AP* Chemistry Big Idea 1, Investigation 1

Analysis of Food Dyes in Beverages

Analysis of Food Dyes in Beverages 1

IntroductionAssume an investigative role and design a valid procedure using spectroscopy and graphical analysis to determine the concentration of FD&C food dyes in sports drinks. The investigation will developand testyour skills in preparing accurate serial dilutions, understanding spectroscopic measure-ments, and extrapolating from graphical data.

Concepts Spectroscopy Wavelength Beerslaw

Absorbancevs.transmittance Consumerscience Solutionconcentration

BackgroundThecolorofasolutionisanimportanttoolusedbyscientiststogaininformationaboutthecomposi-tionofthesolution.Colorisaphysicalpropertythatisusefulforbothqualitativeandquantitativeanalysis. A qualitativemethodyieldsinformationaboutthenatureortypeofcompoundinasample,whereas a quantitative method provides numerical data for the amount of a compound in a sample.

Spectroscopy is the study of the interaction of light andmatter. A spectrophotometer is aninstrument that uses electromagnetic radiation from a selected region of the electromagnetic spectrum such as ultraviolet, visible or infrared light, to analyze the absorption or transmis-sion of radiation by a sample. The basic func-tion of a spectrophotometer is shown in Figure 1. The electromagnetic spectrum (see Figure 2) is the entire range of possiblewavelengths orfrequenciesof electromagnetic radiation. In thisinvestigation a visible spectrophotometerwillbe usedit scans the visible regionof the elec-tromagnetic spectrum, from 380 nm to 750 nm. Typical light sources for visible spectrophotom-eters include xenon and tungsten lamps.

Glass cuvets or test tubesmay be used assample cells for visible spectrophotometers.More specialized spectrophotometers requirequartz cells,which are invisible to and donot absorb ultraviolet radiation. In additionto the energy source used in spectrophotome-ters, a diffraction grating called a monochroma-tor is also incorporated. The monochromator spreads thebeamof light into the lightscom-ponent wavelengths. The desired wavelength is then focused onto the sample cell to detect any absorptionoremissionof lightbyasubstancein a sample.

Spectrophotometryisananalyticalprocedurethatuseselectromagneticradiationtomeasurethecon-centrationofasubstance.Thesuccessofaspectrophotometrictechniquerequiresthattheabsorp-tionoflightbythesubstancebeinganalyzedmustbedistinctordifferentfromthatofotherchemicalspecies in solution. How do scientists select the desired wavelength for spectrophotometry?

Stimulus

Energysource

Systemunderstudy

Analyticalinformation

Response

Figure 1.

Visible light

Increasing wavelength Increasing energy

gamma

greenviolet indigo blue yellow orange red

X-ray ultra-violet

infrared radio, radar, TV

Figure 2.

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Analysis of Food Dyes in Beverages continued

2 Flinn Scientific Advanced Inquiry Labs for AP* Chemistry

Theabsorptionofvisiblelightbyasubstanceresultsfromelectrontransitions,thatis,thepromo-tionofagroundstateelectrontoahigherenergyatomicormolecularorbital.Bothlightenergyandelectronenergylevelsarequantized,sothatthespecificwavelengthoflightabsorbedbyasubstancedependsontheenergydifferencebetweentwoelectronenergylevels.Theoptimumwave-lengthforspectrophotometricanalysisofasubstanceisselectedbymeasuringthevisible spectrum ofthesubstance,correspondingtoaplotofabsorbance(A) versus wavelength (,lambda).JustsevenuniquedyesareapprovedbytheFoodand Drug Administration for use in foods, drugs and cosmetics. These seven FD&C dyes give rise to the entire palette of artificial food colors. Three FD&Cdyes,FD&CBlue1,FD&CRed40,andFD&CYellow5, arediscussed in this advanced inquirylab for the analysis of sports drinks and otherbeverages.

Thestructureof FD&CBlue1 is shown inFigure3. Notice the extensive series of alternating single anddoublebonds (also called conjugateddoublebonds) inthe center of the structure. This feature is characteristic of intensely coloredorganic dyes andpigments. Everydoublebond added to the system reduces the energy differencebetween thebonding andnonbondingmolecular orbitals sothattheresultingenergygapcorrespondstovisiblelight.

