Fluorescence SQ’s & MCQ’s PDF

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Chemistry Department

CHM4483 Assignment topic

‘Fluorescence SQ’s & MCQ’s’ Instructor: Prof. Fatima Jabbar

Submitted by Usman Ghani ID: R2F17BSCH0062 Semester: 8th

Section: CF3-17 Submission Date: 29th Apr Due Date: 289th Apr

Short Questions Why does the ratio of anti-Stokes to Stokes intensities increase wit sample temperature? The scattering processes leading to anti-Stokes lines begin from vibrationally excited initial states, while those producing Stokes lines originate from the ground vibrational state. Since the ratio of the population of excited vibrational levels to the population of the ground vibrational state increases with increasing T (by the Boltzmann distribution), the intensities of anti-Stokes lines consequently increase relative to the Stokes lines. Why is spectrofluorometry is more sensitive than spectrophotometry? The key difference is that spectrofluorometry measures the intensity o fluorescence, while spectrophotometry (absorption spectroscopy) measures the ratio of the intensities of two beams (incident and passed beams). The fluorescence signa is directly proportional to the intensity of the excitation source. F ∝ P0 We can increase the intensity of the excitation source (use a laser!) and gain a subsequent increase in the fluorescence signal—potentially leading to greate sensitivity. Spectrophotometry, on the other hand, is an absorption technique Absorption depends on the ratio of incident to passed light: A = log(P/P0) so simply increasing P 0 also increases P. What is Quenching? Quenching Decrease of fluorescence intensity by interaction of the excited state of the fluorophore with its surroundings is known as quenching and is fortunately relatively rare. Quenching is not random. Each example is indicative of a specific chemical interaction, and the common instances are well known. Quinine fluorescence is quenched by the presence of halide ion despite the fact that the absorption spectrum and extinction coefficient of quinine is identical in 0.5M H 2SO and 0.5M HCl

Terminologies Based Questions: 1. fluorescence

Relaxation of an excited state by emission of a photon without a change in electron spin. This satisfies the selection rule ∆S=0, and so is allowed, rapid and fairly probable, depending on the relative rates of competing relaxation pathways Note that this often involves singlet->singlet transitions in neutral molecules since typically all electrons are paired in the ground state. Strictly speaking though, this could involve any multiplicity (as long as it is conserved in the process) for free atoms, ions, molecular ions, molecular radicals, etc. 2. resonance fluorescence Relaxation of an excited state by emission of a photon of the same frequency as the excitation frequency. This is more common in atoms than in molecules, which have vibrational relaxation pathways that may dissipate a portion of the excitation energy before fluorescence occurs. 3. singlet state A many-electron state in which all electron spins are paired. Total spin angula momentum S=0. This is commonly the multiplicity of neutral molecules, both in the ground state and in excited electronic states reached by spin-allowed optica transitions. 4. triplet state A many-electron state in which two electron spins are parallel. Total spin angula momentum S=1. Typically, not accessible by *allowed* optical transitions from the ground state for neutral molecules. 5. vibrational relaxation Dissipation of excess energy in a molecular excited state among the vibrationa degrees of freedom. This is the means of the Stokes shift in molecula fluorescence (emitted light appears at longer wavelength than the excitation wavelength). 6. internal conversion Intramolecular processes which allow an excited state to relax to anothe electronic state or the ground state without emission of a photon and without a change in multiplicity. This is a consequence of overlap of the vibrationa manifolds of the electronic states. 7 external conversion

Relaxation of an excited electronic state without emission of a photon due to intermolecular processes (collisions with other molecules). This competes with fluorescence, reducing fluorescent quantum yield, and is often referred to as quenching. i) intersystem crossing Processes in which the spin of an excited electron flips. For neutral molecules this is typically a singlet->triplet conversion. 8. quantum yield The ratio of the number of molecules that luminesce to the number of excited molecules. A quantification of the efficiency of luminescence.

Multiple choice questions for fluorescence spectroscopy 1. A) B) C) D)

The main advantage of fluorescence over UV-Vis spectroscopy is Its sensitivity Its compatibility with separation techniques Its compatibility with most analytes None of the above

2. Select the true statement about fluorescence spectroscopy of molecules in the UV- visible region A) Emission usually occurs at energies that are greater than the energies of excitation B) C) D)

Emission usually occurs at energies that are less than the energies Emission usually occurs at energies that are equal to the energies of excitation None of the above is accurate

3. A)

D)

Fluorescence probes are highly fluorescent reagents that are designed to bind strongly and specifically to certain targets are the basis of many very sensitive assays developed biological molecules of interest enable the use of fluorescence for the detection of molecules that are not fluorescent all of the above

4. A) B) C)

Accuracy is defined as The closeness of a measured value to the real value. A measure of how often an experimental value can be repeated. The number of significant figures used in a measurement.

B) C)

5. A) B) C) D)

Which of the following molecule does not show fluorescence Quinine sulphate Chlorophyll Ethidium bromide Nucleic acids

6. Which among the following class of compound shows intense fluorescence? A) Compounds having aromatic functional group B) Compounds having aliphatic carbonyl structures C) Compounds having alicyclic carbonyl structures D) Compounds having conjugated double bond 7. A) B) C) D)

Fluorescence efficiency in most molecules increases with Increasing temperature Increasing solvent viscosity Increasing quencher concentration None of the above

8. A)

The correct path of light in a spectrofluorometer is Source lamp → excitation monochromator → sample holder → emission monochromator → Detector Source lamp → emission monochromator → sample holder → excitation monochromator → Detector Detector → emission monochromator → sample holder → excitation monochromator → source lamp Source lamp → Sample holder → excitation monochromator → emission monochromator → Detector

B) C) D)

9. When molecules are excited in visible region which lamp is used? A. Hydrogen deuterium lamp B. Xenon lamp C. Tungsten lamp D. Mercury lamp 10. A. B. C. D.

Which detector are used in Fluorimetry? Photo voltaic cell PMT Photo tube All of the above...