Candy Lab

Zhi Yang Lin

Lab Partners: Angelina Risi and Kwame Owusu
Chem 053-125
Purpose

November 26, 2014

TA: Stephanie Barros

The purpose of this two week lab is to find which color dyes are in a certain M&M by using TLC
and spectroscopy and then determining the concentration and amount of the dyes in the M&M.
Procedure
Thin Layer Chromatography
Part 1: Extracting Dyes
Five pieces of gold M&M was dissolved in about 6 mL of acetic acid, and once the color came
off, the solution was decanted. A yarn was placed in the decanted liquid and put in a hot water
bath for about 25 minutes to absorb the dye. The bright yellow yarn was removed from the
liquid, washed with distilled water, dried, and then placed in a new test tube with approximately
2.5 mL of ammonia and placed in the hot water bath again. The yarn was removed and the
leftover liquid was concentrated by dumping it into an evaporating dish and heating it until
approximately 0.5 mL was leftover.
Note: The same procedure was done for lilac and maroon but only the data for lilac and gold was
kept.
Part 2: Separating the Dyes
A TLC was ran with red, yellow 5, yellow 6, blue 1, gold, and lilac with a mobile phase of
4:4:1:2 mixture of isoamyl alcohol, ethanol, water, and ammonia. Rf values were calculated.
Spectroscopy Project
Part 1: Spectrometer Calibration
A SpectroVis Plus spectrometer was calibrated with distilled water.
Part 2: Measuring the Sample
One gold M&M is dissolved in 5 mL of distilled water. The dye was transferred to a test tube and
moved to the centrifuge. The sugar sank to the bottom and the liquid was decanted into another
test tube. Data was obtained from the liquid by placing it into the SpectroVis Plus spectrometer.
Note: The same was done for maroon and lilac.
Part 3: Measuring the StandardsYellow 5
Concentration of yellow 5 was 6.562x10-5M. The absorbance was way greater than 1 so dilutions
were done to obtain 4 points that fell within the range of 0.05 and 1.0. A standard curve was
obtained using Excel and shared with the class.

Data and Results

Table 1: TLC Data
Dye

Spot Color (s)

Distance Solvent Travelled

Distance spot Travelled

Red 40
Yellow 5
Yellow 6
Blue 1
Gold

Red
3.5 cm
2.0 cm
Yellow
3.5 cm
1.7 cm
Orange
3.5 cm
1.9 cm
Blue
3.5 cm
2.1 cm
Orange
3.5 cm
2.5 cm
Yellow
3.5 cm
2.0 cm
Lilac
Light blue
3.5 cm
2.7 cm
Mobile phase of 4:4:1:2 mixture of isoamyl alcohol, ethanol, water, and ammonia.

Rf
0.57
0.49
0.54
0.60
0.71
0.57
0.77

Rf equals distance spot travelled divided by distance solvent travelled

Table 2: Standard Dye: Yellow 5
Contents Ratio
Wavelength
Absorbance
Concentration***
1:0 Yellow 5 to DW*
423.7 nm
Greater than 1.000
N/A
1:1 yellow 5 to DW**
423.7 nm
0.972
3.281x10-5M
2:3 yellow 5 to DW
423.7 nm
0.610
2.625x10-5M
1:2 yellow 5 to DW
423.7 nm
0.535
2.187x10-5M
1:3 yellow 5 to DW
423.7 nm
0.389
1.64x10-5M
*This was omitted because the absorbance of this solution is way over 1.000
**DW is Distilled Water.
***Concentration is calculated with the Absorbance=34662(Concentration)
Table 3: Standardization Graph Yellow 5
1.2
1
0.8

f(x) = 34661.54x - 0.22

R = 0.94
Linear ()

0.6

Linear ()
Linear ()

0.4
0.2
0
1.50E-05

2.00E-05

2.50E-05

3.00E-05

3.50E-05

Constant: Wavelength of 423.7 nm and Beers Law for Yellow 5 is A=34662C

Table 4: Standard Equation for all Dyes as sent in the email
Dye
Standard Equation
Blue 1
A = 107906c
Yellow 5
A = 34662c
Yellow 6
A = 18890c
Red 40
A = 22886c
A is absorbance and c is concentration

Wavelength
632.1 nm
423.7 nm
474.5 nm
494.5 nm

Table 5: Absorbance of Samples at Peak Wavelengths

Color
Gold
Maroon*
Lilac**

Peak Wavelengths
Absorbance
409.8 nm
0.670
496.0 nm
0.953
629.4 nm
0.522
528.9 nm
0.522
396.7 nm
0.763
*Information for Maroon was not used as instructed due to problems with TLC.
**Lilac has three peaks so three absorbances and wavelengths were given.
Table 6: Mass of Dyes given Concentration
Color
Gold
Lilac

Yellow 5
Blue 1

Concentration (M)
1.9x10-5
4.84x10-6

Moles in 5 mL
9.5x10-8
2.42x10-8

Mass (g) of dye per M&M

5.1x10-5
1.92x10-5

Calculations
Note: Only one type of each calculation is shown.
Table 1: Calculations
Rf of Red 40
Rf =

Distance spot travelled

2.0 cm
=
=0.57
Distance solvent travelled 3.5 cm

Table 2: Calculations
Calculating concentration given absorbance and standard curve equation
A=34662 c

0.972
=c=3.281E-5 M
34662

Table 6: Calculations
Finding the grams of yellow 5 dye per gold M&M
Absorbance 5 =34662 Concentration

