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Project 2: Food Dyes Analysis in Commercial Products (Daniel Ordonez, Hend Zaid, Ankur Verma, Ashley Piccillo)
Intro
Results
Background
Spectrophotometers are used in forensics for the analysis of unknown compounds at a crime scene.
This is important as medical and legal investigators must confirm what these unknown substances are in
order to determine whether or not it is applicable toward their cases in court. Spectrophotometers are
useful in forensics because it only requires small samples of the unknown. As the unknown substance is
put into the cuvette the spectrophotometer will determine the absorbance value and this is dependent on
factors such as if the substance was digested than the absorbance value would be lower. However, the
unknown substance can be determined because every known substance has a specific absorbance.
Using Beer’s law, forensic investigators can use the absorbance value to determine other values such as
concentration in order to identify the unknown substance.1 This is an example of why the use of
spectrophotometers is important in everyday life and the reason why this research must be conducted.
Theory
The experiment works as the spectrophotometer measures how much light a substance absorbs and
essentially every substance transmits light at a specific wavelength.2 Then using Beer’s law, the
concentration can be calculated A = ?lc where the absorbance is proportional to concentration while the
length and molar absorptivity remain constant for each different dye. Concentration means that there are
more molecules in the substance for light to hit so the more molecules there are, the higher the
concentration will be.2
Hypothesis
The hypothesis of this experiment was that the concentration of dyes in the samples of Gatorade and
mouthwash would be in the same range as the calibration curves for each dye which were red 3 and
green 3.
Objectives
The objective for the first week was to establish a calibration curve for four different dyes using a
spectrophotometer. The second week’s objective was to use those calibration curves to calculate the
concentration of dye in two commercial products.
Week 1 Results
Methods
Week 1 Methods
For week 1, three calibration curves had to be created for three different dyes. Those dyes were red dye
#3, yellow dye #5, green dye #3. First, the spectrometer was calibrated using a water filled cuvette and
an all-black cuvette. Then the cuvette was filled up with the stock solution of the dye to make sure the
concentration was between 0 and 1. The red #3 and yellow #5 dyes had a concentration between 0 and
1 so no dilution was needed. However, the green #3 dye needed to be diluted since the stock solution
was recorded higher than 1. This was done by diluting it by a ratio of 1 mL stock solution to 4 mL of water
(1:4 dilution). The red #3 dye was parallel diluted using a 1:9 ratio of 1 ml of dye and 9 ml of distilled
water were mixed together into 100 ml beaker. A sample of this was put into the cuvette until it was
almost full and afterwards, the cuvette would be put into the spectrometer and the absorbances were
recorded. This process was repeated with the use of 2:8, 3:7, 4:6, and 5:5 ratios with the stock solution
providing an additional data point (if needed). That process was repeated using yellow dye #5 and green
dye #3. The absorbances and the concentrations were graphed in order to find the calibration curve with
the absorbances on the y-axis and the concentrations on the x-axis.
Week 2 Methods
During Week 2 there was a determination of the concentration of food dyes in two commercial products
with the use of the calibration curves that were made the previous week. Specifically, in this case the two
commercial products of Gatorade and Listerine mouthwash were used. The Gatorade contains the food
dye of yellow #53 meanwhile the Listerine mouthwash contains the food dye of green#3.4 First, the
spectrophotometer was calibrated with the use of a water-filled cuvette and a black blocked cuvette.
Next, a sample of the Gatorade was taken and put into the cuvette with a pipette. The cuvette was then
put into the spectrophotometer and a maximum absorbance reading was produced in which it was 0.602.
The same steps were done with the mouthwash and the maximum absorbance reading that was
produced was 0.395. While performing this experiment it is important to note that the absorbance values
of the samples must be within the absorbance range of the calibration curves. Lastly, we took the
absorbance values from the samples and used the linear equation from the calibration curves to
calculate the unknown concentrations.