AsolutioncontainingFD&CBlue1appearsblueundernormalwhite lightblue is thecolorof light transmittedby the solu-tion. The colors or wavelengths of light that are absorbed bythis solution are complementary to the transmitted color. A colorwheel(seeFigure4)providesausefultoolforindentifyingthecolorsorwavelengthsoflightabsorbedbyasubstance.ThebluesolutionabsorbsorangelightandwewouldexpectthevisiblespectrumofFD&CBlue1tocontainapeakinthe600640nm region. The optimum wavelength for spectrophotometric analysis of a dye solution is generally determined from the wavelength of maximum absorbance(abbreviatedmax,orlambdamax).ThevalueoflambdamaxforFD&CBlue1is630nm.

Thewavelengthoflightabsorbedbyasubstanceischaracteristicofitsmolecularorelectronicstructure.Theintensityoflightabsorbeddependsontheamountofthesubstanceinsolution.Generally,themoreconcentratedthesolution,themoreintensethecolorwillbe,andthegreatertheintensityoflightthesolutionabsorbs.Adigitalspectrophotometermeasuresboththepercenttransmittanceoflightandtheabsorbance.Whenlightisabsorbed,theradiantpower(P)ofthelightbeamdecreases. Transmittance (T ) is the fraction of incident light (P/Po) that passes throughthesample(seeFigure5).Therelationshipsbetweentransmittanceandpercent transmittance (% T )andbetweentransmittanceandabsorbance(A) are giveninEquations1and2,respectively.

% T = T 100 = P/Po 100% Equation 1

A = absorbance = logT Equation 2

Theamountoflightabsorbedbyasolutiondependsonitsconcentration(c) as well as the path length of the sample cell (b)throughwhichthelightmusttravel.SeeEquation3,whichisknownasBeerslaw.Theconstantaintheequationisacharacteristicofasubstanceandisknownasthemolarabsorptivitycoefficient.

A = abc Equation 3

orange600640 nm

blue450480 nm

violet400450 nm

green480560 nm

red640700 nm

yellow560600 nm

Figure 4.

N N+

-O3S SO3

- SO3-

Figure 3.

Na+ Na+

Figure 5.

c

Po P

b

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Analysis of Food Dyes in Beverages continued

Analysis of Food Dyes in Beverages 3

Experiment OverviewThepurposeofthisadvancedinquirylabistousespectroscopyandgraphicalanalysistodeterminetheconcentrationofdyeinasportsdrink.TheinvestigationbeginswithanintroductoryactivityforpreparingaseriesofstandarddilutionsofanFD&CBlue1stocksolutionandmeasuringthepercenttransmittanceofeach.Theresultswillbeanalyzedgraphicallytoidentifyanoptimumlinearrelation-ship among various functions (T, % T, log T and A)foraBeerslawcalibrationcurve.Theprocedureprovidesamodelforguided-inquiryanalysisoftheconcentrationoffooddye(s)insportsdrinksandotherconsumerbeverages.Additionaldyes,FD&CYellow5andFD&CRed40,mayalsobeusedforoptional extension or cooperative class studies.

Pre-Lab QuestionsThevisibleabsorptionspectrumforFD&CBlue1isshowninFigure6.Theestimatedconcentrationof the dye was 7.0 M (7.0 106 M).

1

0.8

0.6

0.4

0.2

0400 450 500 550 600 650 700 750

Ab

sorb

ance

Wavelength, m

Absorption Spectrum for Blue 1 FD&C Dye

Figure 6.

1. WhatwouldbeanoptimumwavelengthformeasuringtheabsorbanceversusconcentrationofaseriesofFD&CBlue1dyesolutions?Explainyouranswer.Absorbancemeasurementsaremostaccurateandsensitiveintherange0.21.0.

2. Toconstructacalibrationcurve,aseriesofknownconcentrationstandardsisprepared.UsingtheestimatedconcentrationoftheFD&CBlue1stocksolution,determinetheconcentrationofeach of the following dilutions. Hint: M1V1 = M2V2.

Dye Stock Solution (A)

B C D E F G H

Concentration (Micromolar, M) 7.0 M

Water(mL) 0 2 4 6 7 8 9 10

StockSolution(mL) 10 8 6 4 3 2 1 0

3. UsingtheinformationprovidedinQuestion1,predicttheabsorbanceofeachsolutionAHattheoptimumwavelength.RefertoEquation3intheBackground. The value of a is 130,000 M1cm1 and b = 1 cm.

