Spectrophotometry Materials

Transcription

1 Spectrophotometry Materials Item per Class per Bench Genesys 10UV Spectrophotometer ml test tubes box 7 Test tube racks 6 1 1% Albumin solution 25 ml/one flask 2 ml 0.7% Albumin solution (unknown conc.) 25 ml/one flask 1 ml Sharpies 6 1 Kim wipe boxes ml pipettes 36 6 DI Water Bottles 6 1 Pipette Pumps 6+ (1 ml pipettes) at least 1 Biuret reagents bottles 6 1 Disposable transfer pipettes (droppers) 6 1 0

2 Spectrophotometry Abstract: When you look at a green leaf, it appears green to you because of the leaf pigments ability to absorb red and blue/violet and transmit green visible light. In this lab you will learn about the spectrophotometer, an instrument used to detect light energy that is transmitted by various substances. The spectrophotometer is useful because the absorption and transmission of light energy, the basis for one of the most widely used procedures for determining the concentration of substances in a solution. You will learn to use the spectrophotometer, become familiar with its parts, and use this instrument to determine the concentration of an unknown substance through the construction of a standard curve. You will use this instrument several times during this laboratory course, so make sure that you become comfortable in using this instrument! This lab also includes the principles of the Scientific Method, a series of steps commonly used to make observations, formulate hypotheses, carry out experiments and interpret the data generated from these experiments. Genesys 10UV Spectrophotometer 1. Locate the following on the spectrophotometer. As you complete the lab, come back to this section and record its use: 1

3 2. Identify the following on the spectrophotometer at your bench. a. On/Off Switch b. Measure Blank button c. Set nm button d. Sample Holder Determine the concentration of an unknown substance by constructing a Standard Curve When light of specific wavelength (color) is passed through a solution, some of the light energy will be absorbed. The remaining light energy is detected and expressed on a meter as either percent transmittance (%T) or absorbance (A). The spectrophotometer can be utilized to determine the concentration of a solution using a set of standards of known concentration. A higher concentration means a larger amount of light will be absorbed. By graphing a standard curve using the absorbance you determine for each standard, the unknown concentration can be extrapolated. Follow these directions carefully. Do not rush, or you will end up having to do the entire experiment again! Based upon my understanding of the effects of concentration on absorbance, I hypothesize that tube with the _highest concentration will have the highest absorbance because a higher concentration will have more color and in turn absorbs more light. Furthermore, I hypothesize that the tube with the lowest concentration will have the lowest absorbance because a lower concentration will have less color and in turn absorb less light. SM: The independent variable in this experiment is concentration SM: The dependent variable in this experiment is absorbance 2

4 Turn on the spectrophotometer. 1. Turn on the spectrophotometer, allow it to initialize. Prepare your samples: 2. Obtain 7 test tubes. a. Label one of them U for unknown. b. Label one B for blank. c. Label the remaining tubes #1- #5. (Steps 3-9 pertain ONLY to tubes #1- #5) 3. Add 1 ml of distilled water to tubes #2- #5. 4. To tube #1, add 1 ml of standard protein solution (1% albumin). Record the concentration of the standard protein solution here: 0.01 mg/ml Check your understanding At this point, what is the difference between tube #1, and tubes #2- #5? Tube #1 has protein while tubes #2---#5 only have water. 5. To tube #2, add 1 ml of standard protein solution. Shake gently. 6. With a fresh pipette, remove 1 ml from tube #2 and add it to tube #3. Shake gently. 7. With a fresh pipette, remove 1 ml from tube #3 and add it to tube #4. Shake gently. 8. With a fresh pipette, remove 1 ml from tube #4 and add it to tube #5. Shake gently. 9. With a fresh pipette, remove 1 ml of fluid from tube #5 and discard the fluid. At this point, you should have approximately the same amount of fluid in each of tubes #1- #5. Check your understanding What happened to the concentration of protein in each subsequent tube as you proceeded from step 5 through step 8? (Check with your instructor if you are not sure.) The concentration is reducing in half each step. 10. Add 1 ml of unknown protein to tube U. 11. Add 1 ml of distilled water to tube B. Check your understanding What is the difference between tube B and all of the rest of the tubes? Why will you use this tube as your blank? Tube B is the only tube with no protein in it. 12. To ALL tubes that you have prepared, add 2 mls of Biuret reagent and allow the color to develop for 5 minutes. The blank as well. 3

