3. You receive a mixture of two dyes in lab with directions to determine the quantity of each dye in the solution. You receive standard curves for all four dyes and the absorbance spectrum at the right which shows the maximum absorbance for all four dyes.

a. Based on this spectrum, determine which of the following two-dye combinations would provide the most accurate data for the dye concentrations if the given analytical wavelengths were used for the analyses. Remember that the analytical wavelength should be sensitive to the dye molecule (show a high absorbance) but also NOT be sensitive to the presence of other dye molecules which absorb at the analytical wavelength. PLEASE explain how you got your answer and why!

o Red at 520 nm and yellow at 430 nm

o Blue at 630 nm and green at 630 nm

o Red at 520 nm and green at 630 nm

http://www.vernier.com/innovate/innovativeuse53.html (this is the url to the picture used)

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5.3. For another research project, you have labeled a protein with two different dyes made by ATTO-TEC GmbH, a German company that makes some very nice dyes as alternatives to the Alexafluor series that was discussed in class. For this experiment, you have used the ATTO-520 and ATTO-633 dyes.

You have completed the labeling reaction, purified the doubly-labeled protein so that there is not âfreeâ dye in the solution (i.e. all dye is covalently attached to the protein), and obtained the absorbance data below. From these data you can calculate the concentration of each dye in the solution and the concentration of the protein itself (e_280nm = 72,560 M^-1 cm^-1). Using those numbers, you now need to calculate the labeling efficiency from your reaction, i.e. what fraction of the protein molecules have been modified by each of the two dyes.

This is, in essence, a straightforward Beerâs Law absorbance vs concentration question, but there is an added complication that the two dyes have some absorbance at 280 nm in addition to their main absorbance peaks in the visible region of the spectrum. You will therefore need to find both the extinction coefficients of the dyes, which you can use to calculate their concentrations, and the Correction Factor you need to account for how much dye absorbance there is at 280 nm. In effect, the observed absorbance at 280 nm is the sum of the absorbance from the protein and both of the dyes, and you need to find the protein only absorbance before you can calculate the real protein concentration.

To solve this you need to check out the ATTO-Tec web site at www.atto-tec.com where the Support section site has a document called Data-Table that will be extremely useful for answering the question.

Wavelength Absorbance

280 nm 1.31

517 nm 1.32

630 nm 2.21

Data Analysis for Part A. Use your table of wavelength and colorobserved to answer the follwoing:

1. Over what wavelength rage do you observe Red? Yellow? BlueSummarize these ranges below your table.

Data Anaylsis for Part B.By referring to your table, answer thequestions below:

3a. Examine a set of solutions having the same concentration.Does the wavelength absorbed max change with the anion, the cationor both? what do you conclude is causing the absorbance?

3b. Examine pairs of solutions having the same compound butdifferent amounts of compound in the solution(differentconcentrations). Does the absorbance intensity change withconcentration? Write a sentencesating this and suggest areason.

4.Now consider the relationship between the color of thesolution and the wavelength, (plus and minus 5nm) max, assoicatedwith the greatest absorbance by nswering the followingquestions:

a. by referring to part A, write down the color thatcorrresponds to the wavelength max of the cobalt solution. Comparethis color with the observed color of the cobalt solution. Arethese colors the same or different? Write a sentence summarizingthis and suggest an explanation.( You may find it helpful to reviewthe concept o the colors of light transmitted vs the colors oflight absorbed, which is easily visulaized by drawing out theprimary color wheel).

b. Repeat this analysis for the nickel solutions and write asentence summarizing your findings and suggest anexplanation.

Data Anaylsis for Part C.

5.why do sodium chloride and sodium nitrate produce the samecolor flame?

6.Report they identity of your unknown and your evidence forthis determination.

7.Consider the identity the emission spectra of the cobalt andnickel salts. Write down the approximate wavelength correspondingto the observed colors. Compare these values with those youobtained in Part B.4. Are they the same or different? Can youexplain your observation?

Part A: The relationshiop between color and wavelength Part C. CompoundIon flamecolor

Nm Color LithiumChloride hotpink

700 red CopperIIchloride lightblue

680 redorange Unknown#1 purpleorange

660 orange SodiumNitrate Orange

640 orange StrontiumNItrate Orange-red

620 orangeyellow NickelChloride brightorange

600 lightorange potassiumsulfate orange

580 yellow sodiumchloride brightorange

560 yellow cesiumchloride pinkishorange

540 yellow bariumNItrate lightgreen

520 yellowgreen calciumnitrate red-orange