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.

• Submit Your Data

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 “P_i” 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 “P_f” the final amount of light left after it goes through the sample, then as the sample absorbs some of the light, P_f will be less than P_i. 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 = P_f /P_i

Some chemists use the term percent transmittance, “%T”, such that

%T = 100 × P_f /P_i

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 0.1 nm	1 Å (Ångström)

(milli = one thousand; micro = small; nano = a dwarf)

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 × 10^10–2+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 ×10^14–27 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.

(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:

Thus, in general (note slight variation between the two sources that were consulted):

λ in nm (source #1)	λ in nm (source #2)	approx. color seen when transmitted or reflected
400-435	400-424	violet
435-480	424-491	blue
480-490		green-blue
490-500		blue-green
500-560	491-575	green
560-580		yellow-green
580-595	575-585	yellow
595-610	585-647	orange
610-750	647-700	red

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.

Gathering Equipment

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:

• Spectrophotometer — these should already be out on the lab tables and warming up. If not, you will need to turn on the spectrophotometer and let it warm up for at least 30 min. before use.
• 
• A 1-mL and a 5-mL pipet as well as a pipet bulb or pipet filler. The type of pipet bulb we will be using is called a Brinkmann® “Pipet Helper.” Note that the canisters of pipets have been sterilized, and pipets from those canisters are used by other students and faculty for a variety of tasks in which maintaining sterility is important. Thus, while we don’t need to maintain a sterile environment for today’s lab, we do need to use proper sterile technique when removing a pipet from the canister so that the remaining pipets in the canisters do not become contaminated. To lessen the possibility of contamination in the canisters, you should
  • only open the canisters while they are horizontal (on a table) to decrease the chance of airborne bacteria falling into them — do not place open canisters in a position such that they are open-end-up,
  • when you remove the lid from a canister, set it on its side so that the open end is not facing upward,
  • only touch the top end of the particular pipet you are withdrawing, and immediately withdraw that pipet, and
  • immediately put the lid back on the canister.

• 5 13 × 100-mm (“small”) test tubes and a test tube rack to hold them (note that we have two different sizes of test tube racks: some with prongs spaced for “small” test tubes, and others with prongs spaced for “big” test tubes — make sure you get the right size)
  

• 2 cuvettes for the spectrophotometer – these must be in a PLASTIC (not metal) rack. One pair of cuvettes is needed per spectrophotometer, not per student, and these may already be placed next to each spectrophotometer. Cuvettes are special optical-quality glass, and thus, several precautions are needed when handling/using them:
  • Because we have several different brands of cuvettes in use, you will need to make sure that the two you are using are a matched set (same brand, same color, etc.)
  • Do not touch the lower portion of the cuvettes — only handle them by the top end. This will help to avoid getting fingerprints on the “business end” of the cuvette.
  • To avoid scratching the cuvettes (thereby changing the way light passes through them), they should always be stored only in a plastic rack.
  • Also, to further avoid scratching the cuvettes, you should only use lens paper to polish the bottom end of a cuvette before placing it into the spectrophotometer. Make sure you do not use Kimwipes or other “scratchy” paper for this purpose.
  • When you are done for the day and are cleaning up, as you rinse out test tubes, make sure that you keep the cuvettes separate from the test tubes and put them in the designated rack.
• The lens paper is located in the drawer at your seat. You will also need a Kimwipe to absorb droplets of excess liquid, but make sure you can tell the difference between the two types of paper and use the correct one for the correct function.
• A bottle of stock methylene blue solution (0.018 g/L) – while methylene blue is not very toxic (it is used medicinally) and this is a pretty dilute solution, you should try to not get too much of it on you unless you like blue fingers or blue spots on your white shirt.

• A vortex — This is a machine that will help to mix the contents of your test tubes.

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

• the total amount that the pipet can contain, where it says that, and what it says (Do you have the right size of pipet — how do you know it’s a 1-ml vs. a 5-mL pipet?)
• the presence of a frosted band around the top of the pipet — If the pipet has such a frosted band, it is a serological pipet, and if you are emptying the whole pipet, the last drop of liquid must be blown out to correctly deliver the desired amount.
• an accurate representation of the actual subdivisions which are marked on the pipet (For example, it would not be possible to accurately deliver 1.05 mL of liquid with a pipet that was calibrated in 0.1 mL increments.) How many milliliters does each line represent?
• Also, as you get ready to use the pipets, remember to account for whether you are reading the numbers going up or down. Notice that, if you are using a 1-mL pipet and deliver the liquid from the 0.0 mL marking down to the 0.7 mL marking, you have delivered 0.7 mL of liquid, but if you deliver the liquid from the 0.7 mL marking to the bottom you have only delivered 0.3 mL

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:

