Food Dyes: What Wavelengths Do They Absorb?

Goals

The objectives of this experiment are to:

obtain the visible spectra and record the max of the FD&C dyes: blue
#1, blue #2, yellow #5, yellow #6, red #3, and red #40.
use max values to identify the food dyes added to a set of fruit drinks
to prepare a set of working dilutions of one food dye
to construct a Beers law graph using the set of dilutions prepared.
use data from the Beers Law plot to determine the concentration of
food dye in an artificially colored drink.
to analyze the chemical structures of the food dyes and learn what
makes them colored.

Background
In the experiment Atomic Spectra, you observed that when a certain gas is heated, it
emits light with a few characteristic wavelengths. Just as the energy levels in an element
or compound can accommodate the emission of light, a process called absorption can take
place as well. Simply put, some substances absorb light.
When a substance absorbs light in the ultraviolet-visible range, electrons in the
molecules undergo transitions from lower energy levels to higher energy levels. The
difference in energies between the two levels is the amount of energy absorbed, E, and is
related to the frequency, , or wavelength, , of the absorbed light as follows:

E = h = hc/
where h is Plancks constant and c is the speed of light.
Visible radiation, that is the light that we see, is comprised of the spectrum of colored
light that we know as the rainbow, ROYGBIV. Different parts of this spectrum are
characterized by different wavelengths. A particular substance may absorb some of
these wavelengths, while the rest are either reflected or transmitted. If the absorbing
substance is opaque, like the blue dye in a sweater, then all the light that is not absorbed
is reflected and the reflected color is what we see. If the substance in transparent, like a
clear green solution, then the light that is not absorbed is transmitted (it passes through
the solution) and we see the transmitted color.
Imagine an aqueous solution of copper(II) sulfate which is blue and perfectly clear. The
transmitted color that we see is in the blue part of the spectrum (400-500 nm). Thus the
color that is absorbed by copper(II) sulfate is mainly in the red part of the spectrum (>600
nm).

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Figure 1. Simplified Schematic of a Typical Spectrometer

The type of instrument that is used to measure how much light is absorbed by a
solution is called a spectrometer (more sophisticated versions are sometimes called
spectrophotometers.) A spectrometer compares the intensity of the light that enters the
solution (Io) to the intensity of the light that is transmitted (I). When some of the light is
absorbed as shown in Figure 1, the intensity of the transmitted beam is lower than that
of the initial beam (I < Io). This relationship is expressed as the transmittance (T) or
percent transmittance (%T):

T = I/Io

%T = I/Io x 100

While transmittance indicates how much light passes through the solution, the quantity
absorbance (A) expresses how much light is actually absorbed:

A = -log T = -log I/Io = -log(%T/100)

A simple spectrometer can be used to measure the absorbance of a certain substance at
each and every wavelength in the visible range. The graph of absorbance versus
wavelength is called the absorption spectrum of that substance. A typical absorption
spectrum has one or more peaks in absorbance. The wavelength at which the
absorbance is a maximum is called max, lambda max.
The amount of absorbance is directly related to the concentration of the absorbing
substance in the solution. A higher concentration means more molecules per volume,
resulting in more light absorbed. This relationship is expressed by Beers Law:

A = cl
in which c is the molar concentration of the absorbing substance, is the molar
extinction coefficient, a characteristic property of a given substance that depends on its

nature (how wells it absorbs), and l is the path length in centimeters (the distance that
the light travels in the solution). Usually, the diameter of the cuvette containing the
solution is one centimeter, so l = 1 cm. Since is a constant for a given substance, then
we can say that absorbance is directly proportional to concentration, and a graph of A
versus c would have slope that is numerically equivalent to
? .
Since a Beers Law plot (A vs. c) expresses the relationship between absorbance and
concentration for a given substance, it can be used in analytical chemistry as a calibration
curve to determine an unknown concentration of that substance. In this experiment you
will plot the visible absorption spectrum for a food dye and determine its max. Other
teams will do the same with a different food dye and everyone should share their data.
Then you will plot the visible spectrum of one artificially colored fruit drink and
compared it to those obtained by the class. You will use the max values to determine
which dye(s) were added to the fruit drinks. You will construct a Beers Law plot using
a set of prepared dilutions of the stock food dyes to help you determine the
concentration of food dye added to one of the artificially flavored fruit drinks.

