NUR 590 Assignment: Spectroscopy As A Tool In Biology

Read the lab file very carefully. Please be sure you answer every question in the lab in a very short answer. Including the direction listed on how it will be down below. Keep everything organized. And I have also attached the graphs in the file as well as the data.

Follow the grading rubric to know what exactly the lab will be graded on.

This is how it will be:

*Purpose & Background of the Lab: -( 1-2 paragraph)

*Procedures:

  • All procedures of the lab in your own words (use “we”) example: we did, we

*Results

Explain the results, use examples from the data set tables and the graphs. Make sure you mention the name of the part and table and number of the graph (1-2

Paragraph)

*Graphs:#1

-Explanation:

*Graph #2

-Explanation:

*Calculation and discussion of Unknown concentration

*Discussion:

-Answer all of the questions on part A question 8 here into a discussion do NOT number them.

* Conclusion

-Conclude why we did the experiment? what we did in the experiment and don’t forget to include the results. Use question 8 in part A to help you with part (provide answers of the questions)

Exercise 2

Spectroscopy as a Tool in Biology

INTRODUCTION

In the pre-lab you learned that a spectrophotometer can be used to determine which wavelengths of light are transmitted and which are absorbed by a colored solution. This can be used in a variety of ways in biology laboratories in the characterization of molecules. The extent of absorption at any wavelength is known as the absorbance. By measuring the absorbance of a solution at a variety of wavelengths, and plotting absorbance versus wavelength, the absorbance spectrum for the substance can be determined. The wavelength where a maximum in the absorbance value occurs is known as a peak. This wavelength is generally utilized to measure the maximum absorbance of the material

In this exercise there are two things you would have completed in the lab; you would first use a spectrophotometer to examine the absorption spectra of two different colored solutions, determining the wavelengths where maximum absorbance peaks occur in each of the spectra. One of the stock solutions will then be diluted with water and the absorption spectrum will again be measured to determine the effects of the dilution. 

Finally, the effects of mixing the two original different solutions (in effect diluting one with the other) on the absorption spectrum will be examined. You will be using this spectrophotometer on a regular basis throughout the semester, so it is important that you are completely familiar with its operation and are confident in your ability to use it in your experiments.

In the second part of the exercise you will take one of the stock solutions and make a series of dilutions to create a standard curve of concentration versus absorbance. This curve will then be used to estimate the concentration of an unknown solution of the same molecule.

Since you will not be able to actually do these things in the lab, you will read through all of the steps that would have been done, and you have been provided with a complete data set for each of the activities that you would have done. Normally all of this data would have been entered into your lab notebook.

Normally, each section of this exercise would be written up in your laboratory notebook. All graphs would be produced in Excel, and an original of each graph would be glued into the notebook on the first (white) page. Duplicate pages of the notebook would also have another copy of each graph attached, and all original white pages would be removed from the notebook and collected next week at the beginning of

class.

Since we are not working with a lab notebook in this online class, you will type up this lab and submit it instead of writing it in the lab notebook. Your report should include a Title, purpose, procedure, and the data that you have been provided in the separate document. Then you will follow the rest of the directions at the end of this document to complete the report.

Part A. Determination of the Absorption Spectrum of Red, Yellow, Diluted Red, and Orange Aqueous Solutions.

In this section you will determine the absorption of four different solutions, one red and one yellow, a diluted red, and an orange solution (made by mixing red and yellow) at 10 nm wavelength intervals between 350 and 540 nm.