Discussion
Table 1: Red Dye #3
1st Parallel Dilution
2nd Parallel Dilution
3rd Parallel Dilution
4th Parallel Dilution
5th Parallel Dilution
Stock Concentration
5×10^-4 (M)
—
—
—
—
—
Ratio
(Stock Solution/Water)
1/10
2/10
3/10
4/10
5/10
Final Concentration (M)
0.00005
0.0001
0.00015
0.0002
0.00025
Absorbance
0.580
0.667
0.848
0.82
0.863
`In terms of the theory used behind the spectrophotometer it was applicable as it allowed for the
observation of absorbance and thus the concentration could be calculated. When using Beer’s Law the
effects of the law were present in the formulation of the calibration curves which were later utilized to
determine the commercial product’s concentration during week two of experimentation.
Table 2: Yellow Dye #5
1st Parallel Dilution
2nd Parallel Dilution
3rd Parallel Dilution
4th Parallel Dilution
5th Parallel Dilution
Stock Concentration
5×10^-4 (M)
—
—
—
—
—
Ratio
(Stock Solution/Water)
1/10
2/10
3/10
4/10
5/10
Final Concentration (M)
0.00005
0.0001
0.00015
0.0002
0.00025
Absorbance
0.275
0.424
0.406
0.839
1.259
Table 3: Green Dye #3
1st Parallel Dilution
2nd Parallel Dilution
3rd Parallel Dilution
4th Parallel Dilution
5th Parallel Dilution
Stock Concentration
1.25×10^-4 (M)
—
—
—
—
—
Ratio
(Stock Solution/Water)
1/10
2/10
3/10
4/10
5/10
Final Concentration (M)
0.0000125
0.000025
0.0000375
0.00005
0.0000625
Absorbance
0.231
0.602
0.958
1.137
1.251
Safety
Chemical Name (Chemical
Formula)
Red Dye #3
Hazards
Handling
Not hazardous
Wash hands thoroughly after
handling. Do not eat, drink, or
smoke when using the product.
Personal Protection
Wear gloves, lab coat, and
goggles for protection.
Molecular Weight (g/mol)
Week 2 Results
Sample Calculations
Commercial Product
Absorbance
Concentration (M)
Gatorade (Yellow Dye
#5)
0.602
1.11×10^-4
Listerine Mouthwash
(Green Dye #3)
0.395
1.61×10^-5
N/A
Yellow Dye #5
Irritant
Don’t ingest or take internally.
Avoid creating and inhaling dust.
General chemical storage.
Wear gloves, lab coat, and
goggles for protection.
N/A
Green Dye #3
Not hazardous
Avoid formation of dust and
aerosols. Provide exhaust
ventilation. Don’t ingest.
Wear gloves, lab coat, and
goggles for protection.
808.85
Within part one of the experiment the concentrations of dyes were collected for Yellow 5, Red 40, and
Green 3. The theories and the actual execution of the experiment were very similar for all tests of
concentration and absorbance amongst the different dyes. The absorbances of Red 3 ranged from 0.580
to 0.863. Absorbances for Yellow 5 ended up falling between 0.275 and 1.259. Lastly, Green 3 was found
to have an absorbance between 0.231 and 1.251, the largest range amongst all of the dyes
absorbances. As the results of the absorbance of Yellow 3 in the Gatorade being 0.602 and the
absorbance of Green 3 in the mouthwash being 0.395 do not coincide with the given calibration curves
of the dyes on their own we can say that our hypothesis was incorrect. The scientific theory behind the
concentration, absorbance, and calibration curve of each dye as well as absorbance and concentration
applied to the commercial products allows for an argument that with attention to human error, the
hypothesis may be supported in a repeated experiment with the same dyes and products.
