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Spectrophotometers and
the Standard Curve
Bio 211, Mr.Hoyt
Southwestern College
Introduction Chula Vista,
CA,USA
Spectrophotometers are a standard research
tool used in biology and chemistry labs world wide. Among the many uses
they can follow the progress of an enzymatic reaction (as you will do later
in this course), estimate the number of bacteria in sample and detect contaminating
protein in a DNA sample. Knowing the principles behind the spectrophotometer
and how to use one is a valuable laboratory skill and you will learn the
basics of spectrophotometry in this exercise.
Learning Objectives
Upon completion of this lab you should
be able to:
1. Define
blank
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light wavelength
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Absorbance
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% Transmittance
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serial dilution
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absorption spectrum
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absorbance maximum
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standard curve
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2. Describe how a spectrophotometer works.
3. Use a micropipet and measuring skills
to set up an experiment.
4. How to set up a Spec 20 and collect
scientific data.
5. Determine the absorbance maximum for
a chemical in solution.
6. How to set up a serial dilution series.
7. Construct a line ("connect the dots")
graph of the scientific data using
Cricketgraph™.
8. How to make and use a standard curve.
9. How to use the equation C1V1=C2V2 to
calculate concentration.
Introduction to Spectrophotometers
The spectrophotometer is in principle a
fairly simple instrument. But before I describe it I need to say a couple
things about light. Light is a type of electromagnetic energy and this
type of energy travels as a wave. Just like waves
at the beach, electromagnetic waves have
peaks and troughs. One useful way to
describe light is to measure the distance
between peaks. This distance is known as the wavelength (see Figure
1). For visible light, these wavelengths range from about 380 nm (nanometers)
to about 750nm. You can see that we are talking about very small
waves!
Light Traveling as a Wave
What’s more, various wavelengths of light
are seen by you as the colors of the spectrum. Most of the electromagnetic
spectrum is shown in Figure 2. As you can see, light is only one part
of the electromagnetic spectrum.
As you may already know, white light
is made up of light of many wavelengths. Essentially a white light
source generates all visible wavelengths of light and that is a problem
when using a spectrophotometer. To get clear results, you can only measure
the absorbance of one wavelength of light at a time. Therefore, in spectrophotometer,
a white light is shown on a prism and the prism brakes up the white light
into its individual wavelengths. A resulting single wavelength of light
is then selected by the operator of the spectrophotometer and shown on
a sample.
An experimental sample for use in a spectrophotometer
usually contains a chemical in solution or particles in a suspension which
will absorb light. The amount of light the chemical or particles absorb
is measured by the spectrophotometer. That is very useful because the
amount of light absorbed can be correlated to the amount of that substance
present.
Once the light is shown on a sample and
some of the light is absorbed, the rest of the light passes through the
sample. The light that passes through is collected and measured by a phototube
and the results are displayed on a scale.
The scale on a spectrophotometer is something
else that deserves mention. Look
at a spectrophotometer. You will notice
that there are actually two scales: Absorbance and % Transmittance.
No, these weren’t placed here specifically to confuse you. It turns out
that some experiments or procedures work better with one scale or the other.
But lets bypass that and move on to what
the scales actually measure.
% Transmittance is a scale that measures
the amount light that shines through a solution. For example, if 75% of
the light shining through a sample makes it all
the way through, that would be 75% Transmittance.
If a sample completely blocked
the light shining on it, what would be
the % Transmittance? If you answered 0%
Transmittance, you have the idea.
How about Absorbance? Absorbance measures
the amount of light absorbed
by a sample (i.e. blocked from passing
through). That sounds like the opposite of the definition of % Transmittance
and it is! To further confirm this, look at the scales. They run in the
opposite directions.
The Absorbance scale as you can see is
not linear like the % Transmittance scale but exponential, starting at
zero and ending at infinity. This allows for great sensitivity at the low
end of the scale but poor sensitivity at the high end. For this reason
Absorbance readings above about 1.0 are usually not used. Also note that
the Absorbance scale is only a relative scale and therefore has
no
units.
Before you go on with this exercise, there
are some things about the absorbing molecule (or particle) and the amount
of light absorbed by that molecule (or particle) that you should know.