Procedure
1. Start a new page in your lab notebook and include the usual complete heading. Your
instructor will assign your team one FD&C dye and one fruit drink, but you will need
to record class data to complete this experiment.
2. For this experiment you will actually have to write up your own procedure. Make
sure you take all the needed notes so that you can type up a thorough and detailed
procedure as part of your lab report.
3. Determine the visible spectrum of one dye using the Vernier Spectrometers. You will
need to calibrate the spectrometer with deionized water. Set the spectrometer to give
you an absorbance vs. wavelength spectrum. Find the absorbance maxima of the dye
(max), the point with highest absorbance (peak) and record in your notebook.
4. Design a table to record all the max values, the absorbed colors, and the
transmitted color of each of the five food dyes. Rotate around the lab and check out
the visible spectra obtained by other teams. Roughly sketch each of the five spectra in
your notebook. Make sure to make a note of the location of the observed peaks in
terms of range of color and wavelengths .Record all the relevant information in your
lab notebook.
5. Take a sample of one of the artificially flavored fruit drinks and obtain a visible
spectrum, just like you did for the stock food dye solution. Based on the max values of
the five food dyes and the max value of your teams fruit drink, can you determine
which dye(s) was(were) added to your fruit drink? Note: you may have more than one
dye. Record your conclusion in your notebook and share with the class.

6. You will need to use the stock food dye solution to make a series of working dilutions
so you construct a Beers Law calibration curve. For example, measure 3, 5, and 7 mL
of stock into 10.00 mL volumetric flasks and dilute with deionized water to the mark.
Mix well.
7. Prepare a table in your notebook with one column with a list of the final concentration
of the dilutions prepared and the concentration of the initial stock food dye solution
used. The second column should be used to record the absorbances measured at the
same wavelength (max).
8. Choose the max for your food dye, measure and record the absorbance for your
samples in your table. Always record the absorbance at the same wavelength. You
will have to share this data with the class because another team will need your data.
9. Using Excel plot absorbance vs. concentration in grams/Liter. Find the equation of
the line, the intercept should be very close to 0.0. The slope of the line is characteristic
to the dye.
10. Determine the concentration of the dye in the fruit drink using the line equation, the
absorbance measured for the drink sample, and solving for x (concentration in g/L).
Remember that the slope is provided by the line equation and the y value is the
absorbance obtained from the drink sample.

Apparatus
Cuvettes (at least two)
10 mL volumetric flasks (at least 3)
Measuring pipets ( 1 to 10 mL )
Pipet bulbs
Laptop computer
Vernier Spectrophotometer

Chemicals
Artificially colored drinks
Deionized water in squirt bottles
FD&C Blue 1 & 2, Red 3 & 40, Yellow 5 & 6
0.010 g/mL Stock Food Dye Solutions

Conclusions
Gracefully incorporate the following points into your Conclusions section wherever
appropriate (i.e., do not make it look like you are answering a list of questions!)
1.

What is max for each of the food dyes tested? What color light does each dye
absorb and how does this determine its appearance?

2.

Why is it important to make absorbance measurements at max instead of some

other wavelength?

3.

What is the value of the extinction coefficient for the dye your team tested?

4.

What is the calculated concentration of the dye present in the fruit drink you
tested?

5.

What is the significance of the R2 value in this experiment? What would be the best
possible value for R2 and how does it compare to what you obtained?

6.

How important was accuracy in preparing the standard solutions? What are the
other sources of error in this experiment and specifically how would you expect
them to have affected your final result?

7.

Compare the structures of the 6 food dyes. What do they have in common? Find
out what special feature about their structure is responsible for their absorbance in
the visible range of the spectrum.

8.

Lone electron pairs are missing in the handout with the structures, clearly add dots
to the proper atoms to indicate the location of the missing lone electron pairs.

9.

For each of the structures indicate how many pi bonds are present, how many sp2
hybridized carbons are there? Label any sp3 and sp hybridized carbons in the
structures provided in the handout. Also indicate the hybridization on the nitrogen
atoms.

10. Which atom(s) do not follow the octet rule in the structures provided? Explain.
11. All of these dyes are obviously soluble in water. What portions of the structures
help the overall solubility of these molecules?

Lab Report
The report for this lab will consist of copies of all the notebook pages that are relevant to
this lab. Make sure you complete the heading ( Name, partners name, date, title, and
objective). These pages should also include the detailed procedure, the raw data
tables, observations, and sample calculations.
In addition you should include any prepared Excel graphs and a typed Conclusion
section that includes the information listed above.

