Clinical Chemistry (CHE 221) Experiment # 3 Analysis of rare body fluids by UV-Vis Absorption Spectroscopy

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1 Clinical Chemistry (CHE 221) Experiment # 3 Analysis of rare body fluids by UV-Vis Absorption Spectroscopy Name Date Partners Name(s) Partners Name(s)

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3 Experiment title: Analysis of rare body fluids by UV-Vis Absorption Spectroscopy Reading assignment: Tietz 6 th Edition pages or Bishop 6 th edition pages and the introduction and protocols below Pre-lab questions 1) If a sample has a measured absorbance value of 3.3 why is it necessary to dilute it and measure the absorbance of the diluted solution before attempting to calculate the samples concentration? 2) What is stray light? 3) Does stray light cause more error in dilute or concentrated solutions

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5 Introduction Your instructor has been fortunate enough to obtain body fluid samples from several rare and reclusive beasts: Yeti urine, Chupacabra bile, Big Foot blood, Sasquatch seminal fluid, Boogeyman sweat, and Unicorn spinal fluid. These rare beasts each have a strange colored hormone that is not known in any other organism; they have been tentatively named Hormone Yellow, Hormone Green, Hormone Red, Hormone Blue, Hormone Indigo, and Hormone Purple, respectively. Due to their color each of these hormones can be directly detected by UV-Vis Absorption Spectroscopy. You will be provided with standard samples for each hormone at a concentration of 100 mm. You will also be provided with patient samples from actual Yetis, Chupacabras, Big Foots, Sasquatches, Boogeymen, and Unicorns. For each of the patient samples you will determine the hormone level by comparing its absorbance to that of the standard samples. You will do this for each patient sample by two methods: a graphical method involving a calibration curve and a mathematical method based on Beer s Law. Protocol(s) Part 1. Determination of the most sensitive wavelength for hormone detection by absorbance At lower concentrations samples tend to have lower absorbance values. Detection of the lowest levels of a substance by absorbance is best enhanced by selecting a wavelength for analysis where the substance has the largest value for the Molar Absorptivity ( ). Measurement of absorbance at this wavelength will produce the largest absorbance values for any given sample making detection of lowest possible concentrations possible. Before performing any analysis using UV-Vis Absorption Spectroscopy on a new type of sample the first step in developing a protocol for routine sample analysis is to collect the absorption spectrum. This is done in order to determine the wavelength that the absorbing substance absorbs the most strongly (has the largest value of ) which is the best wavelength to use for routine analysis. For your hormone first collect the complete absorption spectrum from 400 nm to 700 nm of the standard sample provided on the Thermo Scientific Evolution 60 using a cuvette with water as the reference solvent. If the maximum absorbance in this range is larger than 1.0 perform a dilution to adjust the concentration so that the largest absorbance is between 0.10 and 1.0. Save the data of this spectrum in a csv format file, export it to Excel, and prepare a graph of versus wavelength for your hormone. From this data record the best wavelength to measure absorbance for detection of low levels of your assigned hormone below as well as the value of at that wavelength. This information should also be placed on the graph near the highest value of.

6 Hormone analyzed Most sensitive wavelength for detection of low levels of hormone nm = M -1 cm -1 at nm (wavelength determined above) Part 2. Creation of a Beer s law calibration curve After determining the best wavelength at which to analyze a substance by absorption the next step in developing a protocol is to create a Beer s Law Calibration Curve. This is a graph constructed using the known concentrations and measured absorbance values of standard solutions. A standard curve allows the concentration of unknown samples to be determined by measuring their absorbance. In this part of the experiment you will collect all the data to construct a calibration curve for your hormone. Using a Spectronic 20, Spectronic 22, or a Junior 35 measure the absorbance at the wavelength determined in Part 1 for the 100 mm standard sample, and solutions of it diluted to concentrations of 75, 50, 25, 10, 1.0 mm with water. Record this data in Table 1. Prepare a Beer s Law Calibration Curve in Excel by graphing Absorbance versus Concentration for your hormone using the data in Table 1. Part 3. Collection of absorbance data of patient samples Using the Spectronic 20, Spectronic 22, or a Junior 35 measure the absorbance for all the patient samples at the wavelength you used to create the calibration curve. Record this data twice, in both Tables 2 and 3. If the absorbance is larger than 1.0 for any of the patient samples record the measured absorbance in both tables. For samples with absorbance values larger than 1.0 use Beer s Law to estimate how much the sample should be diluted so that its absorbance will be between 0.10 and 1.0. Perform this dilution and enter the value for the dilution factor in Tables 2 and 3. Measure the absorbance of the diluted sample(s) and record it in both Tables 2 and 3. Part 4. Analysis of patient samples Use your calibration curve to determine the concentration of the hormone in each of the patients for which you analyzed samples. If a patient sample had an absorbance value higher than 1.0 when it was initially measured use the calibration curve to determine the concentration of the patient sample based on the initial absorbance value. Also use the calibration curve to determine the concentration of the patient sample based on the absorbance of the diluted sample. For the diluted

