Spectrophotometry Teacher Kit - The Colby College Community Web

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Sam Redstone
January 30, 2014
Title:
The Case of the Stolen Sports Drink Recipe
Appropriate Level: High School Biology
Abstract:
Students will make dilutions from a stock concentration of beet
juice, measure their absorbance values spectrophotometrically
and create a standard curve. This plot will allow them to
determine the concentration of an unknown sample of beet
juice.
Time Required:
40 minute class period.
Objectives:
To gain familiarity with the Spec 20 and concepts of
spectrophotometry, making dilutions, and using Microsoft
Excel to create a standard curve.
Chemical Concepts: Spectrophotometry: The use of light to make quantitative
measurements of a substance. This is done by measuring the
absorbance or transmittance of a given wavelength of light by a
solution. Spectrophotometry is frequently used by biologists to
determine the concentration of pigments such as chlorophyll.
Equipment and Supplies:
• Powdered Beet Juice Extract
• Spec 20
• Test Tubes and Rack for Each
Group (2 students/group)
• 100 mL Volumetric Flasks, Beakers
or Graduated Cylinders
• Plastic Funnels
• Beaker or other container for
waste
• 100 mL of 10.0 g/L beet juice stock
solution from which to make dilutions
• 100 mL of beet juice whose
concentration is “unknown”
• Analytical Balance
• DI Water
Preparation Work:
Instructors will need to obtain beet juice extract. Available as a powder at
most health food stores, beet juice extract should be mixed with water to make a
10.0 g/L stock solution. Due to the presence of dirt and other insoluble impurities in
the extract, it is advisable to strain the stock solution through coffee filters prior to
the experiment. It should also be noted that beet juice will oxidize over time and
change color; therefore, the stock solution should not be prepared more than a day
or two in advance of the experiment, and refrigerated.
1
Sam Redstone
January 30, 2014
Expected Results & Explanation
If students are careful when making their dilutions they should produce a
standard curve with a correlation coefficient value greater than 95% and the
trendline should give them an experimental concentration within within 10% of the
known concentration. Errors that may reduce the accuracy of the standard curve
include losing color during transfer (i.e. beet juice extract remaining in the
graduated cylinder or funnel), not reading the Spec 20 at eye level and not properly
blanking the Spec 20 prior to use.
By using the color wheel, students will also find that the red-purple color of
the beet juice will absorb light at wavelengths of 540 nm the best. This is because
that shade of blue-green is complementary to the color of the beet juice.
Supplemental Teaching Information
Hutchingson, Alex. Beet Juice: How Much and When? Runners World Magazine.
http://www.runnersworld.com/nutrition-for-runners/beet-juice-how-much-andwhen (Accessed January, 2014).
Accommodations for Different Age Levels
• As this experiment involves concepts such as graphing and simple algebra,
it would make a great activity for students in Grades 7-8. If this is the case,
instructors should prepare all of the standard solutions in advance so that the
students only need to measure the absorbance of the standards at a wavelength of
540 nm. They can then practice plotting the date in Microsoft Excel and using the
slope-intercept equation algebraically to determine the unknown concentration.
2
Sam Redstone
January 30, 2014
The Case of the Stolen Sports Drink Recipe
Overview
The goal of this experiment is to use spectrophotometry to determine the
concentration of beet juice in a sample. This will be achieved by creating a standard
curve of absorbance versus concentration using known concentrations of beet juice.
The equation of the best-fit line can be used to solve for the concentration that
corresponds to the absorbance of the unknown sample.
Objectives
To gain familiarity with the Spec 20 and concepts of spectrophotometry,
making dilutions, and using Microsoft Excel to create a standard curve.
Background
Baseball is a highly mental sport, and its players are highly particular about
their routines. There is, perhaps, no better example of this than Andrew Suarez, star
pitcher for the USM Huskies, who is insistent upon drinking beet juice during every
game. Beet juice has been shown to increase endurance in athletes (1), and Andrew
credits this to him being able to pitch more than 15 innings on the final day of the
2013 NCAA DIII National Tournament (2). His opponents know this, so prior to the
start of the 2014 season, a rival team stole his recipe. Digging through his locker
from last year, Andrew found a bottle containing several ounces of his special juice,
and he wants us to be able to analyze the solution to recreate the lucky recipe.