0.670
5
=C=1.9 10 M
34662

1.9 105 mol

x mol
=
; x=9.5 108
1L
0.005 L

534.37 g
1 mol
8
9.5 10 mol 5 5 5 =5.1 105 g

There are5.1 10 g of 5 per 1 gold M M

Discussion
The ultimate goal was to determine what dyes and how much of those dyes were in the
coating of a M&M by using TLC and Spectroscopy. We got different results for TLC and
spectroscopy. For the gold M&M when using TLC, we found that it contained both Yellow 5 and
Yellow 6 components in the coating. However, when we used spectroscopy, we only observed
one peak. This peak was similar to a regular parabola but there wasnt any real vertex: the tip
was flat. This could be because the peaks of the both Yellow 5 and Yellow 6 overlapped. We used
Yellow 5 to calculate because the wavelength of our peak was closer to Yellow 5s wavelength.
For the lilac M&M, only one color was observed using TLC, but when it came to spectroscopy,
there were three peaks at 629.4 nm, 528.9 nm, and 396.7 nm. The shortest peak probably
resulted from sugar interference, as we probably didnt separate the dye all the way from the
sugar. The centrifuge separated the sugar and liquid but when we transferred the liquid to a new
beaker we couldve poured in some extra sugar. 629.4 nm corresponds to Blue1, and 528.9 nm
cant really be considered a peak as it was just a near flat line that connected the two peaks which
we noticed and decided to take down just in case. Though the TLC and spectroscopy data didnt
match up exactly, we can explain why the dissimilarities occurred.
When we calculated our assigned standard, our R squared was off; instead of being
0.9999, it was 0.9405. There was a miscommunication within the group, which probably resulted
in a slight error in the concentration and absorbance of the solution of 2:3 ratio of Yellow 5 to
distilled water. The error most probably occurred when we were measuring out the volumes of
each component.
Advantages for using TLC and spectroscopy together only occurs if both are done
correctly with no error. If so, the data from the TLC would match up with the spectroscopy data,
and they would support each other. However, that was not the case in our lab. The disadvantages
occur when error happened. Often times, we forgot to let the dots on the TLC to dry completely
before placing it in the mobile phase causing the TLC to come out runny. Spectroscopy was
troublesome because of the amount of dilution tests we needed to run to obtain one standard
curve. If one point was incorrectly measured the standard curve equation may be altered. As a
result, if one was done correctly and the other incorrectly, we cannot say with completely
accuracy which is correct since the data dont match up, and even more tests need to be run.

Post Lab Questions

1. a) From lowest polarity to highest polarity: hexanes, acetone, ethanol, water.
b) By tuning the ratio of polar and nonpolar solvent in the mobile phase we change the Rf
value of each individual solution that were running on the TLC plate. For TLC, likes
dissolve in likes, so polar dissolves in polar and nonpolar in nonpolar. If a solution is
nonpolar it will be carried up the TLC plate by the nonpolar components of the mobile
phase; the same is true for a polar solution and the polar component of the mobile phase.
In changing the ratios of the mobile phase, some solutions we are testing will be pushed
up much more than others thus giving us incorrect Rf values. We didnt use water
because it is extremely polar, and the silica gel on the TLC wouldve just absorbed it and
there would be no data obtained from the TLC. There is also a possibility that the water
will elute the solutions on the TLC plate.
c) If we used the mobile phase from the Aspirin Lab knowing that the food dyes are more
polar than salicylic acid, the solutions we are testing for would travel a smaller distance
and thus resulting in a smaller Rf value.
2. By calibrating the spectrometer, you are making sure the readings are accurate and making
sure that the spectrometer doesnt absorb a component of a solution that you do not want to
know. For example, if you have a solution A and B, and you only want the data for solution B,
you would make a blank for solution A. This blank is used to zero out the spectrometer and will
allow you to read only data for solution B. In a sense the blank negates the data of solution A in
the solution containing both A and B.
3. Beers Law states that each chromophore is independent of the others in the same solution and
each molecule in the solution has the same chance of absorbing photons in a light beam. At high
concentrations (resulting high absorbances) these claims fail, because one of the chromophore
will shade over the other. The absorbance of these shades chromophore will be much lower than
normal. This can be seen in the flattening of the graphs in LoggerPro in highly concentrated
solutions. To keep this from happening, we keep the absorbance under 1 for accuracy.
(http://www.researchgate.net/post/Is_a_value_of_absorbance_greater_than_1_theoritically_possi
ble)
4. a)

b) It is most likely that in spectroscopy

of an M&M containing both Blue 1 and
2 the graph of these will overlap and the
peaks will be hard to determine because
of the closeness of the peaks. It will act
like the gold M&M in our lab and
figuring out ratio will be extremely
difficult.

5. 1-Joey shouldve used a more accurate measuring tool than a beaker, maybe a volumetric
pipette or flask. This can cause him to calculate the concentration incorrectly, and change the
standard curve. 2-Joey didnt calibrate the spectrometer which will cause trouble in his
absorbance values. 3-A 50% dilution is done by taking half of the stock green solution and
mixing it with the same volume of distilled water. His solution is not diluted 50%, and when he
calculates the moles he will obtain a different value. 4-Joey shouldve measured the peak at the
same nanometers for both solutions. This will cause a slight change in the standard curve he is
trying to create, but if he is trying to patent something, he should try to be as precise as possible.
5-Joey only used two points when he shouldve used at least 5 points. His R squared value will
not be as accurate. 6-Standard curves are linear not parabolic. This would mean that as his
concentration grows his absorbance will grow much faster than a linear curve.