Use of Laboratory Equipment and Supplies 3 When you have completed this exercise, you will be able to: 1. Use a balance. 2. Use pipettes and graduated cylinders to measure the volume of liquids. 3. Use a spectrophotometer to determine the absorbance of solutions at different wavelengths of light. 4. Prepare graphs of the absorbance spectrum for malachite green and a standard curve for the concentration of malachite green. 5. Use the absorbance spectra to determine the maximal absorbance wavelength of malachite green. 6. Use a standard curve to determine the concentration of an unknown solution of malachite green. 7. Prepare a lab report explaining the results of the exercise. Any laboratory is equipped with specific tools, equipment, and machinery. To do the exercises in this lab manual, you need to know how to use a balance, volumetric glassware, and a spectrophotometer. This exercise will allow you to become familiar with these tools. Volumetric Glassware Many of the analyses you will perform in this class use volumetric glassware that is calibrated to deliver accurate volumes of aqueous solutions. In this exercise you will use graduated cylinders and pipettes to deliver specific volumes of solutions. If you observe tap water in a clear glass, you will note that the surface water adheres to the side of the glass. This makes the surface of the water concave, not flat. This concave water surface, when viewed from the side through clear glass, is called a meniscus. The meniscus appears darker than the surrounding liquid. When you observe water or another fluid in a graduated cylinder or pipette, the meniscus line visible when holding it at eye level is what you use to determine volumes. Volumes have been graduated on the cylinders and pipettes to be used for this exercise. Before you use graduated glassware, it is wise to look carefully at the graduations so that you know exactly what the major and minor tick marks mean for each piece. Liquids must be drawn into pipettes using suction. Devices known as propipettes are used to safely suck liquids into pipettes. Even if you are pipetting water, never use your mouth to suck the liquid into a pipette. You never know what chemicals might be contaminating the pipette you are using, and you would not want to accidentally draw unknown chemicals into your mouth. Spectrophotometry A spectrophotometer measures light absorption at set wavelengths of light. This is a relatively simple piece of equipment, but it has many applications and you will use it to perform several exercises in this manual. Inside the machine is a lightbulb. It produces white light, which is composed of all colors of light. The light goes through a small slit in the machine and hits a diffraction grating, which works like a prism to separate the many colors of white light into individual colors. You select the wavelength or color of light. Only the set wavelength or color of light goes through a second slit and hits the test tube in the sample compartment of the spectrophotometer. Light is either absorbed by the sample or transmitted through a slit on the other side of the sample compartment. The light then goes to the photomultiplier tube. The photomultiplier measures the amount of light that hits it, much like a photographer s light meter. You can read the absorbance on the scale on the face of the spectrophotometer, which shows transmittance on the top and absorbance, reading left to right, on the bottom. You will use only the absorbance scale. Alternatively, your spectrophotometer may provide a digital readout. In this case, you will have to set the controls to display the absorbance. Absorbance reflects the amount of light absorbed by the sample in the sample compartment. If the light completely passes through a sample, the absorbance is zero. If the light is completely blocked by a sample, the absorbance is infinity. There are no units for absorbance. Transmittance is the mathematical inverse of the absorbance, and its units are percentages. If all the light passes through a sample, its transmittance is 100 percent. If the light is completely 13