The amount of light absorbed depends on several factors.
1. The higher the concentration
of the absorbing molecule, the more light is absorbed.
2. The longer the distance that the light
travels through the sample, the greater the absorbance. This factor is
held constant because all of the samples are measured in the same sized
test tube.
3. A substance that absorbs a light of
one specific wavelength very strongly may absorb light of another wavelength
only weakly. For this reason, different wavelengths of light are use to
measure the concentration of various substances. How do you know what wavelength
to use? That is what you will discover in Exercise A!
One more thing before you tackle the
experiments. When an object or a solution is illuminated, some wavelengths
of light are absorbed and some are reflected.
The color of an object or solution that
you see is composed of the wavelengths of light that are being reflected.
The colors that you don’t see are the wavelengths of light that are being
absorbed.
The Experiments
Exercise A: The absorption spectrum
of Biuret reagent
1. Introduction
a. What is Biuret reagent? This
blue colored reagent is commonly used to stain proteins. Proteins by themselves
are usually colorless and therefore it is difficult to measure the presence
of protein without a special kind of spectrophotometer (which happens to
be very expensive). An easy (and inexpensive!) way to remedy this problem
is to stain the protein with Biuret reagent and measure the amount
of absorption of the Biuret reagent.
However you don’t know what wavelength of
light is best absorbed by the Biuret reagent. In Exercise B you are going
to use the reagent to figure out the concentration of a protein solution.
But without the knowledge of which wavelength of light to use you won’t
be able to do that experiment.
Exercise A will determine which wavelength
of light to use in Exercise B.
b. You will be using Blanks in these
experiments. Blanks are useful when there are other substances in the experimental
tube besides the substance you are trying to measure. Since those other
substances are not the chemical that you are trying to measure, they often
interfere with the absorbance reading of the chemical of interest. Remember:
you are interested only in the Absorbance due to the substance you want
to measure.
How do you deal with this problem? One
way would be to remove the offending substances, but this is usually not
practical and often the substances are necessary in the experiment. A much
more suitable way to deal with this problem is to exclude these other substances
from our spectrophotometer reading without removing them from the
experimental tube. The way to do that is to use a blank. A blank
contains all the substances (or substance) in the experimental tube except
the substance that is being measured. Then, before reading your experimental
tube, you place the blank tube in the spectrophotometer, set the spec.
to "0" Absorbance (or 100% Transmittance), then read the experimental tube.
What you have done, is to set the spec. to read only the absorbance that
is due to the substance that you are interested in!
c. Here are some tips to help make your
experiments with the spectrophotometer more successful.
1) Let the spec. warm up for 15 minutes.
2) Tubes need to be at least half full.
3) Make sure that the spec. tubes are clean
and free from scratches.
4) Make sure that all of your readings
are with the same side of the tube forward (most spec. tubes have a vertical
line on the front to help with this)
5) Make sure the solution is free from
bubbles.
6) Recheck the "0" Absorbance reading with
the blank often to make sure your Spec is free from drift.
2. Experimental Procedures
a. Read all of Exercise
A. You should do this before starting any experiment. It allows you to
anticipate what is coming up and lets you avoid nasty surprises.
b. Turn on the Spectrophotometer 20 (left
front knob) and set the wavelength (right top knob) to about 460nm. Let
the machine warm up for 15 min.
c. While the Spec 20 is warming up, collect
2 spectrophotometer tubes and make sure that they are clean and free of
scratches. Now fill 1 tube with Biuret reagent and the other with DI H2O.
The tube with DI H2O will be your blank. Since the Biuret reagent is made
with water, you need a blank for the water in the Biuret reagent.
d. When the Spec 20 is warmed up, set
the range so that your sample will read on the scale. Do this by ........
1) With nothing in the sample
holder and with the cover closed, use the zero control knob (same as the
on/off knob), left front, to set the meter to 0% Transmittance.
2) Insert your clean, fingerprint free
blank in the sample holder. Using the transmittance control knob (right
front) , set the meter to 100% Transmittance.
3) You can now read your sample by inserting
your fingerprint free sample into the sample holder and closing the cover.