5 Determine Peak Wavelength: Each molecule has a wavelength fingerprint, or peak wavelength. The peak wavelength (λmax) is the best wavelength to make measurements since it will yield the highest absorbance values and thus the highest sensitivity; high sensitivity means that lower concentrations of protein can be detected and measured accurately. This can be determined by measuring the absorbance of a substance over a range of wavelengths to create an absorbance spectrum. 1. Place the blank (tube 0) in the sample holder of the spectrophotometer. 2. Enter the lowest wavelength (420 nm). 3. The distilled water blank has no color and should not absorb any visible light. If the absorbance is not zero, re-click set blank to calibrate to zero on the absorbance scale. 4. Remove the blank and replace it with tube 1 (1% albumin). This is the sample you will use to determine the absorption spectrum of albumin. 5. After the meter has stabilized (5-10 sec) read the absorbance value and record the wavelength and absorbance value in Figure 1 below. 6. Remove tube 1 and adjust the filter to the next wavelength in Figure Put the blank back into the spectrophotometer and readjust for zero absorbance at the new wavelength. The spectrophotometer should be recalibrated with the blank often, especially when you change the wavelength. 8. Insert tube 1 and measure its absorbance at the new wavelength. Record the absorbance in Figure Complete Figure 1 for the other wavelengths by repeating steps 1-7 and measuring the absorbance of the contents of tube 1. Results: Wavelength 420 nm 460 nm 490 nm 530 nm 570 nm 610 nm 660 nm Figure 1. Absorption Spectrum for Albumin Absorbance Record the wavelength with the peak absorbance here: 530 nm 4

6 Use the spectrophotometer set at the peak wavelength to find the absorbance (A) for each sample (cuvette). 1. Place the blank sample in the diamond shaped sample holder again. 2. Once the blank is oriented correctly, close the lid of the sample holder and press the Zero button to set the blank to 0.00 Absorbance. Check your understanding If the blank is set to zero absorbance, what does this mean in terms of the percent of light that will be transmitted through the cuvette (%T)?100% T Is there anything in your blank cuvette that should attribute an absorbance value? No 3. Remove the blank test tube from the sample holder and determine the absorbance of each of your other test tube (one at a time) by: (there is no need to the blank again) a. removing fingerprints from the cuvette with a soft wipe b. inserting each test tube into the sample holder c. closing the lid and d. recording the reading from the digital display in Figure 2. Results: Serial Dilution Tube # mg/ml protein Absorbance B (blank) U (Unknown) Figure 2. Comparative concentrations and absorbances in known and unknown samples. 0? 5

7 Note: It is important to note that you must construct a fresh standard curve every time you are determining the concentration of unknowns. You should prepare the standards and your unknowns together. You cannot use a friend s standard curve and expect accurate results for your unknown! To construct your standard curve, you will plot the absorbance of each dilution on the Y- axis against the concentration of each dilution on the X- axis, as shown in Figure 3. You will draw a line of best fit using the data points you plot. By drawing a straight line from (a) to the curve at (b) and dropping from the curve to (C), the concentration of an unknown solution can be determined. Figure 3. Using a standard curve to determine the concentration of an unknown protein sample. 6

8 Absorbance Use the blank graph provided in Figure 4 to create your standard curve by plotting each of the absorbance values that you determined from tubes #1- #5. Make sure that you label both the X axis and the Y Figure 4. Standard curve correlating sample concentration to absorbance. Protein Concentration (mg/ml) Figure 2. Standard curve correlating sample concentration to absorbance. 7

9 Conclusions: What is the concentration of your unknown (include units!)? Points for Discussion: 1. Why are you not able to use a standard curve generated in one lab to assess the concentration of an unknown in another lab? List at least 2 reasons. The spectrophotometer needs to be calibrated every time it is used. Lab conditions may be variable. 2. How does the way you connect the data points affect your conclusions about the concentration of your unknown? If all of the data points lie in a straight line except for one, should you ignore the one point or shift the line to connect the points? The outlier may be ignored or retested to verify an error. 3. Would a hand generated line of best fit or a computer generated line yield the most accurate results? Why? Computer generated line may be more accurate because it can eliminate human error. 8