blocked by a sample, the transmittance is 0 percent. It might seem as if the transmittance scale would be easiest to use, but in practice the absorbance scale is easier because light absorbance relates directly to the concentration of a lightabsorbing chemical in a solution. Therefore, the absorbance scale is used universally to determine the concentration of an unknown. On many spectrophotometers, the absorbance scale reads from right to left, further complicating matters. Take a moment to familiarize yourself with the absorbance scale on your machine so that you can read it correctly. Be sure to ask your instructor for help if you are not sure that you are reading the scale correctly. For research, specially designed sample holders or containers, called cuvettes, are used. Cuvettes are optically pure, and they do not absorb any light when used in the appropriate wavelength range. But because cuvettes are very expensive, you will use disposable test tubes, which are very inexpensive and work adequately for this and other exercises in this manual. Balances Almost every laboratory has a balance. All allow you to determine the mass of objects you place on the weighing pan. Quite often, the things you want to weigh should not be placed directly upon the weighing pan. Therefore, you must first weigh a container, then place the thing(s) to be weighed in the container, and then weigh the objects in the container. To determine the weight of the objects, subtract the weight of the container. Every balance is different, but here are a few general rules for their use: 1. Clean up all spills. 2. Leave the balance clean and dry. 3. Set the scale back to zero grams. This keeps the weights from distorting the balance beams. Your instructor will explain exactly how to use the balances in your laboratory. You will use them often while performing the exercises in this manual. Activity 1: Weight of Water For this exercise, you will weigh water in a beaker. Water weighs 1 gram (g) per milliliter (ml). You will be able to assess your pipetting skills by using the balance. Materials 5-ml serological pipettes 25-ml beakers Distilled water Propipettes Procedure 1. Use a balance to weigh a 25-ml beaker. Record its weight here: Weight of beaker 2. Using a propipette as demonstrated by your instructor, draw some water into a serological pipette. You will transfer 1 ml of this water to the weighed beaker with the pipette. Draw the water past the 1 ml mark and then carefully allow the water to drain from the pipette so that the meniscus is at a gradation that corresponds with a whole milliliter. 3. Transfer the tip of the pipette to the beaker and carefully drain the pipette so that the meniscus is aligned with the next whole-milliliter marking. 4. Practice transferring 1 ml at a time four more times until you have transferred 5 ml to the beaker. You should now feel comfortable using the serological pipette to accurately deliver water. 5. Weigh the beaker with the water in it. In the following spaces, record the weight of the beaker with water and then the weight of the beaker alone. Subtract to determine the weight of the water, and record your answer. Weight of beaker and water Weight of beaker = Weight of 5 ml water How close was the weight of the 5 ml of water to the 5 g weight you expected? Activity 2: Absorbance Spectrum of Malachite Green In this exercise you will use the spectrophotometer to determine the absorbance spectrum of malachite green. Note that the instructions given here are specific for the Spectronic 20 spectrophotometer. Other models will work similarly. Your instructor will advise you of differences in procedure that may be required if other spectrophotometers are used. Materials Six 13 100-mm disposable test tubes (per lab station) 5-ml serological pipettes Distilled water Propipettes 0.0005% malachite green dye Lab wipes One 13 100-mm disposable test tube with a 1 piece of chalk in it (per lab station) Procedure 1. With a lab marker, label six test tubes near their tops: B, 1, 2, 3, 4, and 5. Place them in a test tube rack in the same order. 2. Pipette water and 0.0005% malachite green dye into the tubes as shown in Table 3.1, using two 10-ml serological pipettes. 3. Mix the tubes by holding them near the tops and shaking gently. 4. Set the wavelength of light to 420 nm with the upper right-hand knob. Place the test tube with the piece of 14 Part 1 Basic Laboratory Skills
Table 3.1 Preparation and Determination of a Standard Curve Concentration of ml 0.0005% Malachite Green Malachite Tube (wt/vol) ml water Green Absorbance at nm B 0.0000% 5 0 1 0.0001% 4 1 2 0.0002% 3 2 3 0.0003% 2 3 4 0.0004% 1 4 5 0.0005% 0 5 Table 3.2 Malachite Green Light Absorbance Data Wavelength (nm) Color of Light Absorbance 420 440 460 480 500 520 540 560 580 600 620 640 660 680 chalk in it in the sample compartment. Peer into the compartment to discern the color of light shining on the chalk. You may have to move the tube up and down a bit; it helps to dim the room lights if possible. Note the color you see in Table 3.2. (Some models other than the Spectronic 20, will not allow you to do this part of the exercise. You should use a reference book to find out the colors of the different wavelengths of light.) 