Spectrophotometer Use and Beer’s Law
Note: this protocol was written back when we were using our “old” Spectronic-20 spectrophotometers. More recently, we got “new” Spectronic-200 machines. This protocol has not been edited to reflect that change. Thus, if needed, here is information on how to use our new Spectronic-200 spectrophotometers.
What Does Light Have to Do with Biology?
Light, more specifically how light is absorbed, is very important to living organisms. Perhaps the most obvious example would be that of photosynthesis, in which the chlorophyll in plants absorbs light energy and uses that to make sugar, which in turn, serves as a source of fuel for the majority of the organisms on Earth. Consider, however, that water absorbs light, or more specifically, water absorbs certain colors of light more than other colors. That would affect what sorts of living organisms could live in water, and how deep they could live. A photosynthetic organism (plants, algae) would have to be able to absorb the colors of light that the water did not absorb (and therefore were “left-over” and available for those organisms, plus those photosynthetic organisms could only live as deep in the water as a sufficient amount of light was able to penetrate.
Light absorption affects living organisms in other ways, too. Photosynthesis can only happen as deep in a plant leaf as light is able to penetrate. As light passes into a leaf, the chlorophyll absorbs those colors which are useful for photosynthesis, and the deeper into the leaf the light goes, the less is left. That means there would be a limit to the thickness of a leaf if photosynthesis is to occur throughout the leaf, and that’s probably related to why, in general, leaves are thin and flat, and most plants don’t do photosynthesis in their stems. Turtles sunning themselves on a log in a pond are absorbing infrared (IR) light to warm up their bodies. Our bodies are able to absorb certain wavelengths of ultraviolet (UV) light and use that light energy to transform cholesterol into vitamin D, the “sunshine vitamin.” However, that process happens in our skin, and once that light has been absorbed and put to use, little, if any, of it makes it deeper into our bodies.
Light absorption is also important to biologists as they study living organisms. Chlorophyll could be extracted from a leaf at a known dilution, and a spectrophotometer could be used to compare the light absorbed by that sample with the light absorbed by a standard solution whose concentration is known to determine the concentration of chlorophyll in the sample. Similarly, riboflavin (vitamin B2) is yellow colored, so it would be possible to study how much a person’s body needs/uses by taking a known dose, then saving urine samples, checking those to see how much light they absorb, and comparing that with known standard solutions to determine how much riboflavin was excreted. In a few weeks, we will be doing a lab that involves performing dilutions and growing yeast, and we will use the amount of light absorbed by a yeast suspension as an indicator of the concentration of that suspension (if everyone did the same dilution from the same packet of yeast, everyone’s samples should absorb about the same amount of light).
Additionally, in this lab, you will learn how to use a pipet (used as a noun), or how to pipet (used as a verb). That skill is an extremely important technique which is frequently used in both biology and chemistry, and one which is only mastered by practice. Thus, a big part of this lab exercise will be to practice and learn how to use a pipet to measure and dispense small quantities of liquids. Thus, in this lab exercise, the principle known as Beer’s Law will be used to develop and perfect students’ pipetting skills: if the pipet is used correctly, if the spectrophotometer is read correctly, and if the person’s final graph is constructed correctly, each person’s data should form a straight line graph. Errors in reading the pipet, delivering the correct amount of liquid, and/or reading the dial on the spectrophotometer will lead to a graph which is not a straight line. The challenge, then, is to do it right, thereby getting a nearly-straight line on one’s graph.
Background on Beer’s Law
Before discussing what Beer’s Law is/says, there is a misconception prevalent among freshman biology students that must be cleared up. Dr. August Beer was a German physicist and mathematician who also did some chemistry-related research. He was born in 1825, received his Ph. D. in 1848, and died in 1863. In contrast, the German word for that beverage known in English as “beer” has nothing to do with him! The German word for that beverage is “Bier,” which is totally different than his name.
Pierre Bouguer in 1729, and Johann Heinrich Lambert in 1760, both said that for a solution of a light-absorbing chemical such as methylene blue (shown to the right) or chlorophyll, the thickness (distance) of solution through which the light must pass affects how much light it absorbs. For example, a 2 cm thick “layer” of solution will absorb more light than a 1 cm thick “layer” of solution.
In 1852, Dr. Beer added to that by saying the concentration of
the solution also affects how much light is absorbed. Thus, for example, a
6 M solution will absorb more light than a 4 M solution of the same chemical.
This can be expressed mathematically.