1) Final concentration (M) = Stock concentration x ratio
Ex. Red dye #3 stock concentration x ratio (for 1st parallel dilution)
= (5×10^-4) x (1/10) = 0.00005
2) Concentration of commercial product
A = ?lc is the same as y=mx+b where ?lc = m, x=c, l=1, and A = y
Y = mx+b is given in the charts
Ex. Concentration of Gatorade (use equation given by yellow 5 chart)
0.602=4766x+0.0743
x= (0.602-0.0743)/4766
x=c=1.11×10^-4
Conclusion
The overall purpose of this experiment was to create calibration curves for three different color dyes with the use of
a spectrophotometer.The produced calibration curves were then used to calculate the concentrations of different
food dyes in two commercial products. At the beginning of this experiment it was hypothesized that the
concentration of dyes in the samples of two commercial products, Gatorade and mouthwash, would be in the same
range as the calibration curves for each dye. In this case the dyes were red #3, yellow #5, and green #3. This
hypothesis was incorrect because the concentration values from the samples do not fit into the same range as the
calibration curves produced by the food dyes. For example, the Gatorade sample’s concentration value was
1.1×10^-4 M and mouthwash sample concentration value was 1.61×10^-5 M. These values do not fit into the
calibration curves of the red #3, yellow #5, and green #3 as the concentration value range of red #3 was from
0.00005 M to 0.0005 M, the concentration value range of green #3 was from 0.0000125 M to 0.0000625 M and the
concentration value range of yellow #5 was from 0.00005 M to 0.00025 M. Also, none of the R^2 values from the
calibration curves were above 0.95.There may have been a few sources of error that could have caused incorrect
results such as cross contamination from the other food dye colors in the inserted cuvettes. The Spectrophotometer
that was used during the experiment also gave results that were skewed and this possibly may have occurred from
leaked dye into the spectrophotometer slot. There could have also been incorrect measurements done during the
parallel dilution of the food dyes in the first part of the experiment.
Research Connection
The use of a spectrophotometer and Beer’s Law researchers have conducted field experiments
to test the concentration of nitrates in coastal waters of the North Sea.5 The use of Ultraviolet
spectrophotometers to test nitrate levels were used The objective of researchers in the
Zielinski study was to test the levels of nitrates from 0 to 9?mol/L in short time spans on the
coast using the newer uv method. The importance of testing the concentration of contaminants
in the water of the sea is that nitrates are a common fertilizer for algae and aquatic plants
which, if a higher level of nitrates are present, an imbalance will cause higher levels of oxygen
to be dissolved. The overarching importance is that as global warming occurs less oxygen is
present as dissolved particles in ocean waters which can induce oxygen stress in marine
animals.6 The use of the spectrophotometer in this case allows for researchers to evaluate the
current environment of the surrounding aquatic life in terms of water composition. In the
experiment results were found to be obtained in a time efficient manner due to the methods
chosen which then enabled the collection of data for the south-eastern North Sea.5
References
1) Ramanathan, G. Spectrophotometers: The Leading Choice for Reliable Forensic Analysis.
http://blog.hunterlab.com/blog/color-measurement-2/spectrophotometers-the-leading-choice-for-reliable-forensic-analysis/ (accessed Mar 31,
2020).
2) Chemistry Dictionary. https://www.chemicool.com/definition/absorption_of_light.html (accessed Mar 31, 2020).
3) Gatorade Thirst Quencher Lemon Lime (12 fl oz). https://www.instacart.com/products/3313888-gatorade-thirst-quencher-lemon-lime-12-fl-oz
(accessed Mar 30, 2020).
4) https://www.cvs.com/shop/ingredients/listerine-antiseptic-mouthwash-fresh-burst-prodid-1011553 (accessed Mar 31, 2020).
5) Zielinski, O.; Voß, D.; Saworski, B.; Fiedler, B.; Körtzinger, A. Computation of nitrate concentrations in turbid coastal waters using an in situ
ultraviolet spectrophotometer. https://www.sciencedirect.com/science/article/abs/pii/S1385110111000396?via=ihub (accessed Mar 31, 2020).
6) https://www.usgs.gov/special-topic/water-science-school/science/dissolved-oxygen-and-water?qt-science_center_objects=0#qt-science_center
_objects (accessed Mar 31, 2020).
General Chemistry Laboratory – Group Oral/Poster Presentation Rubric
Group Member Names: A: _____________________
D: _____________________
B: _____________________
C: _____________________
Experiment Number/Title: _________________________________________
Category: Content
4
2
1
Score
Introduction (Includes
experiment objectives
and background)
Group presented clear
objectives and complete
background.
