9/11/20121SpectrophotometrySpectrophotometryof Fof Foooodd DDyyeess&Beer’s Lawffyy1Last Update: 9/11/2012 9:54 AMHow is the absorption/transmission of light measured?What is the quantitative basis for the color of substances?OBJECTIVESOBJECTIVESHow is the intensityof color related to the concentrationof colored solutions?What considerations go into graphing experimental data?Questions2Concepts, TechniquesConcepts:Absorbance AbsorptionAbsorptivity Analytical Wavelength Beer’s Law BlankConcentration Electromagnetic Spectrum Incident IntensitySpectroscopySpectrumSpectroscopySpectrumTransmittance Wavelength Purpose3Concepts, TechniquesTechniques:Visible Spectroscopy Quantitative dilution Graphing Plotting /Curve fittingApparatus:PurposeRecording SpectrophotometerSpectronic 20Pipet/Syringe Cuvette 4For a simple, transparent, COLORED material, e.g.BACKGROUNDBACKGROUNDTransmitted lightIncident white lightI(λ)ROYGBVROYGBVCan plot a graph showing the percent of light of each discrete COLOR (wavelength, λ) transmitted by the solutionVIOLETVIOLET CLEAR SOLUTIONIt(λ)Ii(λ)It(λ)100 XIi(λ)%T(λ) =PercentTransmittance5Transmittance of White Light by a Violet Solution5060708090100NSM ITTEDOr, instead, we can plot the percent of light absorbednsmitted% Transmittance of White Light by a Violet Solution010203040WAVELENGTH (nm)% TRAVioletRedGreenYellowBlue% Tran← UV IR Wavelength (nm)69/11/20122Absorbance of White Light by Violet Solution5060708090100bsorbed% Absorbed of White Light by a Violet Solution% Absorbed = 100% - % Transmitted010203040WA VELENGT H ( nm)Violet RedBlueGreen% AbYellow7VISIBLE AND ULTRAVIOLET:Electronic structure of moleculesINFRARED REGION: What determines the WAVELENGTHSof light that substances absorb?Vibrations of moleculesWhat determines INTENSITYof absorption?EFFICIENCY of INTERACTION between Molecules & Electromagnetic Radiation8Which DEVICE measures this phenomenon?A SPECTROPHOTOMETER measures the RELATIVEabsorption of light of DIFFERENT WAVELENGTHS by a substance SOURCE of light,MONOCHROMATOR –an DETECTOR to measure intensityof light transmittedthrough sample, andELECTRONICS to translate detector output into numbers element to select particular wavelengthsSAMPLE & CONTAINER9Detector output depends on intensity, wavelength & instrumental factors. Other substances may also absorb.Therefore, we always measure RELATIVEoutput of detector, where RELATIVEmeans compared with:EVERYTHING BUT THE SAMPLE ( a BLANKBLANK )• Sample container•Solvent Other reagents etc This insures that measured Absorbance of light is due onlyto the substance of interestBLANKBLANK + SAMPLE SAMPLE-=•Other reagents, etc. % Absorbance% Absorbance % Absorbance10How is the amountof COLOR absorbed by a chemical species related to theBEER-LAMBERT LAW (1760 - 1852)absorbance = constantconstant x path length x concentrationAmount of light absorbed by a transparent solution is proportional to the thickness of the solution & concentration of the absorbing speciesA bCONCENTRATIONof that species?A= εbcA = ABSORBANCE at a specified wavelengthwavelengthε = ABSORPTIVITY, a constantconstant characteristic of the substanceat that wavelengthwavelength(does not depend on the amount or concentration of the sample)b=PATH LENGTH of the light through sample, i.e., cell thicknessc = CONCENTRATION of the absorbing substance11A%T =logI0−−−−ItIt−−−−I100=How is Absorbance related to %Transmittance?Definition of AbsorbanceSmaller It means larger AI0100 A = log −−−− %T= log 100 - log %TA = A = 2.000.. 2.000.. --log %Tlog %T129/11/20123A = ε b cNeed to know:What do we need to know to relateAbsorbance to concentration?ABSORPTIVITY, ε, ANDOR at least, their product, ε bABSORPTIVITY, ε, ANDTHICKNESS OF THE CUVETTE,b 132.) Find WAVELENGTH at hi h th b thth How do we proceed experimentally?Absorbance vs Wavelength0.911.) Determine the substance’s SPECTRUM in the desired wavelength range – i.e. absorbance as a function of wavelengthwhich the substancehasthe LARGEST ABSORBANCE(lowest %Transmittance)The Absorbance will be most sensitive to concentration at this wavelengthThis wavelength is called the ANALYTICAL WAVELENGTH00.10.20.30.40.50.60.70.8350380410440470500530560590620650680710740Wavelength (nm)Absorbance142.) Then we prepare series of solutions of KNOWNKNOWNCONCENTRATIONS of the substance3.) Measure ABSORBANCE of each solution at its ANALYTICAL WAVELENGTHc/8 c/4 c/2 c15Conc (mg/L) Abs0.0 0.0009.7 0.2921.0 0.5229 6075Tabulate the dataBLANK29.60.7540.0 1.054.) Plot ABSORBANCEvsCONCENTRATIONand160.60.70.80.911.11.2AbsorbanceAbsorbance vs ConcentrationRed Dye #3 @ 470 nmSLOPE =25 0= 0.0263 L/mgSUPLSUPL--0040040.658 / 25.01700.10.20.30.40.50 5 10 15 20 25 30 35 40 45 50AConcentration (mg / L)BLANK25.00.65817Note : Absorbance is dimensionless. Slope of Beer’s Law plot is ε X b, whereε = the absorptivityof the substance b = the cell thicknessIf we use identical (i.e., matched) cells for blank & sample, we do not need to know the quantities ε& b separatelyBeer’s Law expression for Red #2 at its analytical wavelength in this cell becomes:A = 0.0263 X C (where C is in mg/L)know the quantities ε& b separately.Unitsof the slope of the Beer’s Law plotare (always) reciprocal concentration(e.g., L / mg)189/11/20124In this exercise, we examine spectrophotometric properties of 7 FOOD DYES7 substances approved by Food and Drug Administration ( FDA ), for use as colorants in foodsEach dye is a single complex organic compound. For simplicity they are called:Blue 1, Blue 2, Green 3, Yellow 5, Yellow 6, Red 3, and Red 40,,We will further examine dyes and their chemical properties next week191. Pair obtains 50 mL of ONE assignedassigneddye solutionPROCEDUREPROCEDUREWORK IN PAIRS Using recording spectrophotometer2. Determine SPECTRUM (abs vs ) of diluted stock solution between 350 nm and 750 nm3 Determine ANALYTICAL WAVELENGTH 203. Determine ANALYTICAL WAVELENGTH, i.e., wavelength of maximum absorbance.4. Prepare dilutions of stock solution of precisely knownconcentration: 3/4, 1/2, 1/4, 1/8, and 1/165. Determine ABSORBANCE of stock solution & dilutions at analytical wavelengthmost dilutemost dilutesolution (1/16) first, 2. All pairs begin by preparing preparing dilutionsdilutions for Beer’s LawProcedure ScheduleProcedure Schedule1. You will be assigned a partner and ONEONEdyedyeWhile doing that:21TAs send one pair at the timeone pair