If we let “Pi” stand for the initial amount or power of the
light which is shining on a sample, the initial amount of light before
it goes through the sample, and “Pf” the final amount of
light left after it goes through the sample, then as the sample absorbs some
of the light, Pf will be less than Pi.
We can, then, talk about the amount of light that is transmitted (the amount
that did get through). This is called the transmittance, “T”,
and these three numbers are related by
T = Pf /Pi
Some chemists use the term percent transmittance, “%T”, such that
%T = 100 × Pf /Pi
Chemists also use the term absorbance (the number we will be measuring in this lab) symbolized by “A,” which is equal to the logarithm of 1/T, or
A = log(1/T)
Notice, by the way, the proper term is absorbANCE, not “absorbency” which refers to (among other things) how much water a baby diaper, for example, can hold.
As previously mentioned, absorbance is related to the length of the path the light must travel through the absorbing medium and the concentration of the solution. It has been found that this is a direct relationship, so that if the length is symbolized by “b” and the concentration is symbolized by “C” (not to be confused with the speed of light, symbolized by “c”), this can be expressed mathematically as
A = bCK
where “K” is a constant value for each kind of chemical. This is called Beer’s Law. Thus, note that for several concentrations, several solutions, of the same chemical, if you make a graph of A versus C, you should have pretty close to a straight line because if all you’re changing is the concentration, then b and K stay the same. It is possible, then, to make use of an instrument called a spectrophotometer (spectro = a sight, the spectrum; photo = light; meter = measure) to study various concentrations of solutions and even predict the concentration of an “unknown” solution using the amount of light the solution absorbs.
Background on Light
As background information for this lab, we need to discuss
several of the properties of light. First, all colors of light travel at a
speed of 3 × 1010 cm/s, symbolized by “c”. Typically,
light is thought of as waves, so each color has its own wavelength (the
distance between any two adjacent crests or between any two adjacent troughs
of the wave), symbolized by “λ” (lambda) and
its own frequency, symbolized by “f.” The wavelength is a measurement
of the length/distance of each wave, and the frequency is how many of those
waves go past a given point in a given amount of time. Since all light
travels at the same speed, that means that the shorter the wavelength of
a particular color, the more waves of that color pass in a given time. Thus,
these three quantities are related to each other in the following manner:
c cm/s = λ cm/wave × f wave/s, which can be shortened to c = λf
Visible light is only a small portion of the electromagnetic spectrum, which also included gamma rays, x-rays, ultraviolet light, infrared light, (ultra = beyond; infra = below, beneath) radio waves, and microwaves. Since the wavelengths of these waves vary greatly, a review of distance measurement names and relationships might be of use.
|1 m (meter)|
|0.1 m||1 dm (decimeter)|
|0.01 m||1 cm (centimeter)|
|1 × 10–3 m||0.1 cm||1 mm (millimeter)|
|1 × 10–4 m||0.01 cm||0.1 mm|
|1 × 10–5 m||1 × 10–3 cm||0.01 mm|
|1 × 10–6 m||1 × 10–4 cm||1 × 10–3 mm||1 μ (micron)|
|1 × 10–7 m||1 × 10–5 cm||1 × 10–4 mm||0.1 μ|
|1 × 10–8 m||1 × 10–6 cm||1 × 10–5 mm||0.01 μ|
|1 × 10–9 m||1 × 10–7 cm||1 × 10–6 mm||1 × 10–3 μ||1 mμ (millimicron)
1 nm (nanometer)
|1 × 10–10 m||1 × 10–8 cm||1 × 10–7 mm||1 × 10–4 μ||0.1 mμ
|1 Å (Ångström)|
Thus, the relationships among wavelength, frequency, and energy of various colors of visible light can be summarized as in the following chart. It is important to notice that as wavelength increases, frequency and energy decrease. Thus, for example, ultraviolet light, with wavelengths of less than 400 nm, has both a higher frequency and higher energy than visible light, while infrared, with wavelenghts of over 800 nm has a lower frequency and energy.
