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Urine tests can help diagnose a range of health conditions including dehydration, pregnancy, illicit
drug usage, kidney or liver disorders, diabetes, and infections. During the Taking Accurate
Measurements (TAM) experiment you were able conduct a simple experiment to determine the
specific gravity (density) of a urine sample. While this provides valuable information, it is not the
only parameter for urine testing. A patient with kidney disease will often have elevated levels of
protein in their urine. How could the concentration of protein in a sample be determined?
To answer this question, the concept of standard curves must first be discussed. Standard curves
are graphs that relate two different (but known) physical properties over a given range. For
example, if you wanted to convert pennies to dollars you would use the conversion factor of
1 dollar per 100 pennies. However, data is not always related via a simple ratio. To convert
between ℉ and ℃, it is necessary to use the equation below. At 0°C there is only 32 degrees
between the C and F scale, however at 100°C there is 112 degrees between the two scales.

℃ to ℉ conversion ℉ = ℃ + 32
Slope equation y = mx + b

Interestingly, this example displays a common slope equation, described above, where ℉ is y, 9/5
is m, ℃ is x, and 32 is b. This is the main premise of a standard curve. The graph below shows the
relationship between absorbance and concentration of copper sulfate as a scatter plot. The best-fit
line, shown as the dashed line below, has the equation indicated in the box at the left. The R2 value,
also in the box at the left, indicates the linearity of the data points. A value of 1 means all data
points fall perfectly on the line. At this course level, standard curves should have an R2 of at least
Absorbance vs Concentration for CuSO4 at 635 nm
y = 14.0095x + 0.0173
0.8 R² = 0.9967

0 0.01 0.02 0.03 0.04 0.05 0.06 0.07
Concentration (M)

Since it is not possible to determine the concentration by a simple measurement (like finding the
mass or volume), the standard curve allows the concentration of copper sulfate to be determined
from the direct measurement of the absorption of the copper sulfate solution via a
spectrophotometer, like a Spec20. According to Beer’s law, absorbance and concentration are
linearly related via the following equation, where A is absorbance, e is the molar extinction
coefficient, b is the path length (typically 1 cm), and c is the concentration. The absorbance value

2 | Concentrations & Dilutions

could be inserted into the Beer’s law equation (where the absorbance is the y value), and the
concentration (x) could be solved for.
A = εbc
Be cautious with standard curves, as standard curves based on absorbance are not universal due to
variance between spectrophotometers, so it is important to complete all of experimentation on a
single spectrophotometer. There are also limitations. The samples must absorb light in the visible
spectrum, and the data must fall below an absorbance of 1 for Beer’s law to hold and the
relationship stay linear.
A standard curve is made by creating several solutions with a known concentration of desired
substrate (like copper sulfate in the example above), measuring the absorbance of each solution,
and plotting the data. There are two main protocols for making a solution in chemistry. The first
is to directly measure out the mass of the solute and add it to a known volume of solvent.
Concentration can be calculated by manipulating the equations below.
Grams Solute
Molar mass =
Moles Solute
Moles Solute
Concentration (M) =
L Solution
The moles of solute in the solution can calculated using the mass of solute measured directly from
an analytical scale. Molarity can be calculated from the moles of solute and the volume of the final
solution. However, sometimes the mass of solute required is too small to accurately measure. In
those circumstances, it is helpful to prepare solutions from a more concentrated solution referred
to as a stock solution. Solutions with the desired concentration can be prepared by dilutions of the
stock solution, using the equation below.
M1 V1 = M2 V2
To utilize the dilutions equation above, it is necessary to understand the different components of
an equation. The easiest way to visualize this is by a dilution diagram, like the one below. One of
these should be drawn every time you make a dilution. M1 is concentration of the solution of the
starting solution. The volume of M1 transferred into a new flask is V1. The total volume of the new
flask (after additional solvent is added to the calibration “fill to” line) is V2. The concentration of
the new solution is M2. Note that this equation only works for dilutions, since it is not changing
the substrate, just the amount of substrate in a total volume of solution.


