SOLUTIONS, OPTICAL ABSORPTION, AND LEAST SQUARES ANALYSIS OBJECTIVE At the end of this lab, you should be able to prepare accurate solutions and serial dilutions for experiments, and measure absorbance of a substance in a spectrophotometer.

LAB 3: SOLUTIONS, OPTICAL ABSORPTION, AND LEAST SQUARES ANALYSIS OBJECTIVE At the end of this lab, you should be able to prepare accurate solutions and serial dilutions for experiments, and measure absorbance of a substance in a spectrophotometer. You should also understand the spectroscopic quantitation of biological substances, linear least squares analysis, and computer-assisted data analysis using Excel to graph a standard curve for an assay. INTRODUCTION The absorbance of a substance is measured in an instrument known as a spectrophotometer. This method is one of the most prominent means of measuring the amount of a biological substance or the enzymatic activity responsible for converting substances. The relative concentration of a pure substance in a cuvette having a width and length of 1 cm is directly proportional to its absorbance of a discrete wavelength of energy. This is defined by an equation: [C] = Abs/mE where [C] is concentration of a substance C Abs is absorbance of a solution of C mE is the molar extinction constant for C Therefore, to determine the concentration of a substance in a biological fluid, measure the absorption of the solution and calculate its concentration using the molar extinction coefficient. A definition of the relationship between the absorbance of a dissolved substance concentration and its molar extinction coefficient can be found in “Appendix B” posted on Canvas. Monochromatic light is made by using filters to restrict the full light spectrum to a narrow range of wavelengths that can be specifically set on the instrument. For example, blue light covers a wavelength range from approximately 400 to 500 nm. Setting the spectrophotometer, or spec, to 415 nm filters the full light spectrum to measure the absorbance of blue light by the substance at that specific wavelength. There are two criteria that must be met for a spectroscopic quantitation to be accurate: 1. The absorbance of the solution at the defined wavelength must be dependent upon the substance in question. If other substances in the solution interfere with the absorbance (background), the accuracy will be diminished (note that most solutions will absorb energy). If this “non-specific” absorbance can be accurately determined, you can subtract it from the total absorbance of the solution to yield a net absorbance that is more accurate for the substance in question. 2. The pathlength of the radiation energy, usually emitted by a light source such as a lamp, must either be factored into the molar extinction coefficient or it must be restricted. In most circumstances, the cuvette holding the solution has a 1 cm width and a 1 cm length; therefore, the pathlength is one. OVERVIEW OF THE METHOD The most common procedure used to determine the concentration of a substance using spectrometric measurements is as follows: 1. First, determine the wavelength that will show the greatest absorbance by the desired substance to be measured and the least absorbance by other undesirable substances that may be present in the biological solution (called background). 2. Create a known standard for the substance. Determine the relationship between absorbance and the molar concentration of the substance by preparing a range of known concentrations of the pure substance and measuring their absorbances. 3. The absorbances of the standards are collected as data and graphed in a range of concentrations from high to low against a range of absorbances between 0.100 and 1.000 for a given spectral wavelength. This defines the concentration range of the substance that can be measured accurately. A least squares analysis of the data generated in step (2) must show a linear relationship between absorbance and the concentration of the standard solution. The standard graph can then be used to determine the concentration of an unknown solution of the same substance when its absorbance is measured. A blank consisting of the solution without the substance is used to “zero” the spectrophotometer. The purpose of the blank is to measure the absorbance of the solution without the substance. This value is subtracted from the solution to obtain an