Just to explain some of the following numbers in case you are wondering what they mean, if for example, we look at the calculations for light with a wavelength of 350 nm:
if, from above,
c = λf, then f = c/λ
thus, f = (3 = 1010 cm/s) ÷ (3.50 × 102 nm/wave × 10–7 cm/nm)
= 3/3.5 × 1010–2+7 wave/s
= 0.857 × 1015 wave/s
= 8.57 × 1014 wave/s
Also, just as a brief explanation of a more complicated physics thing (just so you know these numbers didn’t just come out of thin air, but do have a rationale behind them), the energy of each color/wavelength of light is proportional to its frequency, with the relationship, E = hf, where the “h” is something called Planck’s constant and is equal to 6.63 × 10–27 erg-s. Then, to continue the above example:
8.57 × 1014 wave/s × 6.63 × 10–27 erg-s = 8.57 × 6.63 ×1014–27 wave-ergs
= 56.8 × 10–13 wave-ergs
= 5.68 × 10–12 wave-ergs
typically epressed as just 5.68 × 10–12 ergs
Then, for convenience, physicists and chemists convert from ergs to electron volts (eV) by using the converstion factor of 1.60 × 10–12 erg/eV
5.68 × 10–12 ergs ÷ 1.60 × 10–12 erg/eV = 5.68 ÷ 1.60 × 10–12+12 eV
= 3.55 eV
For the sake of comparison, audible sounds are waves of compressed air that are considerably slower-moving than light, at around 340 m/s. Middle C has a frequency of 262 waves/s, and the A above middle C has a frequency of 440 waves/s. Thus, the corresponding wavelengths would be:
340 m/s ÷ 262 waves/s = 1.298 m/wave (= 4.26 ft)
340 m/s ÷ 440 waves/s = 0.773 m/wave (= 2.54 ft)
Visible light, that which can be seen by the human eye, is only a small portion of a larger spectrum known as the electromagnetic spectrum. Visible light can be further subdivided by what we call color. Note that if “white” light is passed through a solution that absorbs certain wavelengths while others are transmitted, we see only the wavelengths that are transmitted and thus, hit our eyes, not those that are absorbed.
Color & RGB equivalent
|350||8.57 × 1014||3.55||UV||(no RGB)|
|375||8.00 × 1014||3.32||UV||(no RGB)|
|400||7.50 × 1014||3.11||V||(131,0,181)|
|425||7.06 × 1014||2.92||V||(84,0,255)|
|450||6.67 × 1014||2.76||B||(0,70,255)|
|475||6.32 × 1014||2.62||BG||(0,192,255)|
|500||6.00 × 1014||2.49||BG-G||(0,255,146)|
|525||5.71 × 1014||2.37||G||(74,255,0)|
|550||5.45 × 1014||2.26||YG||(163,255,0)|
|575||5.22 × 1014||2.16||Y||(240,255,0)|
|600||5.00 × 1014||2.07||O||(255,190,0)|
|625||4.80 × 1014||1.99||R||(255,99,0)|
|650||4.62 × 1014||1.91||R||(255,0,0)|
|675||4.44 × 1014||1.84||R||(255,0,0)|
|700||4.29 × 1014||1.78||R||(255,0,0)|
|725||4.14 × 1014||1.71||R||(209,0,0)|
|750||4.00 × 1014||1.66||R||(161,0,0)|
|775||3.87 × 1014||1.60||R||(109,0,0)|
|800||3.75 × 1014||1.55||IR||(no RGB)|
(If you’re interested in exploring this further, one interesting Web site I found is a Wavelength to RGB Converter.)
Different chemicals absorb different amounts of light of different colors. The colors of light that are absorbed by a chemical (pigment) are, thus, not available for our eyes to see. The colors that are not absorbed are what’s “left over,” what’s reflected back and available to enter our eyes and be seen. For example:
|Chlorophyll A||428 nm ( ) and 660–675–700 nm ( )||~525 nm ( )||Chlorophyll B||453 nm ( ) and 643 nm ( )||~525 nm ( ) to 550 nm ( )|
|That’s why chlorophyll looks green (and why Chlorophyll A looks more of a blue-green color, while Chlorophyll B looks more of a pea-green color.|
|β–Carotene||451 nm ( )||~600 nm ( )|
|Methylene Blue||668 nm ( ) and 609 nm ( )||~400 nm ( ) to 425 nm ( )|
|That’s why methylene blue looks blue and why, for this lab, we will be setting the spectrophotometer to a wavelength of 609 nm. To reiterate, methylene blue looks blue because that is the light that it is not absorbing. One of the colors of light of which it absorbs the most and which we will be examining in this lab is at 609 nm, in the orange range — what it absorbs is what we cannot see.|
Thus, in general (note slight variation between the two sources that were consulted):
|λ in nm
|λ in nm
|approx. color seen when transmitted or reflected|
Parts of the Spectrophotometer & How It Works
A spectrophotometer has a light source, usually a special light bulb. The light passes through a narrow slit or lens to focus it into a small beam and then through a diffraction grating which disperses the light into a spectrum, similar to the dispersion of light by a prism.
