Spectronic 200 Use


Parts of the Spectronic 200

Spec 200 doors shut Here is what the Spectronic 200 looks like (or should look like) when it has just been obtained from where they are stored or when it is ready to be stored. All the doors on the body are closed and the screen is down.

Screen up Here, the LCD screen has been tilted up so that it can easily be viewed. The screen should be returned to its flat position prior to storage.

Spec 200 doors open In this photo, the lid of the sample chamber has been raised to show the location of the sample chamber, within. Also, if you look carefully, you should be able to see that the doors to the cuvette-storage compartments on the right and left sides have been opened.

turn wavelength dial Across the top of the machine, in front of the screen, is a row of control knobs and buttons. The left-most one of these is the wavelength knob (or dial). Note that the symbol used to represent wavelength is the Greek letter “λ” (lambda). Thus, in this Web page, the wavelength knob will also be referred to as the λ knob. You may turn the wavelength dial to adjust the wavelength by 10-nm intervals until you’re close to where you need to be.

push down and turn for fine Push down on the wavelength dial while turning it to fine-adjust the wavelength by 1-nm intervals.


Also notice in the above photo that the button just to the right of the wavelength knob is the Print Button. As of the writing of this Web page, we did not, yet, have a printer for our Spec 200s, but we are in the process of obtaining one. When that and/or the computer-interface software are installed, you will be able to print a copy of your results.

blank button To the right of the print button is the Blank, or Auto-Zero Button (here, also referred to as  0.00 to help you remember which button is being discussed). Since solvents such as water and ethanol, as well as the plastic of which the cuvettes are made, absorb some light, pushing the  0.00 button will “zero” the machine so it ignores any light absorbed by the solvent and cuvette, and displays just the amount of light that is absorbed by the specific substance being tested.

Home button To the right of the blank/zero button is the Home Button (here, also referred to as ). Pushing this button will send the Spec 200 back to its “home” menu display.

Enter button On the right-hand side of the top of the machine is a group of navigational buttons. The four Arrow Buttons (▲=up, ►=right, ▼=down, and ◄=left) are used to navigate to various menu options displayed on the screen. The cEnter is the Enter Button, used to choose/execute various options, just like on a computer.

sample chamber, cuvette holder Here’s a close-up of the Cuvette Holder within the sample chamber. The Spec 200’s Cuvettes are “square” plastic tubes that fit into the holder. Note in the photo of the detector, below, the top edge of a cuvette in place.

light source The Light Source or Lamp is to the right, and shines its light through the specimen in the cuvette.

light detector To the left of the cuvette holder, there is a Detector which, as its name suggests, detects the amount of light that is still left after passing through the sample. The Spec 200’s onboard computer then compares that with the amount of light put out by the lamp to determine how much light was absorbed by the specimen. Note that, because the machine’s calculations are based on a comparison of the amount of light put out and the amount of light left, it’s necessary to keep stray light out by keeping the lid of the sample chamber shut.

power switch The Power Switch is located on the back of the Spec 200. After the machine has been plugged in, the power may be switched on. A couple-minute wait will be needed for the Spec 200’s onboard computer to boot up. Also, a USB Connector and a Printer Port are located on the back if/when needed (but the Spec 200 can only “talk to” one certain kind of small printer).


Turning on the Spec 200

Warming Up When the Spectronic 200 is turned on, it will take a couple minutes for its onboard computer to boot up.

initial screen After the machine’s initial splash screen, it will display this “initialization” screen which asks you to check to make sure someone else didn’t leave a cuvette in the sample compartment. Do check to make sure there’s not one in there, then make sure the lid is shut — the machine needs that to correctly calibrate itself on bootup. Once you’re sure there in not a cuvette in the chamber and that the door is shut, press the Enter () button.

running initialization As the machine goes through its bootup sequence, it will display this initialization screen to show what it is doing.

choose interface mode When it is done with its bootup, it will display its Home Screen. For this course, we will use the “Spec 200E Modern Interface” Since that is highlighted by default, just press Enter () to go there. After that, there will be several choices you will need to make, depending on how the machine will be used.

Modern Interface screen Here’s what the Modern Interface Screen looks like. From this, you will need to choose the option that is appropriate for the data to be gathered. Note that the default “Application” is Live Display.