accurate absorbance for the desired substance. In other words, when the instrument is calibrated to zero by a blank, any change in absorbance will be due to the presence of the substance. It is important to remember to use the correct solution for the blank: that is, a solution without the substance you want to measure. This could be the buffer you use to dissolve the substance, the buffer plus an acid or base, an organic solvent, or sometimes plain water, as appropriate to your experiment. There is a bottle of Blank Solution premade for these class exercises. ABOUT P-NITROPHENOL P-nitrophenol is a chemical that is yellow in color at basic pH—this characteristic can be exploited as a reporter of enzyme activity. The reason it looks yellow is that the chemical absorbs the violet wavelength of the visible light spectrum, which is in the 400 nm range. The intensity of yellow can be quantitated (absorbance measurement at 415 nm) and correlated directly to the amount of end products resulting from action of the enzyme on a substrate. This type of assay has many real-life, useful applications in research and practical science, including, as an example, tests for soil enzyme activity that indicate levels of healthy microbial growth associated with nutrient cycling in areas being evaluated for crop production. This is particularly important to produce enough food for the peoples living in third-world countries. (Reference: Louis V. Verchot, Teresa Borelli, 2005. Application of para-nitrophenol (pNP) enzyme assays in degraded tropical soils, Soil Biology and Biochemistry 37, 00. 625-633). http://www.worldagroforestry.org/downloads/publications/PDFs/ja05031.pdf SAFETY NOTICE P-nitrophenol is sometimes used as a precursor chemical in manufacturing pesticides; it is toxic and can be absorbed through the skin. A public safety announcement with detailed information is posted in the classroom. The pH is maintained by the addition of sodium hydroxide (NaOH), which is corrosive. These chemicals can be used safely, if handled properly, by wearing gloves, safety glasses and lab coat for personal protection. All liquid waste containing p-nitrophenol must be disposed of into the labeled bottles. You may be working with glass test tubes. Glass tubes are fragile and easily broken under pressure in your hand. Handle glass with care. Report broken glass found anywhere to your teacher. EXERCISE 1—SERIAL DILUTIONS Serial dilutions are normally made in a set of tubes containing different concentrations of a stock solution going from stronger to weaker in a graduated series. They start with a concentrated stock (1:1), which is progressively diluted to produce a series of geometrically weaker dilutions of the original stock. The dilutions are usually made by a preselected factor, for example 2, 5, or 10. In the following example, the dilutions are made by a factor of 10. EXAMPLE: Figure 1. Serial Dilution Scheme To dilute a concentrated solution by a factor of 10, each successive tube in the series is one-tenth the concentration of the one before it. To make these tubes, first pipet 9 mL water into each tube, and pipet 1 mL of the previous dilution for a total 10 mL final volume per tube. Each tube is one-tenth the concentration of the one before it, as well as being a fraction of the concentration of the original stock solution.

Dilutions are often made more quickly by directly pipetting a small fraction of stock into the final volume of water. For example, a 1/100 dilution of any stock can be made using 1 mL of a one-tenth dilution plus 9 mL water, as above, OR by adding 0.1 mL concentrated stock C to 9.9 mL water. However, for dilutions greater than 10−2 or 10−3, serial dilutions by a factor of 10 are much more accurate. In real life applications, serial dilutions are commonly used in microbiological, medical, or pharmacological applications, for example, in testing water for coliform bacterial contamination or clinical blood chemistry analysis. MATERIALS • 1 mL of a 10 mM bromophenol blue stock solution • DI water (DI = deionized) • 1 mL pipettes (micropipette) • 10 mL pipettes (serological pipette) • 5 large test tubes PROCEDURE 1. Calculate the volumes needed to make 5 tubes of serial dilutions of the stock using a dilution factor of 10 (a 1:10 dilution) and a final volume of 10 mL. Enter the numbers into Table 1. 2. Complete the blank cells in Table 1. The first few have been done for you as an example. 3. Make the dilutions in test tubes as shown in Figure 1 on the previous page. Mix well by pipetting up and down several times until the color appears uniform, indicating that the solution has equilibrated, before transferring 1 mL to the next tube. 4. As the concentrated stock is diluted, the change in color is a graphic demonstration indicating the degree of dilution of a substance in solution. Note the color in the Table 1. 5. This table should be added to your Lab Notebook. Table 1. Serial Dilutions by a Factor of 10 Dilution factor: 10 Dilution Ratio Bromophenol Blue Concentration (M) Bromophenol Blue Concentration (mM) Volume (mL) Volume H2O (mL) Qualitative Data: Color of Solution 1:1 (undiluted) 0.01 M 10 mM NA NA 1:10 0.001 M 1 mM 1 mL of 1:1 9 mL 1:100 1:1000 1:10,000 1:100,000 EXERCISE 2—GRAPHING A STANDARD CURVE In this exercise, you will measure the absorbance of several dilutions of a standard solution and determine the range to use for your standard graph. In research, where the absorbance of a biological sample lies within the linear portion of the standard curve, the concentration of the substance can be calculated using the equation y = mx + b, where y equals the absorbance, m equals the slope of the linear correlation, x equals the concentration of the standard and b equals the y intercept (in ideal solutions, b should equal 0, a rare occurrence). Even though it is called a standard “curve,” the graph should be a straight line. For any assay that involves either the disappearance of a colored substrate or the formation of a colored product, the relationship between the intensity of the color as measured on a spectrophotometer and the actual concentration of the colored substance must be determined. This information is graphed as a standard curve. The absorbance of known concentrations of the colored substance is measured under the same conditions employed in the actual enzyme assay. Several concentrations of p-nitrophenol in a range must be measured for this determination to accurately indicate concentration of the experimental samples. The absorbance measurements for these known samples should be in the range of 0.100 to 1.000 to stay within the accurate limits of the instrument and minimize the error inherent in only one or two determinations. The linear plot of absorbance (or optical density) versus p-nitrophenol concentration is called a standard curve, but if it is indeed a “curve,” something has gone wrong (it should be a straight line).