SamebluedyeinM&Mslinkedtoreducingspineinjury

Researchersfindwaytoreducesecondarydamagecausedbyspinalinjuries
CompoundBBGissimilartobluefooddyeusedinsweets,sportsdrinks
Onlysideeffectofintravenousinjectionwasthatitturnedtestratsblue
ResearchersareplanningtoapplytotheFDAforpermissionforhumantests

Findthefollowingarticleat:
http://www.cnn.com/2009/HEALTH/07/28/spinal.injury.blue.dye/index.html
(CNN)ThesamebluefooddyefoundinM&MsandGatoradecouldbeusedtoreducedamagecausedbyspineinjuries,
offeringabetterchanceofrecovery,accordingtonewresearch.ResearchersattheUniversityofRochesterMedicalCenter
foundthatwhentheyinjectedthecompoundBrilliantBlueG(BBG)intoratssufferingspinalcordinjuries,therodentswere
abletowalkagain,albeitwithalimp.Theonlysideeffectwasthatthetreatedmicetemporarilyturnedblue.Theresultsof
thestudy,publishedinthe"ProceedingsoftheNationalAcademyofSciences,"buildonresearchconductedbythesame
centerfiveyearsago.
InAugust2004,scientistsrevealedhowAdenosineTriPhosphate,whichisknownasATPanddescribedasthe"energy
currencyoflife,"surgestothespinalcordsoonafterinjuryoccurs.ResearchersfoundthatthesuddeninfluxofATPkilledoff
healthycells,makingtheinitialinjuryfarworse.ButwhentheyinjectedoxidizedATPintotheinjury,itwasfoundtoblock
theeffectofATP,allowingtheinjuredratstorecoverandwalkagain.
"WhileweachievedgreatresultswhenoxidizedATPwasinjecteddirectlyintothespinalcord,thismethodwouldnotbe
practicalforusewithspinalcordinjuredpatients,"saidleadresearcherMaikenNedergaard,professorofNeurosurgeryand
directoroftheCenterforTranslationalNeuromedicineattheUniversityofRochesterMedicalCenter.
"First,noonewantstoputaneedleintoaspinalcordthathasjustbeenseverelyinjured,soweknewweneededtofind
anotherwaytoquicklydeliveranagentthatwouldstopATPfromkillinghealthymotorneurons.Second,thecompoundwe
initiallyused,oxidizedATP,cannotbeinjectedintothebloodstreambecauseofitsdangeroussideeffects."
Backin2004,Nedergaard'steamdiscoveredthatthespinalcordwasrichinamoleculecalledP2X7,whichisalsoknownas
"thedeathreceptor"foritsabilitytoallowATPtolatchontomotorneuronsandsendthesignalswhicheventuallykillthem.
NedergaardknewthatBBGcouldthwartthefunctionofP2X7,anditssimilaritytoabluefooddyeapprovedbytheFoodand
DrugAdministration(FDA)in1982gavehertheconfidencetotestitintravenously.Itworked.TheratsgivenBBG
immediatelyaftertheirinjurycouldwalkagainwithalimp.Thosethatdidn'treceiveadoseneverregainedtheirmobility.
NedergaardtoldCNNthatthereiscurrentlynostandardtreatmentforpatientswithspinalinjurywhentheyreachthehospital
emergencyroom.
"Rightnowweonlytreat15percentofthepatientswereceivewithsteroidsandmanyhospitalsquestionifthatevenworks
forthat15percent;it'saverymoderatebenefittoonlyasubsetofpatients.Sorightnow85percentofpatientsareuntreated,"
shesaid.Nedergaardsaidtheresearchteamisn'tclaimingthatBBGcancurespinalinjuries,insteadthatitoffersapotential
improvementinpatients'condition.
"Evenamoderateimprovementinfunctionalperformanceofthepatientisabig,bigeventforthesepatients,"shesaid."They
cancontroltheirbladder.Iftheycanjusttakesmallstepsinsteadofsittinginawheelchairallthetime,it'satremendous
benefitforthesepatients,"sheadded.Thedosemustbeadministeredimmediatelyaftertheinjury,beforeadditionaltissue
diesasaresultoftheinitialinjury.ResearchersarecurrentlypullingtogetheranapplicationtobelodgedwiththeFDAto
stagethefirstclinicaltrialsofBBGonhumanpatients.
"Ourhopeisthatthisworkwillleadtoapractical,safeagentthatcanbegiventopatientsshortlyafterinjury,forthepurpose
ofdecreasingthesecondarydamagethatwehavetootherwiseexpect,"saidStevenGoldman,ChairoftheUniversityof
RochesterDepartmentofNeurology.

Chemical Structures of Food Dyes

O

Yellow #5

FD&CO Blue #1

O-

O
N

-O

Yellow #6

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-O

N
O

Red #40

N
CH3

O
S

OH
O-

OH

Red #3

-O

N
S
O

S
O

N
O

O-

CH3

OH

O-

FD&C Blue 2

S
O

O-

O-