As “white” light passes through a diffraction grating or prism, the light is bent. Red light, for example, (lower energy, lower frequency, longer wavelength) is bent less than violet light (higher energy, higher frequency, shorter wavelength), thus a spectrum is created. The spectrophotometer has another fine slit to let only a narrow band of the colored light go through. The color is chosen/adjusted by a knob which focuses a different portion of the spectrum on/through the slit. The light then passes through the sample to a detector (a photoelectric cell) which is electrically connected to the meter on the machine.
Many spectrophotometers, including those here in the biology lab, have both Absorbance (A) and Percent Transmission (%T) on their scales. For this course we will use only the Absorbance (A) scale. Due to factors within the machine itself, and due to the fact that any solvent used (water alcohol, etc.) absorbs light and, in fact, absorbs more of certain wavelengths/colors of light than others, before using the machine, it must be calibrated; the maximum and minimum A must be set to compensate for those factors. When there is no specimen in the machine, it must be “told” that things are totally dark, all the light is being absorbed, no light is getting through to the detector. When there is a specimen of plain, pure solvent (water, alcohol, or whatever is being used) in the machine, it must be “told” to “ignore” any light that is absorbed by that solvent and/or the glass in the cuvette that is used to hold the sample, and “pretend” that all of the incoming light is going through the specimen and is being received by the detector. That way, when samples are tested, the machine will report only the light that is absorbed by the solute in question.
Pipets and Pipetting
There are a number of exercises and experiments in this and your other Biology courses in which you will need to accurately measure a small amount of liquid. This is typically done by using a pipet via the process called pipetting, and thus, one of the goals of this lab exercise is to learn how to use a pipet.
The pipets here in the Biology Lab are serological (sero = serum, whey) pipets, which have a slightly different design than the pipets you may have used in Chemistry Lab. Serological pipets are calibrated such that the last drop of liquid should be blown out, and the markings go all the way to the tip. For this lab, we will be using 1-mL and 5-mL pipets. Note the various markings, bands, and color-coding on each of those sizes. Do not mouth pipet — while the pipets, themselves, are clean and sterile, in the future you will be pipetting solutions that you wouldn’t want in your mouth. We will be using Beer’s Law to test the accuracy of your pipetting technique. You will be making solutions of varying concentrations of methylene blue, using pipets to measure the specified volumes of water and methylene blue.
You should work individually on this lab. Each person MUST learn how to use a pipet and practice using it. We will be doing labs this and next quarter where you will be working individually and will need to know how to pipet! You will need the following equipment:
Carefully examine and draw the pipets and the spectrophotometer. On your drawing of the spectrophotometer, make sure to label all the parts and their functions. On your drawing of each pipet, include
Pipetting and Mixing Solutions
Set your five 13 × 100 mm test tubes in the test-tube rack. Make sure the tubes are clean because sometimes they get put away dirty, and anything in your solution will change the readings you get (Hint: Leave them clean for the next students, which could be you.). Be sure that your tubes are labeled so you know which is which. Add the appropriate amount of distilled water (dH2O), and methylene blue to each tube. If you’re sharing a beaker of methylene blue with someone else, one of you could work on measuring his/her water, first, while the other person works on measuring his/her methylene blue, first.
To use one of the pipet fillers, first notice and draw its parts. There is a small lever that goes up and down, and the liquid in the pipet will go the same direction: move the lever up and liquid will be sucked up, move the lever down, and liquid will be released from the pipet. There is a small spot to push to puff out the last drop of liquid, if needed. Before pipetting, squeeze the bulb to let some air out (if the bulb is totally full of air, the pipet filler will not suck liquid up into the pipet). Fit the desired pipet into the bottom end of the pipet filler, and immerse the tip of the pipet below the surface of the stock liquid to be measured. Use the lever to suck up liquid to a level slightly higher than the amount you need, then adjust downward until the bottom of the meniscus touches the top of the desired line.