You will get a full scale reading in either Absorbance or % Transmittance.
e. Now you are ready to start
taking experimental readings. With the wavelength set to 460nm, insert
the sample into the sample holder and record the reading on the Absorbance
scale. Record your results in Table 1.
f. Reset the wavelength to 480nm, reset
the scale by the procedure described in (d) above and take another Absorbance
reading.
g. Repeat taking readings every 20nm until
you reach 600nm. Remember to reset the scale with the water blank each
time you change wavelengths.
Table 1: Absorbance maximum of Biuret
reagent
Wavelength Absorbance
460nm |_____________
480 |_____________
500 |_____________
520 |_____________
540 |_____________
560 |_____________
580 |_____________
600___|_____________
h. Clean up when finished. Then
plot Absorbance (on the "Y" axis) vs. wavelength (on the "X" axis) as a
line graph ("connect the dots"). Do this by hand on graph paper or use
Cricketgraph™ or other graphing software if available. This graph
will be turned in with the exercise.
i. Answer these questions on a separate
piece of paper
1) What is the absorption maximum
for Biuret reagent and what is the wavelength of light you will use in
Exercise B? Why would it be best to use this wavelength?
2) Why is a blank necessary in this experiment?
3) Why must you use the blank each time
you change wavelengths?
4) If the concentration of Biuret reagent
were changed in this experiment, would the wavelength absorbance maximum
change? Why or Why not?
5) What color light do plants reflect and
which are absorbed and used to drive photosynthesis?
6) How much light is being absorbed by
a sample when a spectrophotometer reads infinity?
7) Why must a spectrophotometer use a single
wavelength of light in measuring Absorbance?
8) Some spectrophotometers use wavelengths
of light in the ultraviolet range. What are the wavelengths of light that
are in the ultraviolet range? Can you think of a situation in which a "UV
spectrophotometer" would be more useful than a visible light spectrophotometer?
Exercise B: Making and Using a Standard
Curve
1. Introduction
a. What is a serial dilution?
A serial dilution is a set of dilutions in which the important reagent
is present in a regularly decreasing concentration. You will be
making this type of dilution in this experiment.
b. What is a standard curve? As
the concentration of a light absorbing chemical in a solution decreases,
the absorbance of the solution decreases. If you make a serial dilution
of the chemical in a solution and then take absorbance readings, you can
construct a graph which shows the relationship between concentration
and absorbance. That is a standard curve and the standard curve has
many applications in the biology and chemistry lab when the concentration
of a solute in a sample must be determined.
2. Experimental Procedure
a. Read all of Exercise
B.
b. Set the Spectrophotometer to ______
nm (You fill in the blank).
c. Collect a test tube rack and 6 spectrophotometer
tubes, making sure that they are clean and free of scratches.
d. You will also need
~15ml of DI H2O
4ml of 1% Bovine Serum Albumin (a blood
protein also known as BSA) ~40ml of Biuret Reagent
1000µl and 100µl micropipetors
and tips (or 1ml serological pipets)
Two 5ml serological pipets
e. Before you make up your experimental
tubes and the blank, lets do a little exercise that will help you to do
this step.
1) Read step f.
2) Notice that you will be making a serial
dilution. Remember that a serial dilution is a set of dilutions in which
the important reagent is present in a regularly decreasing concentration.
3) In Table 2, fill in the column for "Amount
of BSA in ml" for each tube. (Hint: remember that an amount of BSA is removed
from each tube)
4) Make the conversion from ml to µl.
In Table 2, fill in the column for "Amount of BSA in µl" for each
tube.
5) Leave the column "Final concentration
of BSA" empty for now. The reason is that you will be adding other reagents
to the tubes.
f. Make your sample tubes. Since none of the
reagents are sterile, you may use the same micropipet tip for a single
reagent, as long as you do not dip the tip when you deliver the reagent.
Remember to change micropipet tips when you change to another reagent.
When you are finished, all of the tubes should have the same amount of
solution in them. If they don’t, you have made a mistake in measurement
and should correct it by making that tube(s) over again.
1) Label your spec tubes 1 through
5. Using the 5ml pipet, remove 4ml from the stock BSA solution and place
in tube 1.
2) Using the 1000µl micropipetor,
remove 2ml from tube 1 and place in tube 2.