Otherwise, Andrew fears that he will not be able to regain his top form of last year.
Fortunately, Andrew’s beet juice is highly colored, making it easy to
determine the concentration using a technique called spectrophotometry, which
monitors the absorbance of light. Beet juice is colored because it contains biological
pigments, molecules that that absorb visible light. Visible light is a small portion of
the electromagnetic spectrum, which includes all the types of energy that travels
through space as waves. The “white” light from the sun or a light bulb is actually
made up of all the colors of light in a rainbow
(red, orange, yellow, green, blue, indigo, and
violet).
You can predict the color of light absorbed
by a pigment by using a color wheel. Colors
opposite each other on the color wheel are
called complementary colors. In general, a
pigment of a particular color appears to be the
color absorbs light of the complementary to the
color that it absorbs. For example, an orange
pigment absorbs blue light (with which has a
wavelength between 435 and 500 nm). Using the color wheel shown here, predict
the color of light and the wavelength absorbed by beet juice.
3
Sam Redstone
January 30, 2014
Procedure
1. Each group will make their own standards at the following concentrations:
0.4 g/L, 0.6 g/L, 1.0 g/L and 2.0 g/L. You will
Concentration Vol. 10.0 m
make these standards by making dilutions
(g/L)
Stock (mL)
from the 10.0 g/L stock solution.
0.4
4.0
0.6
6.0
2. To make the dilution, you will use a
1.0
10.0
graduated cylinder to measured the desired
2.0
20.0
amount of stock solution into a volumetric
flask and dilute to 100 mL with DI water. Use the table to the right to guide
you with your dilutions. To minimize the beet juice lost during transfer, be
sure to rinse the funnel and graduated cylinder—or any other object used to
assist the transfer—into the volumetric flask.
3. To ensure that the solution is uniform throughout, you should be sure to
invert the volumetric flask several times before adding it to a test tube and
measuring its absorbance with the Spec 20.
4. The Spec 20 must be blanked prior to use (3). To do this, turn the instrument
on and allow it to warm up for 10-15 minutes. Using the top-right knob, set
the instrument to read the samples at the desired wavelength, which you will
have agreed upon by now.
5. When a “blank,” a test tube containing only DI water, is inserted into the
instrument, the front right knob must be adjusted such that the apparatus
gives a reading of 100% transmittance (the top reading on the display).
When the blank is removed and the lid to the instrument is closed, the front
left knob should be adjusted until the apparatus gives a reading of infinite
absorbance, or 0% transmittance.
6. Measure the absorbance of each standard and the unknown solution using
the Spec 20 by filling a test tube with the solution, closing the lid and reading
the top line of the scale.
*Note that when reading a Spec 20, the needle should be looked at from eye
level, and the mirror should be used to ensure that the needle is lined up properly.
To do this, shift your focus until the needle splits the image of the needle on the
mirror.
4
Sam Redstone
January 30, 2014
Data Analysis
1. Once all readings are made, each group should enter the data into an Excel
Spreadsheet . To create a standard curve, plot Absorbance vs. Concentration,
making sure to add a data point at the origin, as you will have zeroed the
Spec 20.
2. Add a trendline, making sure that you include the equation of the line on the
chart. By knowing the absorbance of the unknown solution, you can use this
equation to solve for the concentration of the unknown concentration.
Clean Up
All beet juice is safe to be poured down the drain and the glassware should
be rinsed out with water.
Table to Assist with Data Recording
Standard
Concentration (g/L)
Absorbance
1
2
3
4
Unknown
???
Can you help Andrew recreate his beet juice by telling him the amount of beet juice
to add to obtain a final volume of 1 L?
5
Sam Redstone
January 30, 2014
Literature Cited:
1. Wylie, L.J., Kelly, J., Bailey, S.J., Blackwell, J.R., Skiba, P.F., Winyard, P.G.,
Jeukendrup, A.E., Vanhatalo, A., Jones, A.M. (2013). J. Appl. Physiol. 115: 325336.
2. USM.edu. http://southernmainehuskies.com/sports/bsb/201213/bios/richards_andrew_b6ml?view=gamelog (Accessed January 9, 2014).
3. Use of the Spec 20. Bates College.
http://abacus.bates.edu/~ganderso/biology/resources/spec20.html
(Accessed January 7, 2014).
6
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