7 sample you must take account of the dilution that you performed to determine the concentration of the original patient sample. Record all this data in Table 2. Examination of a Beer s Law calibration curve shows limits of the instruments ability to measure absorbance. Typically the curve will appear to start at absorbance = 0 at concentration = 0 and follow a straight line up to absorbance values of at least 2.0. At higher absorbance values the data usually falls below this line. This is the region where it fails to obey Beer s Law. In the region where Beer s law is obeyed concentrations could be determined either graphically or by solving Beer s Law mathematically. Outside the Beer s Law region the curve can be used to determine concentrations as long as it can be read precisely. As the curve becomes almost horizontal (parallel to the x axis) it cannot be read precisely and will give poor estimates of concentration from absorbance data. Use Beer s law to calculate the hormone concentrations for each of your patients mathematically from each of the measured absorbance values. Use the value of the molar absorptivity at the wavelength that the absorbance values were measured, which you determined when you created the graph of versus wavelength in Part 1 for your hormone. If a patient sample had an absorbance value higher than 1.0 when it was initially measured calculate the concentration of the patient sample based on the initial absorbance value and also calculate the concentration of the patient sample based on the absorbance of this sample after it was diluted. For the diluted sample you must take account of the dilution that you performed to calculate the concentration of the original patient sample. Record all this data in Table 3. c A b

8 Results Table 1. Absorbance and concentration data of hormone standard. Concentration (mm) Absorbance Table 2. Absorbance data and concentration values determined graphically from the Beer s Law calibration curve. Data for Undiluted Patient Samples Data for Diluted Patient Samples Patient Absorbance Concentration of Patient Sample Dilution factor Absorbance Concentration of Patient Sample When determining the concentration of patient samples from absorbance of diluted solutions use the absorbance of the diluted sample and the dilution factor to calculate the concentration of the original patient sample. The concentration of the sample after you diluted is not relevant patient information. The data for diluted samples is only recorded and calculated if the sample had an absorbance value larger than 1.0.

9 Table 3. Absorbance data and concentration values determined by calculation assuming Beer s law is obeyed (c=a/ b). Data for Undiluted Patient Samples Data for Diluted Patient Samples Patient Absorbance Concentration of Patient Sample Dilution factor Absorbance Concentration of Patient Sample When calculating the concentration of patient samples from absorbance of diluted solutions use the absorbance of the diluted sample and the dilution factor to calculate the concentration of the original patient sample. The concentration of the sample after you diluted is not relevant patient information. The data for diluted samples is only recorded and calculated if the sample had an absorbance value larger than 1.0.

10 Post-lab questions 1) For the patient samples that had absorbance values in the range 0.10 to 1.0 do the two values obtained graphically and by calculating (c=a/ b), agree well with each other? Answer yes or no, and explain why you think this is the case. Support your answer by calculating the percent error for one sample assuming the graphical method is accurate. 2) For the patient samples that had absorbance values larger than 1.0 before dilution you have determined the concentration four times (before and after diluting each using the graphical method and the Beer s Law calculation method). Do the four values obtained agree well with each other? Answer yes or no and explain why you think this is the case. Support your answer by calculating the percent error for a few of the samples assuming the graphical method applied to the low absorbance sample is accurate.