Materials (foreachlabgroup)FD&CBlue1stocksolution,50-mL Kimwipesorlenstissues

Blueconsumersportsdrink,10-mL Pipet,serological,10-mL

Water,distilledordeionized Pipetbulborpipetfiller

Beakers,50-mL,23 Spectrophotometerorcolorimeter

Cuvetsortesttubes,13100mm,38 Testtuberack 2014, Flinn Scientific, Inc. All Rights Reserved. Reproduced for one-time use with permission from Flinn Scientific, Inc. Batavia, Illinois, U.S.A. No part of this mate-rial may be reproduced or transmitted in any form or by any means, electronic or mechanical, including, but not limited to photocopy, recording, or any information storage and retrieval system, without permission in writing from Flinn Scientific, Inc.

Analysis of Food Dyes in Beverages continued

4 Flinn Scientific Advanced Inquiry Labs for AP* Chemistry

Safety PrecautionsThe FD&C dyes are slightly hazardous by eye and skin contact. The dyes have been stored with other, nonfood-grade chemicals and are not for consumption. Avoid contact with eyes, skin, and clothing. Wear chemical splash goggles, chemical-resistant gloves, and a chemical-resistant apron. Wash hands thor-oughly with soap and water before leaving the laboratory. Please follow all laboratory safety guidelines.

Introductory Activity1. Turnonthespectrophotometerandallowittowarmupfor1520minutes.

2. Basedonthemaximumabsorbanceofthedyebeingtested,selecttheappropriatewavelengthonthe spectrophotometer.

3. Readtheentireprocedure.Constructanappropriatedatatabletorecordmeasurementsandtheresults of calculations. Note:Aspartofacooperativelabactivity,yourinstructormayassigndif-ferentgroupstoprepareandanalyzedifferentsolutions,andtographtheresults.Eachgroupwillneedtotranscribedataandanalyzetheresultsforallsolutionsinordertocompletetheguided-inquiryactivity.

4. Obtainapproximately50mLofstocksolutioncontainingFD&CBlue1dye.

5. Usingaserologicalpipetforaccuratevolumemeasurements,dilutethestocksolutionasindi-catedinthefollowingtabletoprepare10mLeachofaseriesofstandardsolutions,BH.Carefullymix each solution. Hint: To avoid contaminating the stock solution, first use the pipet to add the requiredamountofdistilledwatertoeachtesttube.Rinsethepipetthreetimeswiththestocksolution.Thenmeasureandaddtherequiredamountofstocksolutiontoeachtesttube.

Solution Stock (A)

B C D E F G Blank (H)

Water(mL) 0 2 4 6 7 8 9 10

StockSoln(mL) 10 8 6 4 3 2 1 0

6. Usetheblanktesttube(H)tosetthe100%transmittancevalueforthespectrophotometeratthe desired optimum wavelength for this study.

7. Measure and record the percent transmittance (% T ) of the stock solution and each standard solution(BG)attheoptimumwavelength.Remembertohandlethetesttubesonlyatthetopandtopolishthetesttubeswithlenstissue.Toavoidspills,donotplacetesttubesinthespec-trophotometeriftheyaremorethan75%full.Removesomesolutionifneededfromeachtesttubebeforeinsertingitintheinstrument.

8. Convert % T to transmittance (T ) for each measurement, and calculate the appropriate values of bothlogTandlogT.Recordallresults.

9. UseBeerslawtocalculatethepreciseconcentrationofFD&CBlue1inthestocksolution.Themolarabsorptivity(a)ofFD&CBlue1is130,000M1 cm1at630nmandthepathlength(b) is 1cm.Recordthemicromolar(M)concentration(1M= 1 106M)inyourdatatable.

10. Prepareseparategraphsof(a)% T,(b)T, (c) 1/T, (d) log T,and(e)logT (on the y-axis) versus dye concentration (on the x-axis) for each solution. Note: Dye concentrations were calculated in the Pre-Lab Questionsusingtheestimatedconcentrationofthestocksolution.RecalculatetheconcentrationsofsolutionsBG,ifneeded,basedonyouranswertoQuestion9.Usethedilutionequation.

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Analysis of Food Dyes in Beverages continued

Analysis of Food Dyes in Beverages 5

Guided-Inquiry Design and ProcedureConcentration of FD&C Blue 1 in Beverages

1. BasedonthegraphsobtainedintheIntroductory Activity, identify the optimum linear relationship or calibration curveforquantitativeanalysisoftheconcentrationofanunknownsolutioncon-tainingFD&CBlue1fooddye.