V2 “fill to” line

M1 M2

Series Dilutions Parallel Dilutions

Concentrations & Dilutions | 3

In the dilution picture above, you may also have notices that there are two different dilution
schemes. Series dilutions, or the three on the left, can make large concentration changes very
quickly. This is useful when you need to work with a very dilution concentration. Parallel dilutions,
or the three on the right, allow for smaller variations in the concentrations, which is especially
useful for a set of standards, or creating a standard curve.
A standard curve is generally made with at least five data points. This means that five solutions
with different but known concentrations of analyte must be created, the absorbance measured, and
the data plotted by inputting the concentration (x-values) and absorbance (y-values) into Excel.
The linear equation derived from the standard curve can be used to calculate the concentration of
a sample with an unknown concentration of the analyte of interest.
Standard curves are not limited to chemicals that you have never heard of. The conversation
regarding the safety of consuming FD&C Red 40—red food dye—has increased over the years
and is likely to be brought in some context over your medical career. For this experiment you will
determine the concentration of FD&C Red 40 in a Kool-Aid® solution that has been prepped
according to the manufacturer’s directions (sans sugar). To do so, it is necessary to make five
parallel dilutions of a stock solution with a known concentration of FD&C Red 40. After obtaining
absorbance values for each solution, a standard curve will be created by inputting the concentration
(x-values) and absorbance (y-values) into Excel. The linear equation derived for the standard curve
can be used to calculate the concentration of FD&C Red 40 in a sample of Kool-Aid® using the
experimentally determined absorbance.
You will be able to further analyze this data by exploring the toxicity of FD&C Red 40 by
comparison to the LD50, or the dose which causes death to half of the participants (typically
obtained via animal studies) that consumed the dose.
4 | Concentrations & Dilutions


In your own words, write the purpose and goal of this experiment in the space below.

Use pictures to illustrate the procedure required for this experiment in the space below.
Concentrations & Dilutions | 5


• 6 – small or medium beaker • 1 – test tube
• 1 – 10 mL measuring pipet • Spectrophotometer (Spec20)
w/pipetman • Plastic dropper (optional)

• ~3.5 x 10-4 M FD&C Red 40 solution • Kool-Aid® sample


Ensure that the spectrophotometer nearest to you is turned on. Spectrophotometers may be shared
between several pairs. Spectrophotometers must be turned on at least 15 minutes prior to obtaining
any readings.
Be sure to wear gloves throughout the experiment, as FD&C Red 40 will stain anything it touches.


Obtain a 250 mL beaker filled with DI H2O, a beaker with ~15 mL stock FD&C Red 40, a 50 mL
volumetric flask, and a 10 mL measuring pipet with an appropriate pipetman. After pre-rinsing the
pipet, carefully pipet ~5 mL stock FD&C Red 40 into the 50 mL volumetric flask. Record the
actual concentration of FD&C Red 40 below. Add DI water to the calibration line, cap, and invert
several times to mix thoroughly. This is Solution 1.
Create a dilution of Solution 1 by pipetting some volume (less than 10 mL) into a 10 mL
volumetric flask, then filling to the calibration line with DI water. Cap the flask and invert several
times to mix. This is Solution 2. Transfer Solution 2 to a labeled beaker. Repeat the process to
obtain four parallel dilutions of Solution 1. To obtain the best calibration curve, start with between
~1 mL and ~9 mL of Solution 1, then fill to the calibration line with DI water.
Initial vol. Final vol. Total vol.
Solutions Observations
pipet pipet solution
Solution 1 50.00 mL
(diluted from Stock)

Solution 2 10.00 mL
(diluted from Soln 1)

Solution 3 10.00 mL
(diluted from Soln 1)

Solution 4 10.00 mL
(diluted from Soln 1)

Solution 5 10.00 mL
(diluted from Soln 1)
6 | Concentrations & Dilutions

After performing appropriate calculations, fill out the following dilution picture with your values.
Remember to show all work in the space provided.


Volume Volume Volume Volume

Solution 1
50.00 mL
Solution 2 Solution 3 Solution 4 Solution 5
10.00 mL 10.00 mL 10.00 mL 10.00 mL

Concentration Concentration Concentration Concentration Concentration Concentration

Show your work below.

