UV-Vis and Fluorescence in Molecules

Question 1

Real deviation

Answer 1

Limitations of Beer’s Law. Ex: Absorbance is best at low levels (0.1-1.0) Ex: Refractive Index, which has a correction factor

Question 2

Chemical deviations

Answer 2

Chemical changes that occur with due to alterations in concentration. Ex: Solvent/Analyte interactions, or dissociation and association with pH.

Question 3

Instrumental deviations

Answer 3

How absorbance measurements are made. Ex: Stray light, polychromatic bands from source, mismatched sample cells, not measuring at max absorbance, and noise.

Question 4

Stray light

Answer 4

scattered or reflected light by surfaces and optics of instrument.

Question 5

Slit width influence

Answer 5

Narrower slit gives greater spectral detail and increased peak height. Quantitative has a large slit for increased intensity Qualitative has a narrow slit width for increased detail

Question 6

Uv Vis Range

Answer 6

~200nm to ~900 (or much higher for near IR)

Question 7

Sources for UV-Vis

Answer 7

Deuterium Lamp: 200nm to 360nm Tungsten Lamp: 360nm to 2500nm (Iodine causes decomposition of W back onto filament increasing the lifetime of the lamp)

Question 8

In Space Double Beam UV-Vis Spectrophotometer

Answer 8

Beam splitter divides beam between blank and sample, which goes to two detectors, where the difference is amplified.

Question 9

In Time Double Beam UV-Vis Spectrophotometer

Answer 9

Mirror rotates to alternate beam between blank and sample, but only one dectector is needed.

Question 10

Diode Array UV-Vis Spectrophotometer

Answer 10

Scan the entire spectrum at once, providing great speed, but poor resolution.

Question 11

Chromophore

Answer 11

Light absorbing groups in molecules that undergo pi-pi* and n-pi* electronic transitions.

Question 12

Conjugation

Answer 12

Delocalization of electron in molecules, so the more resonance structures (lone e- and double bonds) the more conjugation.

Question 13

Bathochromic

Answer 13

shift of spectra to longer wavelenght, or a red shift.

Ex: Increase number of double bonds or aromatic rings

Question 14

Hypsochromic

Answer 14

a shift of spectra to shorter wavelenght, or a blue shift.

Question 15

Hypochromic

Answer 15

decrease in spectral peak height

Question 16

Hyperchromic

Answer 16

Increase in spectral peak height

Question 17

Auxochrome

Answer 17

Nonabsorbing group on the molecule that influences the conjugation, and thus the absorbance, of the molecule. It specifically induces a red shift and an increase in intensity. (Ex: OH)

Question 18

Electronic Transitions

Answer 18

n to pi*, pi to pi*, n to sigma*, pi to sigma*, sigma to pi*, and sigma to sigma*.

Only n to pi* and pi to pi* occur in the 200-900nm range of UV-Vis.

Question 19

Inorganic absorption spectra shows

Answer 19

non bonding e- that are d-d or f-f e- transitions.

Occurs in transition metal ions and complexes.

Question 20

Charge Transfer Complex

Answer 20

redox reaction between a metal ion and a chromophore that results in high sensitivity with large molar absorptivities.

Question 21

Qualitative UV-Vis Spectroscopy

Answer 21

Not sufficient by iteself to identify a molecule, but can help detect certain functional groups in the molecule.

Question 22

Measuring non absorbing molecules

Answer 22

They can be measured indirectly through a reaction with an absorbing species, like a metal ion.

Ex: Glucose reducing absorbing copper into an insolube state.

Question 23

Kinetics

Answer 23

Kinetic region is were absorbance of analyte and/or reagent is changing to products.

Equilibrium region is where the products are all formed and equilibrium has been reached.

Axis of graph is absorbance vs time.

Question 24

Titrations

Answer 24

flat, then increase: only Titrant absorbs

increase, then flat: only prodcuts absorb

decrease, then flat: only analyte absorbs

decrease, then increase: analyte absorbs and titrant absorbs

small increase, then sharp increase: product absorbs, but titrant absorbs more

sharp increase, then small increase: titrant absords, but products absorb more

Question 25

Enzyme kinetics

Answer 25

Measures the affinity of a substrate to an enzyme and how fast the reaction is with the catalyst present. (Velocity is slope)

Question 26

Elisa

Answer 26

Enzyme=antibody 2=analyte=antibody 1=surface

1. wash plate with antibody 1 with sample
2. wash off matrix of sample, leaving analyte bound to antibody 1
3. add solution with antibody 2, tagged with enzyme
4. Wash off excess, leaving only the antibody 2/enzymes that bound to the analye bound to antibody 1.
5. add uncolored subtrate and measure the absorbance of product formed by enzymes still there.

(A nonfluorescent molecule can be in place of the enzyme and chemically altered to become fluorescent)

Question 27

Surface plasmon resonances

Answer 27

How a wave of light osscilates between nanoparticles smaller than its wavelength is determined by the shape, size, and charge of the particle. This inphase osscilation causes diopole momentums, which are changed by characteristics of the particle.

Question 28

Chemiluminescence

Answer 28

emission of radiation by excited species formed in a chemical reaction.

Ex: Luminol + NO = blue light

(Forensic test for blood)

Question 29

Internal conversion

Answer 29

Relaxation between one singlet state to another signlet state

Question 30

Intersystem crossing

Answer 30

relaxation from a singlet state into a triplet state

(Results in phosphorescence instead of fluorescence)

Question 31

Stokes shift

Answer 31

Energy difference between the highest peak in excitation and the highest peak in emission, which is due to energy lost to vibrational relaxation in the excited states.

Question 32

Mirror image spectra: emission and excitation

Answer 32

The molecular and orbital spacing and symmetries don’t change, and the spacing of vibrational levels don’t change, thus the intensity will be the same as what was absorbed.

Question 33

Excitation Spectra

Answer 33

Absorption

Excitation is scanned

Emission is constant

Question 34

Emission Spectra

Answer 34

Fluorescence

Excitation is constant

Emission is scanned

Question 35

To be fluorescent, a molecule must be

Answer 35

1. rigid in structure (prevent intermolecular dissipation of energy via nonradiative rotations and vibrations)
2. have minimal orbital interactions (to prevent energy transfers)
3. low concentrations of paramagnetic species (to prevent collisional quenching)

Question 36

Quenching

Answer 36

nonradiative relaxation of a molecule that results in a reduction of fluorescence intensity

Question 37

Concentration quenching

Answer 37

Fluorescence intensity decreases with increased concentration

Question 38

Chemical Quenching

Answer 38

Chemical changes occur due to alterations in concentration that cause a different or no fluorescence

(Ex: Indicators in altered pH)

Question 39

Collisional Quenching

Answer 39

A decrease in fluorescence intensity due to collisions causing more triplet states to occur.

Question 40

Fluorescent measurements of inorganics

Answer 40

Fluorescence can be used to determine the concentration of an inroganic molecule based on the formation of a fluorescent chelate or quenching of an instrisically fluorescing dye.

Question 41

Fluorescence imaging

Answer 41

Use of fluorescent dyes or intrinsic nature of biomolecules to visualize cellular processes in fluorescent microscopy.

Question 42

Aptamers

Answer 42

DNA or RNA fragments that act as synthetic antibodies that can be used with fluorescent dyes for biological imaging.