Using the Spec 200 to Obtain a Beer’s Law Graph

Change to Scan For the Beer’s Law lab, there is one optional step that may be done first, if desired. (If you are not doing this optional step, skip down to the instructions for setting the Application mode to “Quant.”) Your protocol says to use a wavelength of 450 nm for riboflavin, but do you wonder where that number came from? It really wasn’t just pulled out of thin air. If you’d like to find that number for yourself, use the right (►) and/or left (◄) arrows to change from Live Display to Scan. The rest of what is displayed on the screen changes accordingly. If “Measurement Mode” does not say ABS (which stands for “Absorbance”), use the down (▼) arrow to go down to the Measurement Mode line, and the right (►) and/or left (◄) arrows to adjust it to ABS (for “absorbance”) — it should not say, “%T”.

Then, use the down (▼) arrow to cursor down to Low λ (remember “λ” stands for “wavelength”). The default setting for the lowest wavelength it will use is 400 nm. Use the wavelength (λ) knob and/or the right (►) and left (◄) arrows to adjust that to 350 nm.
Low wavelength 400 default
Before
Low wavelength reset to 350
After

Similarly, use the down (▼) arrow to cursor down to High λ. The default setting for the highest wavelength it will use is 900 nm. Use the wavelength (λ) knob and/or right (►) and left (◄) arrows to adjust that to 800 nm.
High wavelength 900 default
Before
High wavelength reset to 800
After

Click Next Then use the down (▼) arrow to cursor down to Next, and Enter () to go to the next screen.

the resulting display screen The next screen that will come up is an “empty” spectrum that looks like this.

arrow on cuvette insert cuvette The next step is to “blank” the machine. Find a pair of plastic cuvettes in one of the side compartments and because our samples are dissolved in water, put water in one of them. Only touch/hold the cuvettes by their top edges — fingerprints on the area through which the light travels will mess up the readings. If needed, cuvettes may gently be polished with a piece of lens paper. Notice that the top of one side of each cuvette has an arrow () on it. Always insert the cuvette with the arrow to the right (toward the light source).

cuvette is in cuvette holder Make sure the cuvette is all the way down and the arrow is on the right side (toward the light source).

pressing the zero button Then press the zero or blank (  0.00 ) button so the machine can auto-zero itself (also referred to as “blanking” it).

auto-zero message While the machine is performing its auto-zero, it will display an hourglass and a “please wait” message.

sample in cuvette insert into cuvette holder cuvette in chamber

When the machine is done with it calibration, remove the cuvette of water, and as was done with the water, place some of the riboflavin solution from the tube of your most concentrated solution into a second cuvette. Insert the cuvette into the cuvette holder, making sure the arrow points to the right. Make sure the cuvette is all the way in and close the lid. Then, press Enter () to determine at what wavelength riboflavin absorbs the most light.

riboflavin spectrum The Spec 200 will measure and display the spectrum for riboflavin, which should look similar to this. However, the green line that is the Cursor may be over at the far left (or anywhere else on your graph). Use the wavelength (λ) dial and/or right (►) and left (◄) arrows to move the green cursor line until it exactly matches with the top of the highest peak. Notice that, as the cursor is moved, the machine will display what wavelength (λ) it is on and the corresponding absorbance (ABS) for that wavelength. Notice that the highest light absorbance is at (or very close to) 450 nm. That is why the instructions in the protocol say to use 450 nm (hopefully, your data correspond to this). You may photograph this spectrum to include in your lab notebook (or if the new printer is working, print out a copy).

set up quant mode Use the up (▲) arrow to return to the “Application” screen. Change the application from “Scan” to “Quant.” A number of options will appear on the screen. “No. of STDs” means number of standards, and the machine will only allow a maximum of 4. If it does not already say “4,” cursor down to that line and change it to 4. If the “Measurement Mode” does not already say “ABS,” cursor down to that line and change it to ABS. If the “Measurement λ” does not already say, “450” (if you followed the previous instructions, it should “remember” whatever wavelength the green cursor line was on when you left “Scan”) cursor down to that line, and change it to 450 nm. The way we’re expressing concentration (milliliters of riboflavin solution added) doesn’t really correspond to the machine’s choices of “Unit,” so if it’s saying something like “C,” you can just leave it there. Don’t worry about the line that says, “USB Memory.” When everything is properly set, cursor down to “Go,” and press Enter () to continue to the next screen.

