UV-Vis and Fluorescence in Molecules Flashcards
Real deviation
Limitations of Beer’s Law. Ex: Absorbance is best at low levels (0.1-1.0) Ex: Refractive Index, which has a correction factor
Chemical deviations
Chemical changes that occur with due to alterations in concentration. Ex: Solvent/Analyte interactions, or dissociation and association with pH.
Instrumental deviations
How absorbance measurements are made. Ex: Stray light, polychromatic bands from source, mismatched sample cells, not measuring at max absorbance, and noise.
Stray light
scattered or reflected light by surfaces and optics of instrument.
Slit width influence
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
Uv Vis Range
~200nm to ~900 (or much higher for near IR)
Sources for UV-Vis
Deuterium Lamp: 200nm to 360nm Tungsten Lamp: 360nm to 2500nm (Iodine causes decomposition of W back onto filament increasing the lifetime of the lamp)
In Space Double Beam UV-Vis Spectrophotometer
Beam splitter divides beam between blank and sample, which goes to two detectors, where the difference is amplified.
In Time Double Beam UV-Vis Spectrophotometer
Mirror rotates to alternate beam between blank and sample, but only one dectector is needed.
Diode Array UV-Vis Spectrophotometer
Scan the entire spectrum at once, providing great speed, but poor resolution.
Chromophore
Light absorbing groups in molecules that undergo pi-pi* and n-pi* electronic transitions.
Conjugation
Delocalization of electron in molecules, so the more resonance structures (lone e- and double bonds) the more conjugation.
Bathochromic
shift of spectra to longer wavelenght, or a red shift.
Ex: Increase number of double bonds or aromatic rings
Hypsochromic
a shift of spectra to shorter wavelenght, or a blue shift.
Hypochromic
decrease in spectral peak height
Hyperchromic
Increase in spectral peak height
Auxochrome
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)
Electronic Transitions
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.
Inorganic absorption spectra shows
non bonding e- that are d-d or f-f e- transitions.
Occurs in transition metal ions and complexes.
Charge Transfer Complex
redox reaction between a metal ion and a chromophore that results in high sensitivity with large molar absorptivities.
Qualitative UV-Vis Spectroscopy
Not sufficient by iteself to identify a molecule, but can help detect certain functional groups in the molecule.
Measuring non absorbing molecules
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.
Kinetics
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.
Titrations
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
Enzyme kinetics
Measures the affinity of a substrate to an enzyme and how fast the reaction is with the catalyst present. (Velocity is slope)
Elisa
Enzyme=antibody 2=analyte=antibody 1=surface
- wash plate with antibody 1 with sample
- wash off matrix of sample, leaving analyte bound to antibody 1
- add solution with antibody 2, tagged with enzyme
- Wash off excess, leaving only the antibody 2/enzymes that bound to the analye bound to antibody 1.
- 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)
Surface plasmon resonances
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.
Chemiluminescence
emission of radiation by excited species formed in a chemical reaction.
Ex: Luminol + NO = blue light
(Forensic test for blood)
Internal conversion
Relaxation between one singlet state to another signlet state
Intersystem crossing
relaxation from a singlet state into a triplet state
(Results in phosphorescence instead of fluorescence)
Stokes shift
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.
Mirror image spectra: emission and excitation
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.
Excitation Spectra
Absorption
Excitation is scanned
Emission is constant
Emission Spectra
Fluorescence
Excitation is constant
Emission is scanned
To be fluorescent, a molecule must be
- rigid in structure (prevent intermolecular dissipation of energy via nonradiative rotations and vibrations)
- have minimal orbital interactions (to prevent energy transfers)
- low concentrations of paramagnetic species (to prevent collisional quenching)
Quenching
nonradiative relaxation of a molecule that results in a reduction of fluorescence intensity
Concentration quenching
Fluorescence intensity decreases with increased concentration
Chemical Quenching
Chemical changes occur due to alterations in concentration that cause a different or no fluorescence
(Ex: Indicators in altered pH)
Collisional Quenching
A decrease in fluorescence intensity due to collisions causing more triplet states to occur.
Fluorescent measurements of inorganics
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.
Fluorescence imaging
Use of fluorescent dyes or intrinsic nature of biomolecules to visualize cellular processes in fluorescent microscopy.
Aptamers
DNA or RNA fragments that act as synthetic antibodies that can be used with fluorescent dyes for biological imaging.