  • You should have a stock bottle of a red, water-based solution, and a yellow, water-based solution.Make The Following Mixtures The Two Solutions,eachine clean, small beaker. Do not contaminate the stock bottle of thesolution!
  • Using a 10.0 ml pipette, add 5.0 ml of deionized water to the first beaker. Then use a clean pipette to add 5.0 ml of the red stock solution to the beaker containing the water.Mixthesolutionbygentlyswirlingthe Beaker until the color throughout is uniform. This will become solution D listed below.
  • Using a 10 mL pipette add 5.0 ml of the red stock solution to a second, clean beaker. Using a clean pipette, add 5.0 ml of the yellow stock solution to the same beaker. Mix the two solutions by gently swirling the beaker until the color throughout is uniform.
  • Fill 5 clean and dry Spec 20 round cuvettes with the following solutions (you can label each of the tubes as A,B,C,D,& E by placing a small piece of tape near the very top of each tube and writing your label on the tape.
  • Deionized water
  • Red stock solution
  • Yellow stock solution
  • Diluted resolution
  • Mixture of red and yellow solutions.
  • Set the first wavelength and then use the water tube to blank the Spec 20 following instructions and blanking the instrument from the pre-lab exercise.
  • Measure the absorbance of each of the solutions (b through e) at that wavelength, and record the data in a laboratory notebook.
  • Change the wavelength setting of the instrument by 10 nm and re-calibrate the spectrophotometer using the deionized water blank. Read the absorbance of each of the solutions at the new wavelength.
  • Repeat this procedure until all wavelengths have been
  • Create a graph in Excel that shows the absorbance spectra of all four solutions on a single graph. This graph should be part of your report. Be sure to add a descriptive title to the graph.
  • Each of the following questions should be answered as part of your discussion and conclusions in your report. (these questions are listed in a logical order to help lead you through a discussion of this exercise). These answers should be written as a logical discussion and conclusions, not numbered as individual questions. The questions are here to help guide you to the discussion and conclusion of the key points of the exercise.
  • Where do maximum absorbance peaks occur for the red and yellow solutions? Are the peaks at the same or different wavelengths?
  • Are the absorbance values of the peaks the same for each of the two solutions? What, if anything, does this tell you about the concentrations of the two solutions as related to each other?
  • How does the wavelength of the maximum absorbance peak of the diluted red solution compare to the original red stock solution? What Is the effect of dilution of the solution on the position of the peak?
  • What is the effect of diluting the red stock solution on the absorbance? Is there a mathematical relationship between the absorbance of the red stock solution and the diluted red stock solution? How does this relationship relate to the relationship of the concentration between the two solutions?
  • If equal volumes of the red diluted solution and water were mixed,predict the absorbance you would expect at the peak in the spectrum. (Give a mathematical example that shows how the numbers would work out). What would this tell you about the concentration of this further diluted solution?
  • Do the wavelengths where peaks occur for the orange solution match the peak positions in the spectra for the red and yellow solutions? (Be careful about this one! The peaks will not be as obvious here as they were for the original solutions).
  • Do the absorbance values at the peaks in the spectra for the orange solution match the absorbance values for the corresponding peaks in the spectra for the red and yellow solutions (remember, that red is diluting yellow and yellow is diluting red; therefore, at a specific wavelength, the absorbance will be composed of the diluted absorbance of red at that wavelength + the diluted absorbance of yellow at that wavelength).
  • Speculate on the relationship between the absorption spectrum for the orange solution, and the spectra for the red and yellow solutions (remember to take the previously described dilution effects into account). 
  • How do you account for the spectrum of the orange solution in light of your data for the red and yellow solutions? Give a mathematical example that shows how the spectra of the red and yellow solutions that are diluted by each other end up resulting in the spectra that you see for the orange solution.
  • How does all of this relate to Beer’s Law? How does this relate to the ability to use a spectrophotometer to calculate the concentration of a solution of unknown concentration?

Part B: Preparation Of A Standard Curve And Determination Of The Concentration Of An Unknown Solution.

In this section of the exercise you will be preparing a series of dilutions from the red stock solution that you will use to create a standard curve of the red solution of concentration vs absorbance.

  • Using 50 ml beakers, label the 5 beakers B through F. Then make a series of dilutions of the original red stock solution(A)asshowninthefollowingtable.For each dilution that is prepared, mix the beaker by swirling gently until the mixture is thoroughly mixed.
  • Label 6 spec20 cuvettes A through F, and one “blank”. Add 4 ml of DI water to the blank tube. Add 4 ml from each of the labeled beakers to the corresponding labeled spec 20tube.
  • Label one additional spec20 cuvette “unknown”. Add 4 ml from the bottle labeled “unknown” to this cuvette.
  • Determine the peak absorbance wavelength for the red solution from Part A of this exercise. Set your Spec20 to this wavelength and blank the spec using the DI water blank tube. ( Your data was determined at 520 nm wavelength).
  • Read the absorbance of each of the 6 diluted tubes.YOU HAVE BEEN GIVEN THE

RESULTS OF THESE ABSORBANCE READINGS ON THE DATA SHEET FOR THIS EXERCISE.

  • Graph the tubes A through F on a plot of dilution (X-axis) vs absorbance(Y-axis). Remember, the dilutions on the X-axis are not linear! So you need to make sure that you plot the points at the appropriate distances on that axis (hint: they will NOT be equidistant along the X axis!) 
  • The easiest way to plot this curve is in terms of percent concentrations. If sample A is 100%, then Sample B would be 50%, Sample C would be 25%, Sample D = 12.5%, Sample E = 6.25% and Sample F = 3.125%. These values would be plotted on the X axis, and the associated absorbances on the Y axis.

Then In Excel, set a best-fit line through the plotted points using the linear regression function. Label the equation of the line.

  • Determine the approximate % concentration of the unknown using the equation of the standard curve.
  • Discuss this process, the graph and discuss your results in your report.