5. With the sample compartment empty and closed, set the light transmission to zero on the %T (transmission) scale, using the left-hand knob. There is no light hitting the photomultiplier tube so this effectively calibrates zero in the machine. 6. Using the lab wipes, wipe the fingerprints off tube B. Place tube B in the sample compartment. Be sure to close the sample compartment. Zero the spectrophotometer on the absorbance scale using the lower righthand knob. All the light that goes through the sample and hits the photomultiplier tube is not absorbed by the water. This zeros the absorbance for your experiment. Your malachite green solutions are in water, and you want to subtract the absorbance of water and only measure the absorbance of the malachite green. 7. Replace the B tube with tube 5 (after wiping off the fingerprints). Be sure to close the sample compartment. Determine the absorbance of malachite green at this wavelength and record it in Table 3.2. 8. Reset the wavelength to 440 nm and repeat steps 1 through 6 for all the wavelengths noted in Table 3.2. 9. As part of your lab report, prepare a graph of wavelength versus absorbance for malachite green. Draw or label the colors of light that correspond with the different wavelengths of light you set. Activity 3: Preparation of a Standard Curve A standard curve works on the principle that the absorbance of light at the wavelength of maximal absorbance is linearly related to the concentration of the solute. That is, as the concentration goes up, the absorbance goes up as well. Knowing this, we can plot a line graph of concentration Exercise 3 Use of Laboratory Equipment and Supplies 15
(x-axis) versus absorbance (y-axis). We can use it to determine the concentration of an unknown by first measuring the absorbance of that unknown. When you find that absorbance on the x-axis of the standard curve, follow across the graph to the standard curve line, and follow horizontally to the y-axis to find the concentration. You will use standard curves often in this class, but this is the only time you will prepare one yourself. Be sure you understand how they are used and prepared. Materials 6 test tubes prepared as in Activity 2 (per lab station) Lab wipes 1 test tube filled with 5 ml of unknown concentration of malachite green (per lab station) Procedure 1. Determine which wavelength of light provides the greatest absorption with malachite green by looking at Table 3.2. Set the spectrophotometer to that wavelength. 2. With the sample compartment empty and closed, set the light transmission to zero on the %T scale using the left-hand knob. 3. Place tube B in the sample compartment and zero the spectrophotometer on the absorbance scale using the lower right-hand knob. Be sure to close the sample compartment. 4. Replace the B tube with tube 1. Be sure to close the sample compartment. Determine the absorbance of 0.0001% malachite green at this wavelength and record it in Table 3.1. 5. Replace tube 1 with tube 2. Record the absorbance in Table 3.1. 6. Continue to determine the absorbance of tubes 3, 4, and 5, recording each absorbance in Table 3.1. 7. Determine the absorbance of a tube of malachite green of unknown concentration. Record your unknown s code letter and its absorbance here: Unknown letter Absorbance Lab Report Your lab report may be written in standard lab report format (see Appendix A) and should include two graphs and the concentration of your unknown sample of malachite green. Alternatively, your instructor may require that you use the lab report form provided at the end of this exercise. 16 Part 1 Basic Laboratory Skills
Name Lab Report 3 Use of Laboratory Equipment and Supplies 1. Prepare an absorbance spectrum graph for malachite green dye. Draw or label the colors of light that correspond with the different wavelengths you set. 2. Prepare a standard curve for your dilutions of malachite green dye. The graph should be concentration (%) on the horizontal (x) axis versus absorbance on the vertical (y) axis. This should be a line graph. Draw a straight line through zero and all your data points. If the line does not fit perfectly with every point, align it to provide the best fit for the most points. Exercise 3 Use of Laboratory Equipment and Supplies 17
3. Use your standard curve to determine the concentration of the malachite green in your unknown. Follow across from the absorbance you measured for the unknown to the line of your graph and at a right angle down to tell you the concentration of your unknown. What is the percentage concentration (wt/vol) of your unknown? 4. How does absorbance relate to concentration? Could you compare colors to estimate a concentration? Explain. 5. What did your 5 ml of water weigh? How close was that to what you expected? List probable sources of error. 18 Part 1 Basic Laboratory Skills