Group somewhat presented
clear objectives and
complete background.
Group did not present clear
objectives and complete
background.
Experimental Details
(Includes safety)
Group described
experimental procedure in
detail. Safety protocols
were described.
Group somewhat described
experimental procedure in
detail. Some safety
protocols were included.
Group did not describe
experimental procedure in
detail. Safety details were
not included.
Results (Includes
interpretation of
results)
Group presented results in
a logical manner and with
clear interpretation.
Group somewhat presented
results in a logical manner
and with clear
interpretation.
Group did not present
results in a logical manner
and results were not clearly
interpreted.
Discussion and
Conclusion (Includes
possible ways to
improve experiment for
future attempts)
Q’s and A’s
Group discussed
experiment in detail and
provided insight on how to
improve the experiment.
Group discussed
experiment with some
detail and provided little
insight on how to improve
the experiment.
Group did not discuss
experiment with detail and
did not provide insight on
how to improve the
experiment.
Group answered all
questions correctly.
Group answered some of
the questions correctly.
Group did not answer any
of the questions correctly.
Category: Delivery
4
Eye Contact/ Clear
Speech
Lots of eye contact,
projection, clear speech,
not reading from notes or
board.
Group had a very creative
presentation and engaged
the audience regularly.
Some reading of notes,
mostly clear speech and
projection.
Group had some
creativeness and engaged
the audience sometimes.
Difficult to hear,
consistently reading from
notes, difficult to discern
speech.
Group did not have any
creativeness and never
engaged the audience.
Time (Not including
Questions and Answers)
Collaboration /
Teamwork
10-12 minutes
Not including Q and A
7-10 minutes
Not including Q and A
< 5 minutes or >15 minutes
Not including Q and A
Group showed evidence of
collaboration. Group
members communicated
well among each other
(synergy and no
interrupting each other).
Group did not show any
evidence of collaboration.
Group members did not
communicate well among
each other (no synergy and
interrupted each other).
Overall Quality of Oral
Presentation
Outstanding
Group somewhat showed
evidence of collaboration.
Group members somewhat
communicated well among
each other (some synergy
and no interrupting each
other).
Satisfactory
Creativeness/Audience
Engagement
2
1
Unsatisfactory
Total:
Individual Contribution
Evaluated by TA’s ONLY
Group member assisted other group member(s) with
preparation (out of 5 pts)
Group Member A
Group Member B
Group Member C
Group Member D
Note: Write any comments on the back of this page.
/ 40 pts
Group member participated equally
during oral presentation (out of 10 pts)
Analysis of Red Dye #40 and Green Dye #3 in Commercial Products
Prema Kallepalli, Swetha Mukalel, Alyssa Rill, and Shalini Subramanian
Results
Introduction
Background
Understanding and identifying the concentrations of certain substances within a solution is a necessary skill needed in various areas of
scientific research and everyday life. Chemical reactions regarding producing pharmaceutical drugs is an example. A major factor of
distributing such medicine is understanding what the best concentration of a specific drug given should be that which will allow it to be most
effective in treatment. In most cases, a prescribed amount is given based upon certain traits of a patient, however certain drugs require
constant monitoring at every dosage in order to ensure the most effective and safest concentration of the drug is present within the
patient’s body1. The wrong concentration of the medicine can lead to very harmful consequences. Hence, in terms of administering
pharmaceuticals, processes such as Therapeutic Drug Monitoring are utilized as to ensure the correct concentration of a potentially harmful
drug is given in a way that the positives outweigh the negatives1. Although monitoring the concentrations of potentially harmful substances
used within medicine has been well-established, the same supervision has recently been placed upon commercial products, specifically
those made for consumption. In recent times, food dyes utilized in food products have come under harsh scrutiny due to studies finding that
almost all commonly used food dyes are carcinogenics2. Red 40, for example, contains benzidine (a carcinogenic) which causes an increase
in risk of cancer when ingested. Since FDA tests however measure only free benzidines and not those that are bound, people can be
exposed to more carcinogens than claimed by the FDA2. Despite the growing worry, institutions such as the Food and Drug Administration
claim that the dyes are always distributed in a safe amount and a need to turn to possible alternatives is unnecessary 2. Investigation
continues into the extent of which certain commercial products are concentrated with these particular food dyes and whether this level of
concentration is harmful to consume.