Next, the Spec 200 will ask you to insert a cuvette of “blank solution” (in this case, water) into the cuvette holder and press the Auto Zero button. Refer to the instructions, above, for proper insertion of the cuvette. When the machine is done with that, it will display a screen with spaces for the four standards to be tested. Use your plain water (again) as your first standard (0 riboflavin should absorb 0 light), as well as your 0.2 mL, 0.7 mL and 1.0 mL tubes for the other three standards.

For each standard, cursor up or down until you’re on that line. Use the right (►) and left (◄) arrows to set the concentration for that sample (to 0.0 or 0.2 or 0.7 or 1.0 as appropriate). Then, press Enter () to analyze that sample. The Spec 200 will put the absorbance reading into the last column. As you do each of your samples, write down the absorbance reading obtained for each specimen into your lab notebook. Then, cursor down to the next sample and repeat the process. In between samples, you may remove the cuvette, pour the sample that’ in the cuvette back into its test tube, then pour the next sample into the cuvette, insert it back into the sample chamber, close the lid, and take a reading. Do not rinse the cuvette with water in between samples as any remaining water droplets in the cuvette will dilute the sample that’s in the cuvette, thus causing the absorbance reading to be wrong.

Beer's Law graph When you have measured all four of those samples, the machine will give you the option to press Enter () to display the Beer’s Law curve. Note that it will calculate a best-fit straight line from your data, but the “object of the game,” here, is to be so good at pipetting that all of your data points fall exactly on the line, not a bit to one side or the other. Notice that the points on the graph in this photo do not all fall right on the line: some are above and some are below. How close to a straight line did you come? You may photograph (or print if available) your graph to include in your lab notebook.

To analyze your 0.1 and 0.4 mL samples, one at a time, place each of those into a cuvette and into the Spec 200. Note at the bottom of the display screen, it says “Measure Sample ←.” Press Enter () to analyze that sample. The machine will display its absorbance (please write that down in your lab notebook), and a concentration reading (as though that sample was an “unknown”) based on the best-fit line that it calculated (so unless you had phenomenal pipetting technique, that will not exactly match with 0.1 or 0.4 mL — but, hopefully, will come close). Determine the absorbance of those two samples by that means.


Using the Spec 200 to Obtain a Spectrum
(Photosynthesis Lab)

Since there are usually about six samples to be tested in this lab exercise, one way of doing this that has worked well in the past is for the class to divide up into six (or however many pigments there are) groups, and each group “adopt” a pigment and a spectrophotometer. That way, the spectrum from each pigment can be displayed on its own spectrophotometer, and all students can, then, go from machine to machine examining (and photographing) the spectra. (However, if/when we do get a printer and/or computer interface that is/are only hooked up to one Spec 200, that will change what works best.)
Due to the way our old Spec 20s worked, the lab protocol for the Photosynthesis Lab includes instructions to obtain absorbance readings for all pigments at 350 nm, then to change to 375 nm, re-blank, and test all pigments there, then 400 nm, etc., but with these new Spec 200s, all of that is no longer needed, and by following the instructions, below, it is actually quicker and easier to obtain the data needed to construct a spectrum for each pigment, one by one.
For the spectra of photosynthetic pigments, start by setting the Spec 200 to scan mode. That is the same mode described above, to find the absorption peak for riboflavin, so many of the directions here are identical to the directions given there.

Change to Scan As above, use the right (►) and/or left (◄) arrows to change from Live Display to Scan. The rest of what is displayed on the screen changes accordingly. If “Measurement Mode” does not say ABS (which stands for “Absorbance”), use the down (▼) arrow to go down to the Measurement Mode line, and the right (►) and/or left (◄) arrows to adjust it to ABS (it should not say, “%T”).

