9/17/20131Separation and Identification Separation and Identification of Food Dyes byof Food Dyes by(A TEST EXERCISE 105 pints)(A TEST EXERCISE -105 points)1Last Update: 9/17/2013 10:01 AMWhat is the quantitative basis for chromatographicseparationWh t i t l diti ti iWhat properties can be the basis for separationof mixtures ?OBJECTIVESOBJECTIVESLearning:What experimental conditions optimizechromatographic separation?With what With what accuracyaccuracy can you identify the can you identify the food food dyes in dyes in an unknown mixture an unknown mixture by by paper paper chromatography?chromatography?Performance/Assessment:2105 ptsConcepts:capillary action chromatographycomponents interactions mobile phase migration rate mixture originrates resolutionretention factor solvent frontsolvents spotstationary phase3Techniques:preparing chromatogramoptimizing resolution by solvent choicemeasuring spotsApparatus:solvent chamber (beaker/watch glass)rulerChromatogramtoothpick4BackgroundBackgroundDecantationC st lli tiPrecipitationSi iSeparationSeparationHow areHow are substances separated substances separated from one another?from one another?TechniquesTechniquesCrystallizationDistillationEvaporationFlotationExtractionMagnetic SeparationChromatography5SievingSublimationZone RefiningSedimentationDryingCentrifugationElectrophoresis……..Chromatographic separation involves TWO PHASES, (i.e., states of matter).MOBILE PHASE (gas or liquid)STATIONARY PHASE (liquid or solid)Technique has been developed in many different ways:BackgroundBackgroundBackgroundThe mobile phases pass through the stationary phase by CAPILLARY ACTIONqpyyIn this exercise:MOBILE PHASES = Solvents (H2O, etc.) STATIONARY PHASE = Chromatography paperColumn, gas phase, gel, paper, etc.69/17/20132CAPILLARY ACTION:Spontaneous rising or lowering of a liquid level in a narrow tubeCOHESIVE ADHESIVE Caused by a BALANCE BETWEENCOHESIVE FORCESADHESIVE FORCESandThose that keep a liquid together and try to minimize its surface areaForces of attraction (or repulsion) between a liquid and its container Capillary Action7How does chromatographic separation work?How does chromatographic separation work?QUALITATIVELY:Different solutemoleculesinteract differentlywith the stationary phasehave varying solubility in mobile phase, andThe interactions can be based on:Therefore, DIFFERENT SUBSTANCESgenerally haveDIFFERENT INTERACTIONSMolecular size Electrical Charge etc.PolarityBonding8In paper chromatography of dyes, primary basis for separation is interactioninteractionbetween dye dye moleculesmoleculesand paperpaper. Paper consists primarily of CELLULOSE, a high molecular weight polymer of the carbohydrate GLUCOSE (C6H10O5)n OHO O O H O H O C H 2 O H O OH O C H 2 H O O H .....Three water-like hydroxyl unitscan HYDROGEN BONDto water or to IONIC dye molecules 9Migration Rates Migration Rates -- QuantitativeQuantitativeFor a given: Solvent FrontOrigindsolventdspot• component, • stationary phase, and• mobile phase, we candefine the:Distance moved by COMPONENTRf= −−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−− Distance SOLVENT FRONThas movedThe RELATIVE MIGRATION RATEand therefore, Rffor a given substance with a given stationary phaseand given solventis REPRODUCIBLEOriginAll distances are measured from the ORIGIN• Rf(Retention Factor)Solvent Level10In this exercise, we will work with:•a FIXEDstationary phase (Chromatography Paper)The mobile phasemobile phasealso affects migration ratesmigration rates(i.e, Rfvalues)•a RANGEof solvent mixtures of varying polarity*Because of their differing chemical properties, the Rf’s f th s f d d s ill sid bl f Ideally, we seek a SINGLEsolvent that clearly disperses( RESOLVES) all seven dyes most effectively. (Pre-lab Exercise #3)of the seven food dyes will vary considerably from one solvent to the next* A polarsolvent is one which tends to dissolve ionic substances better than molecular substances11ProcedureProcedure1. Working in groups of threegroups of threeYou will be given authentic samples of the 7 FDA dyes and three solventsO tdt f h l t !WaterSalt Water Isopropyl AlcoholOne student for each solvent !Objective: to determine which solvent provides best separation - (i.e., RESOLUTION) of the 7 food dyesGroup Prepares 3 Chromatograms129/17/20133X1 = R40 + G3 X3 = R3 + B2The The Chromatogram Chromatogram for Part 1for Part 1Smith, J H2O/NaClX2 = B1 + Y5ShowSolvent13X cmChromatogram Layout DimensionsChromatogram Layout DimensionsWe want to be able to track7 + 4 = 11 spots141 cmX 12cm….Applying the Dye Spots on the ChromatogramApplying the Dye Spots on the ChromatogramDye spots are applied using a toothpickDye spots should be small but concentratedconcentratedAllow spots to dry before a second Allow spots to dry before a second toothpicktoothpick--ful is applied!ful is applied!DON’T MIX TOOTHPICKSDON T MIX TOOTHPICKS15Construction of the Paper CylinderConstruction of the Paper CylinderChromatogram is rolled and stapled into a cylinder.Stapled edges must not overlap or be touching.Place cylindrical chromatogram in solvent chamber.Chromatogram should not touch walls of chamber.16Replace cover on solvent chamber as quickly as possible.Solvent rises through the chromatogram. Observe the SOLVENT FRONT carefully.At appropriate time(when solvent front is 1½ cm from top),• carefully remove chromatogramfrom solvent chamber• watchas solvent front continues to movetowards top of paper•just beforesolvent front becomes just beforesolvent front becomes invisible, trace its location with pencil.Measure Rf’sof each of the dye spotsFrom three chromatograms, select solvent that gives best resolution.Be sure to give reason for your choice on data sheet.17PART 2 - UNKNOWNYou will eachbe given an unknown. It may contain 2 or3 or4 food dyesPrepare a chromatogram as in Part 1 with spots of each of the 7 Food Dyes. In the three positions labeled X1, X2 and X3, you WORKINGALONEFROM THIS POINT FORWARD!p,,yshould place spots of your unknown withThis will insure that if you have light spots (e.g., the yellow dyes), you will be able to see their presence in the developed chromatogramincreasing concentration.yellowyellow18ANDAND9/17/20134PART 3 – YOUR FOODSTUFFIn position labeled X4, place a concentrated spot of dye from your foodstuff.If you use M&M’s or similar recommended items - simplest