2. Whichgraphwouldprovidethemostaccuratemeanstodeterminetheconcentrationofanunknownsolutionwhosetransmittancehasbeenmeasuredspectroscopically?ExplainintermsofBeerslawandgiveanexampleofhowtheanalysiswouldbecarriedout.

3. Consulttheingredientslabelforablue-coloredsportsdrinkorotherconsumerbeverage.Identifythedyesthatarepresentandexplainwhetherthebeveragecanbeanalyzedusingthecalibrationcurvedescribedabove.

4. ObtainthenecessaryspectroscopicdataforthebeveragecontainingFD&CBlue1fooddye.RecallthatabsorbancemeasurementsaremostaccurateintherangeofA values from 0.2 to 1.0. Treatthebeveragesampleifneededtomakesurethedataareintheacceptablerange.

5. Determinetheconcentration(micromolar,M)ofthedyeinthebeverageandcalculatetheamount(mass)ofdyeinmilligramsperliterofthebeverage.ThemolarmassofFD&CBlue1 is 793 g/mole.

Opportunities for InquiryAbsorbance Spectra and Analysis of Dyes

FD&Cdyesareorganicmoleculeswithchemicalstructurescontainingmultiplecarbonringswithdoublebonds(seeFigure3onpage2).Whenmoleculeshaveaseriesofdoublebondsseparatedbysinglebonds,thebondingpatterniscalledconjugation.Thispatternofbondingresultsinareducedseparationbetweenthegroundstateandtheexcitedstateoftheelectrons.Theenergydifferencecorrespondstotheenergyofphotonsinthevisibleregion.Astheamountofconjugationincreases,theenergyoftheabsorbedphotondecreases.FD&CBlue1dyeshouldabsorblightthatisleastenergeticofthethree,followedbyRed40andYellow5withhigherabsorbedenergies.MeasurethevisiblespectraofRed40andYellow5andidentifythewavelengthoflightthatresultsinthemaxi-mumabsorbancevalue,max,foreachdye.Plotthevisiblespectraofallthedyesononegraphtocreateanoverlay.Comparethedyesandmakeobservations.PreparesolutionsandgenerateBeerslawplotstoanalyzetheconcentrationsofthesedyesinvariousbeverages.ThestructuresandmolarmassesofFD&CRed40andYellow5areshownbelowinFigure7.

NNaO3S

CH3

N

SO3Na

HOOCH3

NaO3S N N NN

SO3Na

NaO2C

HO

FD&C Red 40 FD&C Yellow 5 Molar mass = 496g/mole Molarmass= 534g/mole

Figure 7.

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Analysis of Food Dyes in Beverages continued

6 Flinn Scientific Advanced Inquiry Labs for AP* Chemistry

AP Chemistry Review QuestionsIntegrating Content, Inquiry and Reasoning

1. Calculate the value of % TforanabsorbancevalueA =1.5.Usingthisresult,explainwhyabsor-bancemeasurements> 1maynotbeaccurate.

2. Spectrophotometricstudiescanbeconductedonanycoloredcompound.Thetransitionmetalgroupoftheperiodictableexhibitsawidearrayofdifferentcoloredcompounds.Inaqueoussolu-tions, Cu2+ionswillbondtofourwatermoleculesinasquareplanargeometry.Thesolutionisalightbluecolor.Thewatermoleculescanbedisplacedbyammoniamolecules,whichformmorestablecomplexesthanwater.Thecomplexiontetraamminecopper(II)containsfourammoniamoleculescovalentlybondedtoacopper(II)ion.Theappearanceoftheintensedarkblue-violetcolor of [Cu(NH3)4]

2+ ions is often used as a positive test to verify the presence of Cu2+ ions.

a. Writeabalancedchemicalequationforthereactionofcopper(II)sulfateandconcentratedammoniatoproducetetraamminecopper(II)sulfate.

b. [Cu(NH3)4]2+solutionsexhibitadeepblue-violetcolor.Howcanyouusespectropho-

tometry to confirm that this reaction has occurred and that the product formed is in fact tetraamminecopper(II)sulfate?Wouldyouexpectthewavelengthofmaximunabsorbance(max) for Cu(NH3)4

2+tobegreaterthanorlessthanmax for Cu(H2O)62+? Explain.

3. Theelectrontransitionsresponsibleforthecolorsoftransitionmetalionsinvolved d transi-tions.Whyarezincionscolorlessinaqueoussolution?

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