Then, use the down (▼) arrow to cursor down to Low λ. The default setting for the lowest wavelength it will use is 400 nm. Use the wavelength (λ) knob and/or the right (►) and left (◄) arrows to adjust that to 350 nm.
Low wavelength 400 default
Before
Low wavelength reset to 350
After

Similarly, use the down (▼) arrow to cursor down to High λ. The default setting for the highest wavelength it will use is 900 nm. Use the wavelength (λ) knob and/or right (►) and left (◄) arrows to adjust that to 800 nm.
High wavelength 900 default
Before
High wavelength reset to 800
After

Click Next Then use the down (▼) arrow to cursor down to Next, and Enter () to go to the next screen.

the resulting display screen Again, the next screen that will come up looks like this.

arrow on cuvette insert cuvette The next step is to “blank” the machine. Find a pair of plastic cuvettes in one of the side compartments and put ETHANOL (do you know why you should use that and not water?) in one of them. Only touch/hold the cuvettes by their top edges — fingerprints on the area through which the light travels will mess up the readings. If needed, cuvettes may gently be polished with a piece of lens paper. Notice that the top of one side of each cuvette has an arrow () on it. Always insert the cuvette with the arrow to the right.

cuvette is in cuvette holder Make sure the cuvette is all the way down and the arrow is on the right side.

pressing the zero button Then press the zero (  0.00 ) button so the machine can auto-zero itself.

auto-zero message While the machine is performing its auto-zero, it will display an hourglass and a “please wait” message.

Parsley extract Then it’s ready to analyze your samples, so put one in. For example, here is a sample of parsley extract, ready to be put into the sample chamber.

please wait As was done with the ethanol, pour some of the solution of the pigment you wish to study from the test tube into a second cuvette. Insert the cuvette into the cuvette holder, making sure the arrow points to the right. Make sure the cuvette is all the way in and close the lid. Then, press Enter () to display the absorption spectrum for that pigment. The machine will display the message, “Scanning...” with an hourglass.

spectrum of parsley Here is the resulting spectrum for the parsley extract used above. Note that the cursor is on 350 nm.

cursor moved to 400 The wavelength (λ) knob and/or the right (►) and left (◄) arrows may be used to move to another part of the spectrum. Here, the cursor is on 400 nm, and the machine says that, at that wavelength, the absorbance is 1.63.
Your protocol book instructs you to graph the data from the pigment(s) you tested. To do that, you will first need to make a chart of the numbers you will need to construct the graph. Start with a list, going down the page, of all the wavelengths to be used (350, 370, 400, 425 nm, etc.). Then another column will be needed (down the page) for each pigment tested. Use the wavelength (λ) knob and/or the left () and right () arrows to move the green cursor line over by 25-nm increments (350, 375, 400, 425 nm, etc.), up to 800 nm. At each of those wavelengths, students should copy the reported absorbance into their lab notebooks. The protocol book explains how to use those absorbance numbers to construct a graph. Your graph should look similar to the spectrum that is displayed on the Spec 200’s screen, with the big difference that, on your graph, you will know what the actual numbers are.

absorbance at 415 nm Here to the left, the cursor has been moved to what appears to be a peak at 415 nm, and the machine says that, at that wavelength, the absorbance is 2.01. This actually is a bit tricky because this is a parsley leaf extract that contains a mixture of pigments: chlorophylls A and B, carotenes, xanthophylls, etc., overlapping peaks so what is seen here is an additive spectrum of all those put together (similar to the diagram on the right). Thus, while 415 nm appears to be a peak, it may, in fact, be due to the additive effect of several pigments whose peaks are actually at slightly different wavelengths: while their peaks are at other places, the “sides” of those peaks may add up to give an apparent peak here.

cursor on peak at 665 nm Here, the cursor has been moved to the other peak at 665 nm. The machine is saying that, at that wavelength, the absorption is 1.13. Use the wavelength (λ) knob and/or the left () and right () arrows to move the green cursor line to the highest point on each of the absorbance peaks (hint: watch for the highest “ABS” as the green cursor line is moved back and forth). Make note of each of those wavelengths and absorbance readings in your lab notebook. Those numbers may then be compared with published max/min numbers.

cursor at 525 nm This method can also be used to determine areas of minimal light absorbance. For example, here, the cursor has been moved to 525 nm where there appears to be a minimum. The machine is reporting that the absorbance is only 0.02 at that point. However, it’s difficult to tell whether the cursor is at the absolute lowest point. One way to determine that is by slowly moving the cursor back and forth while watching for the lowest absorbance (ABS) reading.