Theory
In this project, an investigation was conducted in order to determine how concentrated two specific commercial products were with
two food dyes that have been deemed carcinogenic, Red #40 and Green #33. This was done by first producing a calibration curve for
each dye. Calibration curves are produced by measuring the absorbance of a set of solutions with known concentrations of the dye at a
wavelength that provides the strongest reading4. Serial dilution, a method that involves diluting a number of solutions utilizing a
constant dilution factor, was used to create a set of solutions with decreasing concentrations of dye 5. The absorbance of each of the
solutions is found using a spectrophotometer, which produces a light beam that passes through a cuvette holding the solution and
measures the amount of light absorbed6. It’s important to note however, that the spectrophotometer is most accurate when producing
an absorbance value between 0.1 and 17. When values fall outside of this range, absorbance and concentration are no longer
correlated linearly. If the absorbance value falls outside this range, it’s an indication that the solution needs to be further diluted. A
calibration curve, where concentration is the x-value and absorbance is the y-value, will then be produced from this data. This in turn
will provide a linear equation that reflects the direct proportional relationship between concentration and absorbance as seen in Beer’s
Law4. This relationship is caused by the fact that a higher concentration correlates to a higher number of solute molecules within the
solution that which absorbs the light8. Therefore, to find the unknown concentration of a food dye within a commercial product, one
needs to utilize the linear equation produced from the calibration curve. Since the linear equation places absorption as the y-value and
concentration as the x-value within the formula y=mx+b, by comparing this formula to the Beer’s law formula, A=elc and understanding
the molar absorptivity (e) equates to absorbance divided by concentration, one can infer that the slope of the calibration curve
equates to the molar absorptivity, or the m in y=mx+b. Thus, the linear equation is sufficient in determining the unknown
concentration of food dye within the product9. However, in order to solve for the unknown concentration, one needs to find the
absorption of the commercial products at the wavelengths in which the absorption readings were recorded for the set of solutions of
each dye. Therefore, the spectrophotometer is employed once more to collect the absorption reading of the commercial product.
Once this value has been obtained, it is simply plugged into the linear equation and, utilizing the molar absorptivity derived from the
calibration curve, one can solve for the unknown concentration of the food dye within the commercial product.
During Week 1 to lower the measured absorbance of green dye #3 the dye had
to be diluted. To make 10 mL solution of green dye #3 with an absorbance of
0.9 it was calculated that .4186 mL of green dye would need to be diluted with
9.58 mL of water as shown by Calculation 2. However, when this was carried
out it yielded an absorbance that was still greater than 1.0. Therefore, a
dilution of 2 mL of dye with 8 mL of water was tested. The absorbance of the
diluted solution was within the range and could be used during serial dilution.
Hypothesis & Objectives
In this investigation, it is predicted that a linear proportional relationship will exist between the concentration and absorbance of both Red
#40 and Green #3 according to the relationship depicted between absorbance and concentration within Beer’s Law. Moreover, this prediction
is also based upon the knowledge that the more solute molecules present in the solution, the more light it will absorb. In terms of the two
commercial products that will be investigated, it is expected that a low concentration of the food dyes will be found in each of the products
based on the theoretical values of molar absorptivity given for both dyes, which are extremely high. When calculating concentration based on
the linear equation and the absorbance values is divided by the molar absorptivity it is expected that a very small number will result. In
addition, due to claims from the FDA on the regulations they have for making sure food dyes are present in a safe amount in products, it’s
expected that the food dyes’ concentration are low in the Fruit Punch Gatorade and Listerine Mouthwash.
In week one, the primary objective of the experiment will be to develop the calibration curve for Red #40 and Green #3. This will be
accomplished by utilizing serial dilution and finding the absorbance reading of each diluted solution with a known concentration using a
spectrophotometer. Once at least five points are achieved, they will be graphed onto a concentration versus absorbance graph which will
produce a calibration curve. This calibration curve will then be utilized to form a linear equation that describes the relationship between
absorbance and concentration for each of the dyes. In week two, the main objective will be to investigate the concentration of either Red #40
or Green #3 in two commercial products. This will be completed by employing the Beer’s Law formula and comparing it to the linear equation
of each dye from the graph in order to make the inference that the unknown concentration can be found by utilizing the known absorbance
of the commercial product and the molar absorptivity found from the slope of the calibration curve. The absorbance reading of the
commercial product will then be measured and placed into the linear equation along with the derived molar absorptivity in order to calculate
the unknown concentration of the dye within the product.
Methods
Week 1:
1. After obtaining green dye #3 and red dye #40, 1 ml of each dye was placed
in a cuvette to observe the absorbance reading of the spectrophotometer.
2. For stock solution of the green dye #3, 2 ml of the stock dye was diluted
with 8 ml of DI water inside a 50 mL Erlenmeyer flask. Using a standard
pipette, the diluted dye was transferred to a cuvette and filled to the top. The
cuvette was placed in the spectrophotometer and the peak absorbance for the
wavelength was recorded and used for the rest of this dye.
3. For red dye #40, a small sample was transferred into a cuvette and placed in
the spectrophotometer. Since the peak absorbance was between 0.1 and 1,
the stock solution didn’t need dilution and this value along with its wavelength
was recorded.
4. 6 ml of the stock solution in the flask and 4 ml of DI water was transferred
to test tube 1 with pipettes. A small sample of the solution in test tube 1 was
transferred to a cuvette with a pipette and filled to the top. The cuvette was
placed in the spectrophotometer to record the absorbance.
5. Then 6 mL of test tube 1’s solution was placed into test tube 2 and mixed
with 4 mL of DI water. A small sample of the solution in test tube 1 was
transferred to a cuvette with a pipette and filled to the top. The cuvette was
placed in the spectrophotometer to record the absorbance.
*This process continued for each dye until a total of 5 absorbance
readings were achieved.*
Week 2:
1. Two commercial products, Fruit Punch Gatorade(red dye #40) and Listerine
mouthwash(green dye #3) were obtained.
2. The red Gatorade was diluted by using pipettes to add 5 ml of the red
Gatorade and 5 ml of DI water in a 50 mL Erlenmeyer flask. 1 ml of this diluted
solution was placed in the spectrophotometer using a pipette and the
absorbance was recorded.
3. A standard pipette was used to obtain a small sample of the Listerine
mouthwash and filled to the top of the cuvette with. This cuvette was placed
in the spectrophotometer and absorbance was recorded.
Discussion
The calibration curves created from the data displayed in Table 2 & Table 3 produced equations that represented
the relationship between concentration and absorbance. While the R^2 value for the calibration curve of green dye #3 is
.9663, this value only reflects how well the data points are to the fitted regression line. It does not show the accuracy of
the actual molar absorptivity value and the measured absorptivity value of green dye #3. Although the actual molar
absorptivity value is at a wavelength of 635 nm and the absorbance values were measured at 650 nm the difference in
molar absorptivity should not be so vast. As shown by Calculation 1 this creates a percent error of 82.80%. Similarly, for
red dye #40 the percent error between the actual molar absorptivity and experimental molar absorptivity was 91.17%.
Using the equation created from the calibration curve will not provide reliable data on the concentration of a known
substance in an unknown sample. Therefore, the calculated concentration of the dyes in the commercial products is not
accurate. During Week 2 of the experiment it was essential to choose a commercial product that only contained red dye
#40 and a commercial product that only contained green dye #3. Fruit Punch Gatorade only contained red dye #40 while
the Listerine Mouthwash only contained green dye #3. To figure out the concentration of the food dyes in the
commercial products the absorbance of the products was needed. If the measured absorbance of Gatorade was greater
than 1.0 then it would have to be diluted. However, if the absorbance value was smaller than 0.1, making the product
more concentrated would involve a complex process of evaporating the water from the product through a vacuum filter.