3) Using the same tip, remove 1ml from
tube 2 and place in tube 3.
4) Using the same tip, remove 0.5ml from
tube 3 and place in tube 4.
5) Using the same tip, remove 0.25ml from
tube 4 and place in tube 5.
6) Using the same tip (or for better accuracy,
using a 100µl pipet), remove 0.125ml from tube 5 and discard it.
g. Using the 1000µl micropipetor and
a new tip, place the indicated amount of DI H2O in the correct tube.
Tube 1: no DI H2O
Tube 2: 1ml DI H2O
Tube 3: 1.5ml DI H2O
Tube 4: 1.75ml DI H2O
Tube 5: 1.875ml DI H2O
h. Using a clean 5ml pipet, place 5ml of Biuret
reagent in each of the 5 experimental tubes and mix well. Remember that
when you are finished,
all of the tubes should have the same
amount of solution in them.
i. Make a blank tube with 2ml
of DI H2O and 5ml of Biuret reagent. This is the blank that you will use
to set the 100% Transmittance on the spec in this experiment.
j. Zero and set the range on your spectrophotometer
with your blank (see Exercise A, p.6 if you need to refresh your memory).
k. Now you are ready to start taking experimental
readings. With the spec wavelength set to the Absorbance maximum for Biuret
reagent (discovered in Exercise A), insert tube 1 into the sample holder
and record the Absorbance reading. Place this reading in Table 2.
l. Repeat until all five tubes and the
solution which contains an unknown concentration of protein have been read
and recorded.
m. Now it is time to figure out the final
concentration of BSA in each of the experimental tubes. The simple equation
that follows will allow you to do this.
C1V1=C2V2
C1 is the concentration of the
original solution
C2 is the concentration of the final solution
V1 is the volume of the amount transfered
V2 is the volume of the final solution
For each of the five experimental tubes
you already know C1, V1 and V2. All you need to do is plug in the numbers
and solve for C2. Put these results in the "Final concentration of BSA"
column of Table 2.
Table 2: Exercise B experimental tubes
| Tube |
Amount of BSA |
Amount of BSA |
Final concentration |
| # |
in ml |
in µl |
of BSA in mg/ml Abs. |
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| 2
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| 3
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| 4
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| 5
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| Unk. |
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n. Clean up. When finished, graph
a "best fit" line graph of your results by hand on graph paper or using
Cricketgraph™ if available. The Y" axis should be the Absorbance
scale and the "X" axis will be the Concentration scale. This graph of a
standard curve will be turned in with the exercise.
o. Determination of the concentration of
an unknown sample is simple once you have a graph of a standard curve for
that solute. Refer to Figure 3, which is a hypothetical a standard curve
for Atropine, a medicinal compound derived from plants, as you read the
following explanation.
1) Locate the absorption value
of the unknown on the Absorbance scale( "X" axis) (see A in Figure
3).
2) Draw a horizontal line at this value
across the graph until it intersects with the standard curve (see B
in
Figure 3).
3) At this point, draw a line vertically
down until it intersects the Concentration scale ("Y" axis) (see C in
Figure 3).
4) The concentration value at this point
will be the solute concentration of the unknown solution.
p. Answer these questions on a
separate piece of paper
1) Why is a blank necessary in
this experiment?
2) What is the purpose of a standard curve?
3) Once a standard curve has been made
for a compound, could it be used to determine the concentration of another
compound? Why or why not?
4) Once a standard curve has been made
for a compound, could it be used to determine the concentration of the
same compound in a solution different from the solution used to make the
standard curve? Why or why not?
5) Can you think of a laboratory procedure
that might utilize a standard curve?
6) Apply your knowledge in making a serial
dilution: You needed to make 10 fold (or 1 in 10) serial dilution of a
0.5 M solution of arginine (an amino acid). Describe how you would make
this serial dilution if the most dilute tube is to contain 5 mM arginine.
If you need help, review p. 10.
This lab exercise was developed in
part with the support of National Science Foundation (Division of Undergraduate
Education) grant # DUE 9552290 and California Community College Chancellor’s
Office (Curriculum and Instructional Resources Division, Special Projects)
grant # FII 95-621-001.
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