zoomed in around 525 nm The Spec 200 has another feature that can help in this situation. Notice in the photo, above, at the bottom of the screen, it says to push the down (▼) arrow to zoom to a higher magnification. Here, with the cursor on 525 nm, the down (▼) arrow was pressed, with the result that the machine is displaying only the section from 400 to 550 nm. The disadvantage to using zoom is that there is no “un-zoom” feature, and the only way to get out of zoom view is to press the up (▲) arrow to go all the way back to the Scan menu, then ask the machine to regenerate the spectrum.

change to Live Display The Spec 200 also gives us a means of “seeing” what color of light corresponds to a given wavelength. Here, with the cursor left on 525 nm (or whatever wavelength you wish to examine), the up (▲) arrow was pressed to go back to the Scan Menu, and back from that to the Application Menu. There, “Scan” may be changed to “Live Display,” as shown here.

press GO Then, use the down (▼) arrow key to cursor to where it says, “GO” and press the Enter () key to do it.

Live Display of 525 nm Here is the resulting display for 525 nm. It’s still saying that the absorbance at 525 nm is 0.20. Notice the “rainbow” (spectrum) across the bottom of the screen, and especially notice where the arrow (▼) is pointing. The color of the spectrum at that point indicates (as best as possible on an LCD screen) the color of light which corresponds to that wavelength: 525 nm is green light, which plants do not absorb (which is why they look green to us).

back to Application menu You can also check the color that corresponds to other areas of the spectrum, especially including the peaks. If you have written down the wavelength(s) of the peak(s), you can use the wavelength (λ) dial and/or the right (►) and left (◄) arrow keys to go to that wavelength. If you do not know or remember the wavelength whose color you wish to determine, you will need to go back to the Application Menu, back into Scan, re-scan the pigment (re-do the spectrum), move the cursor over the desired wavelength, and then go back to Live Display.


428 nm
665 is red
665 nm

Here are the displays (for the same parsley solution) for 428 nm (which your lecture textbook says is the absorption max for Chlorophyll A) and 665 nm (the location of the smaller peak. According to this display, 428 nm corresponds to blue-violet, and 665 nm corresponds to red (your lecture textbook probably mentioned that, too). This way, you’ve seen it for yourself, rather than just reading about it.
Again, because we currently do not have printers available, it is, thus, not possible (yet) to print out a copy of the spectrum graph as displayed by the machine. Thus, students who have a camera or cell phone with built-in camera should be encouraged to photograph the screen showing the spectrum for the pigment they tested (hint: if six groups of students each test one of the pigments on a different spectrophotometer, it would be very easy for everyone to go around to all the spectrophotometers and take a picture of the spectrum for each of the six pigments, so everyone could have the whole set), and include a print-out of it/all of them in their lab notebook. However, that is not a substitute in place of learning the proper method for construction of a graph, so a properly-done, hand-drawn graph should be constructed.

If a second (or more) pigment is to be tested, just remove the first cuvette of pigment and insert the new cuvette/pigment to be tested, then press the Enter () button to generate a spectrum for the new pigment. As above, these absorbance readings can be added as another column on the chart/table students have created in their lab notebooks and subsequently used to create a graph for that pigment.

Depending on how your chromatography turned out, your class should have test tubes of the redissolved, individual pigments: carotene, xanthophyll “1,” xanthophyll “2,” Chlorophyll A, Chlorophyll B, and the “center” spot that did not move. Obtain an absorption spectrum for each of those pigments and any others which your class may be testing (and photograph for inclusion in your lab notebook or print out if printer is available). In your lab notebook, record the wavelength (nm) of the absorption maxima (tips of peaks) and minima (bottoms of valleys) for each of the pigments. Be very careful in your observations — for example, Chlorophyll A will probably show a peak around 425 to 428 nm, and that is, actually, significantly different from carotene’s peak at around 450 nm.


Things to Include in Your Notebook

Refer to the lists at the end Beer’s Law and Photosynthetic Pigments lab Web pages for lists of items that should be included for each of those labs.


Copyright © 2015 by J. Stein Carter. All rights reserved.
Chickadee photograph Copyright © by David B. Fankhauser
This page has been accessed Counter times since 17 Aug 2015.