distance travelled by a component over Distance travelled by the mobile phase

  • ComponentDistance Travelled (cm)Mobile Phase / Adsorbent (70% Isopropyl Rubbing Alcohol)7.25Red Food Coloring (In Mixture) 7Yellow Food Coloring (In Mixture) 6.8Blue Food Coloring (In Mixture) 7.1Green Food Coloring (Yellow Component) 2.3Green Food Coloring (Blue Component)7.1Spinach Juice (Yellow Component 1) 7Spinach Juice (Yellow Component 2) 4Spinach Juice (Green Component) 3.5Pomegranate Juice (Orchid Purple Component) 6.1Pomegranate Juice (Light Salmon Pink Component) 4.6

  • ComponentRfRed Food Coloring (in Mixture)0.97Yellow Food Coloring (in Mixture)0.94Blue Food Coloring (in Mixture)0.98Green Food Coloring (Yellow Component)0.32Green Food Coloring (Blue Component)0.98Spinach Extract (Yellow Component 1)0.97Spinach Extract (Yellow Component 2)0.55Spinach Extract (Green Component)0.45Pomegranate Extract (Orchid Purple Component)0.84Pomegranate Extract (Light Salmon Pink Component)0.63

  • Rf = Degree of AdsorptionVariances caused by Intermolecular ForcesBetween Solute and Mobile Phase Solute carried further by Adsorbent / More adsorption when the forces between them are greaterForces between Solute and Stationary PhaseMore adsorbtion onto Stationary Phase when the forces between them are greaterIn Both Cases:Polarity Major FactorH-bonding Larger Factor but capability limitedLondons Dispersion Forces Weak

  • Mixture of Food ColoringMolecular Formulas:Red: C18H14N2Na2O8S2Yellow: C16H10Na2O7S2N2Blue: C16H10N2O2 H-Bonding PatternRelatively Similar PolarityLong Hydrocarbon Chain = Low PolarityCellulose Chromatography Paper

    Green ColoringYellow and Blue ComponentsYellow: C16H9N4Na3O9S2Blue: C16H10N2O2 Same Blue Dye; Different Yellow DyeHigher Polarity Due to Presence of SodiumHigher Number of Nitrogen Atoms

    Component (in Food Coloring Mixture)RfRed Component0.97Yellow Component0.94Blue Component0.98

    Component (in Green Food Coloring)RfYellow Component0.32Blue Component0.98

  • Spinach ExtractMolecular Formulas:Yellow 1: Carotenes: C40H56Yellow 2: Xanthophylls: C40H56O2 Green: Chlorophyll B: C55H70O6N4Mg H-Bonding commonCarotenes Not-PolarXanthophylls O2

    Component (in Spinach Extract)RfYellow Component 10.97Yellow Component 20.55Green Component0.45

  • Pomegranate AnalysisNot Exact / QuantifiableNot Definite Colours SubjectiveComponents found in mixture cant be re-usedNeither Preparative nor AnalyticalOther Types such as Gas Chromatography provide the components in usable formPrior Knowledge Required of ComponentsOnly Identifiable if knownIncorrect Identifications

  • All Rf Values are RelativePaper & Some Types of Thin Layer ChromatographyOther MethodsDistribution Constant and Concentration More ReliableFreundlich Equation for AdsorptionKovats Retention IndexVan Deemter EquationBelow: More sophisticated methods of quantifying chromatography results

  • Safety ConcernsSafety Goggles must be worn at all timesAny contact with the alcohol must be avoidedAlcohol is Very Flammable and requires a well-ventilated areaModificationsMore Sample PigmentsVarious AdsorbentsThe more data, the more definite the results

  • Thin-Layer ChromatographyGas Chromatography (In Column)Liquid Chromatography

  • Thin-Layer ChromatographyMobile Phase: Solution; Stationary Phase: Flat Sheet of Adsorbent (e.g. Silica Gel)Analyzing organic reactionsComponents are separated using TLC platesThey can then be scraped off to be analyzed Other Applicationsassaying radiochemical purity of radiopharmaceuticalsPlant pigmentsdetection of pesticides or insecticides

  • Click Next Example to View Gas Chromatography Demo

  • Gas Chromatography (In Column)Mobile Phase: Gas; Stationary Phase: Solid/LiquidBlood alcohol analysis in forensicsBlood analyzed using capillary columnsColumn Pressure 20 psiHydrogen is mobile phaseOther ApplicationsEnvironmental monitoringDrug, bomb detection

  • Liquid Chromatography Mobile Phase: Liquid; Stationary Phase: Solid/LiquidHPLC, NPLCTest for Water PollutionAnalyze metal ions + organic compounds in waterOther Applicationspurification of a drug product

  • 2 Dimensional2 separation stagesGas / Liquid ChromatographyStationary Phase Rotated; 2 Mobile Phases Used RPLCPolar Mobile Phase UsedReverse Rf Analysis

  • This concludes the presentation.

    *Title SlideTitle Slide*********************Notice the nitrogen/oxygens follwo pattern for h-bonding**Device similar to a spectroscope would be useful to accurately analyze the colour and match to known composition/elements

    *Other methods of chromatography have other methods of quantifying the results. These are more accurate in many cases.***Travels by Capillary ActionSolid matrix inside large metal tube (packed)*Travels by Capillary ActionSolid matrix inside large metal tube (packed)Travels by Capillary ActionSolid matrix inside large metal tube (packed)**

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