Be very careful to not suck liquids up into the pipet filler. There is a filter between the body and the bulb of the filler, and if that gets wet, it clogs up and becomes disfunctional (not to mention possible contamination of your solution as well as future pipettings). If solution gets into the pipet filler, you can assume that both the pipet filler and the solution within your pipet are contaminated. You will need to give the pipet filler to the lab staff to be disassembled and cleaned out, your solution will need to be dumped out, and you will need to start over again.
As you transfer your sample in the pipet from the stock solution to a test tube, the pipet should be held horizontally to prevent dripping, but it should be held vertically when delivering the solution into the test tube. Never hold the pipet upside-down, as the contents could run into the pipet filler or on to your hand, which if the contents are supposed to be sterile, would contaminate them (as well as clogging up the pipet filler).
Notice that the total amount of liquid, the total volume, in
each tube is 4.0 mL. Allowing for some slight variations in manufacture of
individual test tubes, if all of your tubes contain the correct amounts of
water and methylene blue, the final volumes in your filled test tubes should
appear equal on visual inspection. If you look at your tubes, and your
volumes do not all appear to be about the same “height,” your pipetting
technique was incorrect, and for good results, you should re-mix any tubes
that are off.
After you have the correct amounts of liquid in all of your
tubes, then use the vortex to mix them thoroughly. Hold each tube gently
but firmly, by its sides, near the top, and press it down onto the vortex
to mix it. Do not push down on it from the top because your grip isn’t as
good that way, and the tube could get away from you, and also because there’s
an increased chance of breakage that way.
Solutions should not be mixed by inverting the tube with your thumb on top,
both because chemicals from your thumb could dissolve in the solution and
change your readings, and because some chemicals could damage your thumb
(methylene blue won't hurt you, though).
Use of the Spectrophotometer
After you have mixed all five of your samples, adjust the spectrophotometer, and read the absorbance at 609 nm (often written as “A609”) for each of your samples as follows:
Analyzing Your Data
Make sure that as you do the experiment, you take notes on all procedures, supplemented with illustrations where helpful. Remember to record all absorbance measurements, correlated with corresponding milliliters of methylene blue added, both in your notebook and the computer.
Do a rough comparison of the absorbances of your samples as follows. Since the second dilution contains twice as much methylene blue as the first one, your absorbance reading for the second dilution should also be close to 2× that of the first dilution. Similarly, the third should be 2× that of the second, the fifth 10× that of the first, etc. The accuracy of your results is an indication of your pipetting technique, so if you did not pipet carefully enough, and your results are “off,” you may wish to try again for any of the solutions for which you got less than satisfactory results. You are encouraged to repeat your efforts until you get satisfactory results (Yes, all of the data would go into your notebook). High-quality results come from careful pipetting, and now is the time to develop proper technique.
Once you are satisfied with your results, enter the data from your best results into the data Web page. When data are all entered, you may print out a copy of the class results for your notebool.
Graph your data. The concentration (the amount of methylene blue) is the independent variable (X-axis), and the absorbance, which depends on the concentration, is the dependent variable (Y-axis). Refer to the graphing protocol to construct your graph using proper technique, because knowing how to properly construct a graph of one’s data is something every scientist should know how to do. In this case, if your pipetting technique was good, your graph should be close to a straight line. In this type of graph, do not connect from “dot to dot,” but rather “eyeball” the best-fit straight line (use a straightedge to draw it) that best represents your data. Thus, for data points that don’t fall exactly on the line, the line should be placed such that the distance of points above the line and the distance of points below the line should be about equal. Also, keep in mind that since we told the spectrophotometer that plain water absorbed zero (0) light (because it had zero (0) methylene blue in it), the line on your graph should pass through the 0,0 point. In terms of best use of a notebook page for your graph, let every two lines across the page in your notebook equal 0.1 mL (from 0 to 1.0) of methylene blue added, and every line up the page equal 0.020 absorbance units (from 0 to 0.800). Make sure you use equal-sized units on your axes. For example, if you’re using 0.02, 0.04, etc., then 0.12 (NOT 0.20!) follows 0.10. Label (title) the axes of your graph.
Other Things to Include in Your Notebook
Make sure you have all of the following in your lab notebook: