Chem 521 Chp 14, Chp 15

how does the slit width affect UV-visible absorption spectroscopy

changes the effective bandwidth--must have minimal slit widths (loss of detail as slit width increases) 2. peak absorbance values increase as the slit wieth ecreases. (areas under the peak are the same with both wide and narrow widths, but since the wider peak is over a larger x, it's intensity is smaller.)

where do the lines (which make up an absorption band) come from?

comes form the transition of an electron from the ground state to one of the vibrational and rotational energy states associate with each energy state. these differ only slightly

resonance radiation (resonance fluorescence)

absorbe radiation is reemitted without a chagne in frequency (emit the same wavelengths they absorb)

auxochrome

functional group that doesn't absorb in teh uv region itself but has the effect of shifting chromophore peaks to longer wavelengths as well as increasing their intensities. 2. they do this because they have at least one pair of n electrons capable of interacting with teh pi electrons of the ring, stabilizing the the pi* state. results in red shift.

how does temperature affect florescence

increase temp, decrease quantum yield due to increase in frequency of collisions and therefore increase in external conversion

internal conversion how does this affect fluorescence

intermoecular prcesses where a molecule passes to a lower energy electronic state without emission of raiation. (s2 to s1, of course, must be singlet to singlet or triplet to triplet) particulalry goo when there's an overlaip in vibrational energy levels. 2. WITH FLUORESCENCE--internal conversion trhough overlaippin birational levles is usually more probably than loss of energy fy fluorescenf from a higher excited state. therefore, an absorption from S0 to S2 will result in internal converstion (S2-S1), loweringthrough vibrational relxation, an only then an emission to the fluorescnce, making the wavelength emitted longer (lower energy ) than wavelength absorbe.

how does the solvent affect fluorescence

lower solvent viscosity increases the likelihood of external conversion due to increase in frequency of collisions. 2. heavy atoms in solvent (or other solutes)--orbital spin interactions result in an increase in the rate of triplet formation an corresponding decrease in fluoresce (increase phosphorescence)

FRET

mechanism describing energy transfer between two light-sensitive molecules (chromophores).[1] A donor chromophore, initially in its electronic excited state, may transfer energy to an acceptor chromophore through nonradiative dipole-dipole coupling.[2] The efficiency of this energy transfer is inversely proportional to the sixth power of the distance between donor and acceptor, making FRET extremely sensitive to small changes in distance.[3] Measurements of FRET efficiency can be used to determine if two fluorophores are within a certain distance of each other

concentration of fluorescence intensity

plot of fluorescne radiatn power of a solution verses concentration of teh emitting speices should be linear at low concentrtions. 2. secondary absorption. (when a wavelength of emission overlaops an absorption band--fluorescence is decreased because the emission traverses the solution and then reabsorbe by other moelcules in solution. (analyte or other species in solution)

how does the absortpion of UV or visiblke radiation occur in absorption meas of organic molecules ? what does this mean?

they result from excitation of bonding electrons. (and so wavelengths of the bands can be correlate with the types of bonds in teh species. --fin functional groups. and 2. quantitative etermination of compouns containing abosrbing groups.

stokes shift

when molecular fluorescence or phosphorescence bands center at wavelengths longer than the resonance line (and therefore have a smaller energy transition)

effect of pH on Fluorescence

when the molecule is deprotonated, there are more resonance strucutres--leads to a more stable first excite state, and therefore stronger fluorescence

what changes the wavelength maxima an the peak intensity of a chromophore

1. solvent 2. other surrounding characteristics of the molecule 3. conjgation between two ore more chromophores (longer wavelengths) 4. vibrational effects broaden absorption peaks, so its harder to find the precise peak wavelength.

other types of quenching besides dynamic

1. static quenching--quencer and the groun-state fluorophore forma complex calle ddark complex. --where no fluorescence is seen. lifetime not affected. 2. long-range--energy transfer occurs without collisions, caused by dipole-dipole coupling, also, non-linear fluorescence

principle sources of bakgorun in a fluorimeter

1. stray light from excitation source 2. raman scattering 3. detector's dark current

differncew between spectrophotometry and flourimetry

1. Spec is funamentally linear, and has good quantitative capabilites, but Flu is fundamentally nonlinear 2. Spec--everything absorbs, but not everything fluoresces well 3. Spec--small changes in a large signal shot noise is limited, but near zero backgorund is possible in Flu 4. in Flu you do tricks to get more infor than what is possible in absorption.

process of absorption of raiation by a atomic or molecular speicies M (2 steps)

1. electronic excitation of the species (M+hv goes to M*) 2. relaxation of the excited state by any available method (like conversion of extra energy to heat.)

qualities of the fluorometers an spectrofluorometers

1. emply ouble beam optis to comepnsate for fluations in radiant power. 2. upper sample beam first passes through a filter or monochromator, which transmits radiation that creates fluorescnce but exlcues or limitas raiation of the fluorescne emissio wavelength. flouresnce is emitted ina all direactions but is observe at right angles to the excitation beam in order to minimize contributesion from sattering an form the intense source raiation. 3. the emission passes through an emisison wavelgnth selector (filter or monochromator) that selects teh flourescne emssion. the isolated raiation then strikes aphototransucer, an there converted into an electrical signal. 4. lower reference beam passes trhoguh an attenuater that reuces its power ot approxiamtes that of the fluorescen raiation, then strikes a secon ransducer and is convereted to an electrical signal. 5. the computer then processes the ration of fluorescne emssion intenstiy to the excitation source intenstiy to create teh spectrum.

sensitivity depneds on teh fraciton of the fluorescent light emitted that you detect, How do you maximize that fraction?

1. get a detector that has a good quantum efficiency 2. strong flourescent signal already (due to sample) 3. collection optics (what fraction of light is collects by the "lens" 4. more excitation power--with limitations. at very high laser powers, n2 approaches g2g1 *n1 and the flourescence intensity becomes independent of excitation power. --the transition is saturated.

ligt source requirements

1. good wavelength coverage (200-880 for general coverage) 2. high intensity 3. good stability

types of relaxation methods of an excited species

1. heat emission 2. photohemical processes like ecomposition of M* to form new species 3. reemission of fluorescence or phosphorescense.

charge transfer absorption

1. important because molar absorptivities are unusually large, which leas to high sensitivity. 2. charge transfer complex is made up of 1. an electron donor group bonds to 2 an electron acceptor. When this product absorbs radiation, an electron from the donor is moved to an orbital largely associated with the acceptor. example: Iron 2+ (Fe III) 3. when there's a metal ion, the metal serves as the electron acceptor.

why are luminescence methos (fluorsecense an phosphorescen) are good to use? what are disadvantages?

1. inherent sensitivity (more sensitivie than absorption) 2. large linear concentration ranges(again, better than absroption) DIS--because excite states are susceptilbe to being eactivate by collisions oan other processes, many molecules don'nt fluoresce or phsohrese at all. --strong interference effects. --chromatography an electrophoreseis help with this. 2. not as good for quantitiative analsysi tahn absorption because more species absorb uv that exhibit photoluminensece when radiation is abosrbe in this region of the spectrum.

eleemnts in a spectrophotomoetry

1. light sourece 2. wavelength selector 3. sample holder 4. detector

characteristics of absorption by organic compounds

1. most studied are based on transitions for n or pi to pi* state because the absorption bands into the UV-visible regions. These require an unsaturuated functional group to provided the pi orbitals.

deactivation processes (excited molecule returning to its ground state)

1. photoluminesce (radiation processes) 2. vibrational relaxation 3. internal conversion 4. external conversion 5. intersystem crossing

the magnitude of molar absorptivities relies on

1. probability for an energy absorbing transtition to occur 2. capture cross section of the species. (sq cm per molecule) deals primarily with quantum mechanically allow transitions.

variables that affect fluorescence an phosphorescence (10 things)

1. quantum yield 2. transition types in fluorescence (pi* to n or pi* to pi) 3. quantum efficiency an transition type 4. fluorescent structure 5. temperature 6. solvent effects 7. pH 8. concentration 9. dynamic quenching 10 other types of quenching

quantum yield-def and how it affects fluorescence

1. ratio of the number of molecules that luminescence to the total number of excite molecules. 2. this is determined by the relative rate constants for each process by which the lowest excite singlet state is deactivated. (processes like intersystem crossing, internal conversion, etc)--(higher rate constants=quicker reactions, then the more likely the process is to occur. 3. higher quantum yield--stronger fluorescence

proces of using a spectrophotometer

1. record dark current (adjust knob to get 0) 2. put in blank, record at Io 3. put in sample, record at I1 both I o an I must be measured at every wavelength if you have (Io is going to be differenty at every awvelength)-- if lamba fluxauate, an intensity--bad inaccurate ratio --if variatble light source, the intensity between Io and Is isn't accurate

neat tricks to get more infor than waht is possible in absorption

1. single molecule detection (single molecule can give off a burst of hundres of photons, single molecules in a flowing stream, images of single molecules on a surface, and requires fast optics (high collection efficiency, an teh moecule you're interested in can't photobleach 2. FRET to determine if proteins interact--one protein has one flourophore connected the other, and when they combine, they create a different color-[= 3. STED microscopy--creates super-reslution images by selective deactivation of fluorophores, minimising the area of illumination at the focal point to increase resoltuion.

difference between singlet and triplet excited states

1. singlet--at ground state all electron spins are paired, and no splitting of electronic energy levels occurs when teh molecule is exposed to a magnetic field. (doublet state is the ground state for a free radical because there are two possible orientation for the od electron in teh magnetic field an each imports slightly different energies to the system.) 2. EXCITED STATES--whe one of a pair of eLectrons is excited to a higher energy level, a singlet or a triplet state is formed. in teh excited singlet state, the spin of the excited electron is still paired with the with the ground-state electorn. IN THE TRIPLET STATE, THE SPINS OF THE TWO ELECTRONS HAVE BECOME UNPARIE AND ARE THEREFORE PARALLEL, and each orientation has slightly different energy levels. 3. THE EXCITED TRIPLET STATE IS LESS ENERGETIC THAT THE CORRESPONDING EXCITE SINGLET STATE.

Why does absorption measurements create a minimal distrubancce in the system under study an what is the exception?

1. the M* concentration is neglible becuase its lifetime is so short. (at any instant) 2. the thermal energy is so small. photochemical decompostiion is the hexception.

dual beam in time

1. the beam is split between the blank and the sample, 2. synchronized sector mirrosrs are placed at the transition points between the blank and the sample beams. they alternate between the two so it alternates between I0 an I 3. instead of two detectors, there's only 1, and the sample beam is integrated. 4. not truly simultaneous by eliminates low frequency noise and long term drift.

what structural components contribute to stronger florescence? (4 things)

1. those with more pi to pi* states, like in aromatic compounds, and to a lesser extent, carbonyl structures. 2. increase degree of condensation (more double bond character) or number or rings 3. heavy atom effect for halogens promotes intersystem crossing 4. higher structural rigidity (less rigidity may cause internal conversion rate)

difference between fluorescence and phosphorescence an how are they alike

ALIKE: excitation is brough about by absorption of photons. in many cases, occurs at wavelengths longer than that of the excitation radiation. DIFFER: flurosecen 's electronic energy transitions don'nt involved a change in electron spin. thus, the excite states are short lived.

vibrational relaxation an how does this affect fluorescence

collisions between molecules of the excite species an those of the solvent. lead to rapid energy transfer with a slight increase in temp of the solvent. This happens when the electron is excited to a vibrational level. 2. because vibrational relaxation so efficient (the rate is so much smaller than that of the excited electronic emission), fluoresce form solution can only occur at the lowest vibrational level of an excite electronic state. Energy transfer takes the path of least resistance and vibrational relaxation, in this case, it the path of least resistance.

what is the difference between emission an excitation spectra? Which is closer to the absorption spectra?

excitation spectra--measured luminescence intensity (emission)at a fixed wavelength while teh exciation wavelength is varied emission spectra--involved excation at a fixed wavelength while the emission wavelength is varied. exciation spectrum is closer to absorption spectra because the first step in generating fluorescence emission is absortption of radation to create teh excited states.

external conversion how does this affect fluorescence

interaction an energy transfer between the excite molecule an the solvent or other solutes. limit the external conversion by limting interactions (lower temp and high viscosity) lead to stronger fluorescnce

sted

interrupts fluorescence process before photon is release. the excited electron is force to relax into a higher vibration state than teh fluorescence transition would enter, causing the photon to be released to be red-shifted. b/c the elctron is going to a higher vibrational state, the energy difference of the two states is lower that the normal fluorescence difference, this lowering of energy raises the wavelength an causes the photon to be shifted father into teh red end of teh spectrum, an differentiates the two types of photons an allows the stimulate photon to be ignored. Because the electron is going to a higher vibrational state, the energy difference of the two states is lower than the normal fluorescence difference. This lowering of energy raises the wavelength, and causes the photon to be shifted farther into the red end of the spectrum. This shift differentiates the two types of photons, and allows the stimulated photon to be ignored. To force this alternative emission to occur, an incident photon must strike the fluorophore. This need to be struck by an incident photon has two implications for STED. First, the number of incident photons directly impacts the efficiency of this emission, and, secondly, with sufficiently large numbers of photons fluorescence can be completely suppressed.[6] To achieve the large number of incident photons needed to suppress fluorescence, the laser used to generate the photons must be of a high intensity. Unfortunately, this high intensity laser can lead to the issue of photobleaching the fluorophore. Photobleaching is the name for the destruction of fluorophores by high intensity light.

quantum efficiency

is the ratio of the number of carriers collected by the solar cell to the number of photons of a given energy incident on the solar cell. The quantum efficiency may be given either as a function of wavelength or as energy. If all photons of a certain wavelength are absorbed and the resulting minority carriers are collected, then the quantum efficiency at that particular wavelength is unity. The quantum efficiency for photons with energy below the band gap is zero. in orer for your flourimitry to be more accurate, your etector must have a good quantum efficiency.

dynamic quenching

nonradiative energy transfer from an excite species to other molecules. it requires contact between teh excite species an teh quenching agent 2. rate is temp an viscoity depdent. an quencher concentraiont myst be high enough that there's a high probability of a collison between teh two during the liftime of the excite state. 3.redueces fluorescence quantum yile an fluorescence lifetime.

inter-system crossing

process where there's a crossover between electronic states of different multiplicity (most common is singlet 1 to triplet 1). Probability of this corssing is enhance when vibrational levlels of the two statels overlap. 2. more common in heavy atom-containing molecules.

quantum efficiency and transition type.

quantum efficiency (quantum yield) is greatest for pi to pi* types. this is because the pi, pi* states exhibit relatively short average lifetimes (Kf is larger) an because the deactivation processes that compete with fluorescence are less likely to occur.

how does the solvent around the species affect the absorbance spectrum? HOw does this compare to the gaseous phase?

solvent or the condensed state limit the specie's freedom to rotate and so lines due to diferences in rotational energy levels disappear. 2. when solvent molecules surrond the species, energies of the various vibrational levels are modified in a nonuniform way and the energy of a given state in a sample of solute appears as a single, broad peak. more pronouced in polar solvents (compared to gaseous phase, where each molecule is fairly separated from one another and they are free to vibrate and rotate freely, and the spectrum shows each individual absorption lines)

describe the postfilter effects

some radiation is absorbed when leaving the sample (the middle solute molecules may emit photons, but the solute or solvent molecules may "grab" that energy before it leave the sample and hits the detector.

dual beam in sapce

split beam so you can measure blank and sample at the same time, the cells an detectors and beam splitter.

describe the prefilter effects

the amount of radiation reaching the sample decrease with increasing concentration (the middle of the sample may not get as much radiation because the solutes at the edge are so concentrated, that it can't pass through.

why do nitrogen hetreocyclics don't fluoresce?

the lowest energy electronic transition is an n to pi* system system that rapidly converts to the triplet state.