Study Ouestions 1. The cuvette for this exercise is made of quartz. Why cannot w
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Question
Study Ouestions 1. The cuvette for this exercise is made of quartz. Why cannot we use plastic cuvettes? 2. Give examples of ions that can quench fluorescence. fe1 Fe 3. Predict the type of error (positive or negative) if the determination of quinine is not performed in acidic solutions. Explain why 4. Quinine has two nitrogen atoms. Which of these is protonated by the mild acid? Rationalize your answer. 5. An interesting protein, commonly used in biochemical studies, is the green fluorescent protein (GFP). Show the basis of its fluorescent property.Explanation / Answer
1) Mostly, quartz is used for analyzing samples in spectrometer and can be used several times with proper cleaning and care. Typically, quartz is totally transparent in UV (wavelength below 380 nm) than glass materials (wavelength from 380 to 780nm ) because glass is less transparent due to presence of ions, which eventually interact with solution, affect UV spectrum.
The plastic cuvettes are come under UV range, and therefore absorb some light. We will not get a correct result and moreover, it might react with something in the solution or solvent which would cause false results. These are the reasons, mostly cuvette is made of quartz.
2) A wide variety of ions are used as quenchers for fluorescence. For example, heavy atoms such as halogens ions are the mostly used and some metals are also used as quenchers such as Cu2+, Pb2+, Cd2+, Fe3+. Quenching by these metal substances involve in the donation of an electron or charge transfer from fluorophore to quenchers.
3) Mostly, quinine compound is treated with acid (for ex., H2SO4) to form quinine sulphate, which fluoresces more strongly. However, if we treat the quinine other than acid, for example, Cl- ions from salt, deactivate the fluorescent property of quinine by forming a complex which is non-fluorescent in nature. Therefore, the result is negative if quinine is not treating with an acid.
4) Quinine compound has two nitrogens, one is aromatic heterocyclic nitrogen and another one is aliphatic cyclic non aromatic nitrogen. The aromatic heterocyclic nitrogen attributes lower basicity than aliphatic nitrogen. Therefore, if we treat the quinine with mild acid, the aliphatic nitrogen gets protonated, moreover it also forms hydrogen bonds readily because of non-localised lone pair electrons.
5) Green fluorescent protein (GFP) possesses a typical chromophore that is mainly responsible for its fluorescent nature. This typical chromophore is formed by tri-peptide motif, which is present in all GFP proteins structure. After interacting with oxygen anaerobically, GFP exhibit fluorescence character.