Part 1Since the UV-Vis method outlined in Question 1 above is not a standard method of analysis of the vitamins youwill have to compare your UV-Vis method to a standard HPLC or GC method. In your reading you found thatalthough vitamin B1 absorbs UV light at 240 nm, it is best converted to thiochrome by the scheme shown in Figure2 before analysis by HPLC with fluorescence detection, using an excitation and emission wavelengths of 360 nmand 450 nm, respectively.ононNH:K, Fe(CN,)NaOHFigure 2: Oxidation of vitamin B1, thiamine to thiochrome.?(a) Explain why fluorescence spectroscopy gives a more accurate determination of vitamin B1 thanabsorption spectroscopy.(b) Provide an annotated sketch of the excitation/emission spectrum as well as an annotated energy profilediagram of the vitamin B1 derivative, thiochrome. Part 2Since the HPLC is down, your company would have to outsource the fluorescence method outlined in Part 1above to one of its competitors which may be detrimental to your place of employment and your job in the future.Upon further investigation, you found a GC method for analysis of vitamin B1; and fortunately, the GC is fullyfunctional. Both of the 2Bs vitamins are highly water soluble. In order to quantify vitamin B1 in the supplement,you must first convert thiamine to the less polar derivative, 4-methyl-5-(2-hydroxyethyl)thiazole, shown in Figure3 below, with subsequent extraction and cleanup, before GC analysis. The procedure yields the derivative in highpurity (~90%), provided a suitable extraction and cleanup procedure is followed.H3CHOFigure 3: Structure of 4-methyl-5-(2-hydroxyethyl)thiazole(c) Describe a suitable extraction and cleanup procedure for the GC method described in Part 2 above.In your response include justifications for the extraction solvent chosen.(d) The derivative mixture was analyzed by GC on a 10m capillary column. The solvent eluted after 75seconds. The derivative and a second unknown peak eluted at 407 seconds and 420 seconds,respectively. The width of the peaks at half height for the derivative and unknown are 8 seconds and10 seconds, respectively.(i)Calculate:the column resolution.the number of theoretical plates required for a resolution of 1.5(ii)Without changing the GC column, explain how you can adjust your GC method to improveseparation of the two compounds in (d) above.

Since you have asked multiple questions, we will solve the first question/part 1 for you. If you…

Answered: (a) Explain why fluorescence…

Biochemistry

6th Edition

ISBN:9781305577206

Author:Reginald H. Garrett, Charles M. Grisham

Publisher:Reginald H. Garrett, Charles M. Grisham

Chapter19: The Tricarboxylic Acid Cycle

Section: Chapter Questions

Problem 5P: Understanding the Action of Fluoroacetate on the TCA Cycle In a tissue where the TCA cycle has been...

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Transcribed Image Text:Part 1 Since the UV-Vis method outlined in Question 1 above is not a standard method of analysis of the vitamins you will have to compare your UV-Vis method to a standard HPLC or GC method. In your reading you found that although vitamin B1 absorbs UV light at 240 nm, it is best converted to thiochrome by the scheme shown in Figure 2 before analysis by HPLC with fluorescence detection, using an excitation and emission wavelengths of 360 nm and 450 nm, respectively. он он NH: K, Fe(CN,) NaOH Figure 2: Oxidation of vitamin B1, thiamine to thiochrome.? (a) Explain why fluorescence spectroscopy gives a more accurate determination of vitamin B1 than absorption spectroscopy. (b) Provide an annotated sketch of the excitation/emission spectrum as well as an annotated energy profile diagram of the vitamin B1 derivative, thiochrome.