So if the measured absorbance value was lower than 0.1, it would still be recorded. The initial absorbance of the
Gatorade as measured by the spectrophotometer was 1.2. Through trial and error it was found that a 5-part Gatorade or
5-part water (in mL) dilution would provide a satisfactory absorbance value of .986. Plugging in .986 for y in the linear
equation of red dye #40’s calibration curve gave a concentration value of .00028 M. However, since the Gatorade was
diluted by a dilution factor of 5/10th, the actual concentration of food dye would also be 5/10th of the calculated
concentration. Therefore, the concentration of red dye #40 in the Gatorade was .00014 M. The Listerine Mouthwash
had a measured absorbance of .073, and although this is below the accepted range of absorbance values since it can’t be
altered it was used as y. When plugging in .073 as y for the linear equation, the calculated y-value was -.000022 M.
However a negative value for the concentration of green dye #3 in Listerine Mouthwash is impossible to have. This
discrepancy is due to the absorbance value of the mouthwash being smaller than the range used to create the
calibration curve. The concentration values disagree with the hypothesis because the experimental molar absorptivity
for both dyes should have been much higher meaning concentration of dyes in the commercial products should have
been significantly lower. The unreliability of the data doesn’t allow a proper conclusion to form on the concentration of
dyes in the two commercial products.
The main sources of error that could have greatly affected the reliability of the results include improper calibration
(systematic) and propagation error during serial dilution (random)11. When the spectrophotometer was first set up it was
calibrated with only the light cuvette and not the dark cuvette, which meant that when it read absorbance values it
wouldn’t have the standard of what the highest absorbance the spectrophotometer can read. To increase the reliability
of the data, proper calibration of the spectrophotometer should be done before the initial absorbances of the dyes are
done and then again before the next set of values of absorbance are recorded for the next type of dye. In addition, it’s
important to make sure that the cuvette has no fingerprint marks on them since these can cause the spectrophotometer
to read a higher absorbance value. Ensuring that the cuvettes are wiped by a Kimwipe and held at the open-ended top
side when placed into the spectrophotometer will increase the reliability of the measured absorbance values. Although
the calibration curve for green dye #3 produced a linear line for the first 3 sample absorbance values; the final two
values showed a very small change in absorbance thereby causing the calibration curve to no longer have a linear shape.
This is due to random errors when transferring dye from one test tube to another. When transferring the dye from one
test tube to another, a pipette was used and assumed to be a standard 1 mL pipette. However, the tip of the pipette also
contained the dye which was transferred into the test tubes and this meant that more dye was transferred into each test
tube. This meant that the change in absorbance from test tube to test tube was smaller which is why a linear calibration
curve wasn’t achieved. In addition, the dilution factor (6/10th) for serial dilution could’ve been too small resulting in
close absorbance values as concentration decreases. This is one of the main reasons why the calibration curve wasn’t
linear for red dye #40. For green dye #3 only samples A, B, and C were used to create the calibration curve. Using values
from samples D and E would’ve created a curve shape very similar to that of red dye #40’s. The stock solutions’
absorbance and concentration values also had to be used due to the lack of points available for the calibration curve.
When reproducing the experiment, the dye solutions in the test tubes will be mixed for longer periods of time and
transferred after accurately measuring out in a beaker the amount of dye to be transferred to the test tube. Lastly, the
serial dilution will be changed to 4 mL of food dye mixed with 6 mL of water. This will increase the rate at which each
test tube is diluted, and the absorbance values should have a greater jump as concentration decreases.
Conclusion
The purpose of this experiment was to identify concentrations of different food dyes found in commercial
products. It was hypothesized that a linear proportional relationship would exist between the absorption and
concentration of the food dyes as explained through Beer’s Law, and that there would be a low concentration of
dye in the commercial products. From the data gathered, the results would disagree with the hypothesis. This is
due to the calibration curves found for the commercial dye products not being complete linear proportional lines,
but rather had a curve to them, therefore proving the hypothesis wrong. However, this curvature to the line is
most likely from sources of error that may have made the data found to not be completely reliable. Errors may
include not calibrating