Concentrations & Dilutions | 7

Once your solutions are prepared, obtain the absorbance values for Solutions 1–5. Begin by
obtaining a small test tube and filling it ~3/4 full with DI water. Place the test tube in the Spec20.
Ensure that the Spec20 is set to the maximum absorbance for FD&C Red 40, 504 nm. Zero (blank)
the instrument, then remove the test tube. Be sure to note the direction you place the test tube,
because accurate measurements require that the test tube be placed the same direction every time.
Empty the test tube, rinse it with a small amount of the least concentrated Solution, then fill the
test tube ~3/4 full with the Solution. Place the test tube in the Spec20 and record the absorbance
value. Empty the test tube, then repeat the procedure with the remaining Solutions, from least to
most concentrated, ending with Solution 1, the most concentrated solution. Record all absorbance
values below. Transfer the concentrations you calculated above to the appropriate box in the table.
Solutions Concentration of FD&C Red 40 Absorbance at 504 nm
Solution 1
(diluted from Stock)

Solution 2
(diluted from Soln 1)

Solution 3
(diluted from Soln 1)

Solution 4
(diluted from Soln 1)

Solution 5
(diluted from Soln 1)

Question 1. Why is it important to blank (zero) the instrument before taking absorbance


Obtain ~30 mL of an assigned Kool-Aid® flavor in a small beaker. Record the flavor and prep
directions in the following table. If the red color appears to fall within the range of your standards,
obtain the absorbance value from the Spec20 (be sure to blank the instrument with water before
obtaining the absorbance). Your absorbance must be smaller than your highest standard and larger
than your lowest standard. If your Kool-Aid® flavor appears too dark or falls above your most
concentrated standard, you will need to dilute it. Make an accurate dilution (be sure to draw a
dilution picture in the space below) using a graduated pipet and a volumetric flask. There is not
one correct way to do this, you just need an absorbance that falls within the range of your standards.
Draw dilution picture here.
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Prep __________________ g per
Directions 2.000 L solution
Initial vol. Final vol. Vol. Added Final total vol. Absorbance
pipet pipet (calculated) (volumetric flask)
(if measured)
Dilution 1
(if needed)
Dilution 2
(if needed)
Dilution 3
(if needed)

Show your work below.

Question 2. If you made a dilution of your Kool-Aid® and accidently added DI water
above the calibration line on the volumetric flask, what type of error would have occurred?
How would this error have affected your results?


1. Create a standard concentration curve for the FD&C Red 40 data you obtained in lab. To do
so, enter the concentration and absorbance data from Part A into Excel. Highlight only the
numerical values, select insert plot, then select the scatter option. Do NOT connect your data
points with a line, but instead insert a trendline by right clicking on any data value on the plot
and selecting “insert trendline”. Format the trendline so that is shows the linear equation, R2
value, and an appropriate title and axes labels. Do NOT set your trendline through zero. Take
a screenshot of your standard curve and upload it with your report.
2. To obtain a more accurate slope and intercept, type =slope(Y-values,X-values) into a Excel
cell. “Y-values” is all of your absorbance values, and you should use your cursor to select all
of the appropriate data. “X-values” is all of your concentration values. Write the numbers
below with at least 4 significant figures for each number.

𝑦 = ______________________________ 𝑥 + _______________________________
a. Use the equation of the line to calculate the concentration of FD&C Red 40 in your
Kool-Aid® sample (or dilute Kool-Aid® sample, if you made a dilution.

b. If you made a dilution, use the dilution equation to calculate the concentration of
Kool-Aid® in the original sample. This sample was prepped according to
manufacturer’s directions, and can be considered “drinking strength.”

c. Use the concentration of drinking strength Kool-Aid® to calculate the g/L of FD&C
Red 40 in your drinking strength Kool-Aid® sample.

10 | Concentrations & Dilutions

3. Using the LD50 determined in the Prelab, calculate the number of liters of Kool-Aid® a 50 lb
child would need to consume before exceeding the LD50.

a. Convert the liters of Kool-Aid® into 8-oz cups, if 1 L = 4.227 cups. Is this a
reasonable amount of Kool-Aid® for a child to consume?

4. Based on your experimental results, write a conclusion for this experiment. This should be in
paragraph format and include the purpose, your results, and an explanation of the concentration
of FD&C Red 40 in Kool-Aid® in comparison to the LD50. It should also include an any errors
that may have affected your experimentation. Hint: Be sure to reference your R2 and note how
the FD&C Red 40 concentration in Kool-Aid® depends on the standard curve.

Upload all procedure and data pages (pages 4–10) as a single PDF document to Canvas within 24 hours from the
end of lab. A PDF image of each page can be obtained using an app on your phone, a scanner at the library, or
another method of your choice.