MATERIALS AND EQUIPMENT • 10 mL 50 mM sodium citrate buffer, pH 4.8 • 1 mM p-nitrophenol stock solution • 15 mL 0.1 N NaOH • Deionized water • 5 microfuge tubes • 5 assay test tubes, 13 × 100 mm • 2 cuvets • Spectrophotometer PROCEDURE 1. For this experiment you will: calculate a series of dilutions of p-nitrophenol (p-np) to use as standards of known concentration, use sodium citrate buffer to dilute the 1 mm p-np stock solution, and enter the volumes in Table 2 on the following page. Your instructor should verify the calculations for the class before you begin making them. Use the formula CiVi = CfVf to calculate the volume of stocks to use for each concentration in Table 2. See Appendix F on Canvas for a more detailed explanation of the formula. EXAMPLE: Make 1 mL of p-NP dilution with a final concentration of 0.7 mM, using 1 mM p-NP stock and 50 mM sodium citrate buffer as a diluent. Ci is 1 mM p-NP, Vi is the volume to add, Cf is the final concentration desired of 0.7 mM, and Vf is the final volume desired of 1 mL. Vi = (Cf  × Vf) / Ci = (0.7 mM × 1 mL) / 1 mM = 0.7 mL = 700 μL Table 2. p-Nitrophenol Standard Volume Calculations Final Concentration of p-NP (mM) 1 mM p-NP Stock Solution Volume (mL) 50 mM Sodium Citrate Buffer Volume (mL) Final Volume (mL) 0.05 1.0 0.1 1.0 0.2 1.0 0.3 1.0 0.4 1.0 1. Make the p-nitrophenol solutions needed for the experiment in 5 separate microfuge tubes, according to the volumes in Table 2. Make 1 mL each concentration: 0.05 mM, 0.1 mM, 0.2 mM, 0.3 mM, and 0.4 mM. NOTE! YOUR INSTRUCTOR SHOULD VERIFY THE CALCULATIONS BEFORE YOU BEGIN MAKING SOLUTIONS. 2. Rinse two cuvettes well using DI water to remove traces of prior solutions. Collect rinse wash in a plastic beaker and dispose in the labeled waste carboy for p-nitrophenol waste (not the sink!) at the end of the lab activity. Assay tubes may also be reused when cleaned this way. 3. Set up 5 assay test tubes (not microfuge tubes) in a rack and label them. Add 1 mL of the standard buffer solution (50 mM citrate buffer, pH 4.8) to each of the 5 assay tubes. 4. To assay the samples in tubes 1–5, pipet 1 mL of 0.05, 0.1, 0.2, 0.3, and 0.4 mM p-nitrophenol, respectively. 5. Add 3 mL 0.1 N NaOH to each tube and mix well. Adding the NaOH will turn them various shades of yellow. These are your assay standards. 6. Set the wavelength on the spectrophotometer to 415 nm. 7. Pipet 1 mL blank solution into a cuvette, to use as the blank. Place the blank into the spectrophotometer to calibrate it to zero. 8. Pipet 1 mL of the 0.05 mM p-nitrophenol assay standard into a clean cuvette. 9. Measure the absorbance of the standard and record the data in Table 3. 10. Repeat steps 8 and 9 for the rest of the p-nitrophenol assay standards. 11. Clean and rinse all tubes and cuvettes when you are done for the next class to use. Wipe tables and put away lab tools. 12. Tables 2 and 3 should be added to your Lab Notebook.