1. Set the desired wavelength, 609 nm, with the upper right-hand knob.
   

2. Without any sample in the spectrophotometer’s chamber, adjust the machine to read infinite (∞) absorbance (= 0% T, but we will not be reading percent transmittance) at the left end of the scale by rotating the zero-adjust (left-hand) knob. This tells the machine that, when it’s totally dark inside, all of the light, an infinite amount, is being absorbed, and none of it is left to be detected. As with the pH meter, if you are looking directly at the needle, you will not be able to see its reflection in the mirror behind it (remember parallax error?).
3. Two cuvettes should be sitting in a plastic test tube rack next to the spectrophotometer. If they are not there, obtain two of them, looking at them from the top to make sure they appear to be the same color (some appear more greenish than others – make sure to get a “matched” set) and making sure they are clean. Each person does not need his/her own set of cuvettes, but rather one set per spectrophotometer is sufficient. Handle the cuvettes only by the top edge and place only in a plastic rack. Partially fill one of the cuvettes with dH₂O (about 1.5 in or 4 cm in height or about 4 mL is enough, and the measurement of this does not need to be super-accurate). Because cuvettes are special optical-quality glass, it is imperative to avoid scratching them, and thus, should be PLACED IN A PLASTIC TEST TUBE RACK ONLY. Gently polish off fingerprints with lens paper only — anything else (including Kimwipes) would be too rough and might scratch the cuvette, interfering with your readings — just before each time you insert the cuvette into the machine to take a reading. Again, hold the tube by the top edge only because fingerprints can change your readings. Insert the cuvette of plain water into the spectrophotometer with the line on the cuvette lined up with the raised line on the lip of the specimen chamber on the spectrophotometer. Gently push the cuvette in as far as it will go (this photo shows the cuvette sticking out so you can see its line, and it’s not pushed in all the way) and close the lid.
4. Water does absorb certain colors of light — underwater pictures all look blue because many of the other colors have all been absorbed. Glass also absorbs certain colors of light. However, in this experiment, the light absorbed by the water and the glass is irrelevant, and the spectrophotometer needs to be told to “ignore” the light they absorb, so we can measure only the light absorbed by the methylene blue. We can compensate for the light absorbed by the glass and water and read just what light the sample absorbs by first “blanking” the machine. Note that if another solvent than water is used, that solvent must be used to “blank” the machine. Use the lower right knob on the spectrophotometer to adjust the absorbance to read 0.000 absorbance (= 100% T, right-hand knob for the right-hand end of the scale). This tells the machine that the glass and water absorb “none” of the light and “all” of the light is being transmitted through the blank sample.
5. Remove the blank from the machine and place it back into the plastic rack for future use. Put the sample from your tube #1 into a second, matched cuvette, polish the cuvette with lens paper, place the cuvette into the machine with the lines matching as before, and close the lid.

6. Read the absorbance (bottom scale — not top %T scale). Any absorbances less than 0.7 must be read to three decimal places — remember to interpolate the third place. Any absorbance readings greater than 0.7 should be read to two decimal places. Note that the absorbance scale goes RIGHT TO LEFT and is a logarithmic scale (Thus, the scale intervals get smaller as the scale goes farther left.). Remember to record your reading in your lab notebook.
7. Return the solution to its test tube. Gently blot — do not rub or wipe — the solution from the RIM ONLY of the cuvette with a Kimwipe, but do not rinse out the cuvette, because when measuring a number of solutions of increasing concentration, you will dilute each successive one less if the droplets left in the cuvette are from the last solution than if they are of water. Do not use paper towel on the cuvette, do not use Kimwipes except to blot the rim, do not use test tube brush or any other “scratchy” item in or on the cuvettes because scratches can change the spectrophotometer readings.
8. As before, fill the cuvette with the second solution, polish the cuvette, measure the absorbance, and record your data in your lab notebook. Optionally, your results may be better if you double check the machine with the plain water blank in between readings, although this may not be needed. Repeat these steps to obtain absorbance readings for the rest of your solutions, checking the blank in between as needed.
9. Clean up after yourself. Empty the cuvettes and rinse thoroughly with dH₂O, blot the lip of each, and replace them upside-down in the designated plastic rack to drain. Reminder: do not use a brush to clean cuvettes! Clean all test tubes and place them in the proper, designated location. When you are cleaning up, do not mix cuvettes and regular test tubes together, but rather, please keep the cuvettes separate. Place the used pipets in the designated “used pipet” receptacle (located near the sink), not back with the clean ones. If yours is the last lab section for the day to use the spectrophotometers, they should be turned off and put away. If another lab is to follow, leave the spectrophotometers on.

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:

• all handout pages (in notebook or separate protocol book)
• all notes you take during the introductory mini-lecture
• all notes and data you gather as you perform the experiment
• properly-constructed graph of your personal data
• print-out of class data (available online)
• drawing (yours!) of spectrophotometer with detail of the scale on the meter face, functions of each knob indicated
• drawing (yours!) of cuvette with markings
• drawing (yours!) of 1-mL and 5-mL pipets with detail of all markings on the pipets and pipet bulb
• drawing (yours!) of vortex with detail of controls
• optionally, color drawing showing what your mixed solutions look like
• answers to all discussion questions, a summary/conclusion in your own words, and any suggestions you may have
• any returned, graded pop quiz