Table 3. Absorbance Data for p-Nitrophenol Standards [p-Nitrophenol] Added, mM Absorbance (Aλ = 415 nm) [p-Nitrophenol] Final, millimoles* 0.05 0.1 0.2 0.3 0.4 *Concentration of p-nitrophenol after addition of NaOH NOTE! USING EXCEL OR A SHEET OF GRAPH PAPER, PLOT [P-NITROPHENOL] FINAL, MM VS. ABSORBANCE. THIS IS THE STANDARD CURVE FOR P-NITROPHENOL, WHICH YOU USE FOR THE FOLLOWING EXPERIMENTS. YOU SHOULD INCLUDE A COPY OF THIS FIGURE IN YOUR LAB NOTEBOOK. EXERCISE 3—DETERMINING THE CONCENTRATION OF UNKNOWN SOLUTIONS OVERVIEW In this activity, you will determine the concentrations of p-nitrophenol in unknown samples using the standard curve you made in Exercise 2. ADDITIONAL MATERIALS AND EQUIPMENT • 2 mL each of unknown solutions 1, 2, and 3 • Graph of your standard curve made in Exercise 2 PROCEDURE 1. Obtain unknowns 1, 2, and 3 from your instructor. Note that your instructor may elect to divide this work up amongst the lab groups so follow your instructor’s directions. 2. Rinse two cuvettes well using DI water to remove traces of prior solutions. Collect rinse wash in a plastic beaker and dispose in the labeled waste carboy for p-nitrophenol waste (not the sink!). Assay tubes may also be reused when cleaned by rinsing in this way. 3. Pipet 1 mL blank solution into one cuvette to use as a blank. 4. Pipet 1 mL of the standard buffer solution (50 mM citrate buffer, pH 4.8) into a clean assay tube. 5. Pipet 1 mL of unknown solution 1 into the assay tube. 6. Add 3 mL 0.1 N NaOH to develop the yellow color and mix well by pipetting (total volume 5 mL). 7. Pipet 1 mL of this solution into a clean cuvette. Measure the absorbance in the spectrophotometer and record the data in Table 2-4. PROCEDURE, CONTINUED 8. Repeat steps 4 through 8 for a duplicate reading of unknown 1. 9. Repeat this procedure to obtain duplicate readings for unknowns 2 and 3. 10. Use the equation from your standard curve to calculate the concentration of p-nitrophenol in the unknown solutions using the absorbance data you’ve collected. 11. Use a computer program such as Excel to determine mean and standard deviation of the classroom data. 12. Report all values to your instructor. 13. The data you collect for Table 4, as well as your determination of the unknown should be added to your lab notebook.

Table 4. Unknown Sample Data Duplicate Sample # Absorbance (415 nm) Concentration (mM) Unknown #1 1 2 Unknown #2 1 2 Unknown #3 1 2 WRAP UP, CLEAN UP, LAB NOTEBOOK FOR LAB 2 Your post lab activity will use the data you collected in lab today, so make sure you have the information needed. Table 5. Waste Disposal Lab Item Proper Disposal Site Microcentrifuge Tubes Empty contents into waste beaker. Empty tubes are disposed in waste flasks on bench. Glass Assay Tubes Empty contents into waste beaker. Empty tubes may be washed to reuse during lab. When lab is complete, place empty assay tubes in labeled bin. Cuvettes Empty contents into waste beaker. Rinse cuvette well with distilled water. Place cuvette to dry in the labelled “Cuvette Drying Rack” Waste Beakers Carefully dispose of liquid waste when the beaker is not yet overflowing in the liquid waste container. Wash beakers well and place to dry on the back bench. Please disinfect your workspace and put all instruments back in the correct location. Don’t forget to wash your hands before you leave. LAB NOTEBOOK: Report the results of all your experiments in scientific format, stating: Hypothesis -what do you predict will happen in these experiments, in an if/then statement(s) Purpose – what are you learning/accomplishing in these sets of experiments/ Materials and Methods – what did you use and how did you use it? Results – this is where you include your completed data tables, calculations, and graphs (these may be completed on the computer and pasted into your notebook if you prefer but must be completed by you) Discussion and Conclusion – This is where you can interpret your results and you can address any problems you encountered during the lab. Include all tables and figures in your lab notebook as noted in the handout and your instructor’s directions. *Please note that you will use your standard curves again so make sure your lab notebooks are detailed and complete! See you next week!

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