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What is Derivative Spectrophotometry and why is it used?
- The Derivative Spectrum is obtained by plotting first-order or higher order derivative of absorbance with respect to wavelength as a function of wavelength. Results in degradation of signal to noise ratio.
- 1) reveals detail lost in the original spectrum.
- 2) can be used to conduct concentration measurements of analytes when two or more components are present.
Name 4 Applications of Derivative Spectra.
- 1) Qualitative identification of species.
- 2) Distinguishing between compounds with overlapping spectra.
- 3) The simultaneous determination of two or more components in a mixture.
- 4) The extracting of spectra of analytes in turbid solutions to reveal fine structural details.
Discuss Colorimetry versus Spectrophotometry.
- Colorimetry: Quantification of color based on three component theory of color vision.
- Human eye has receptors for three primary colors (red, blue,and green) and all colors are mixtures of these primary colors. Three components are referred to as X-Y-Z coordinates.
- 1) Employs three photocells to see color similar to that of the human eye
- 2) Used in production and quality control applications
- Spectrophotometry: measures spectral reflectance of an object at each wavelength on the visible spectrum continuum.
- 1) Uses many more sensors (40 or more) to separate beam of light into its component wavelengths.
- 2) Highly accurate and used for research and color formulation applications.
List the 3 Types of Fluorescence Quenching.
involves nonradiative energy transfer from excited species to other molecules resulting in reduction in fluorescence intensity.
- Types of Quenching:
- 1) Dynamic quenching: requires contact between excited species and quenching agent. High quencher concentration is necessary for high probability of collision.
2) Static quenching
: quencher and ground-state fluorophore form a complex called dark complex. Fluorescence of only the unbound fluorophore is observed.
3) Long-range (Forster) quenching
: occurs due to dipole-dipole coupling between excited fluorophore and quencher; energy transfer occurs w/o collisions between molecules.
List the 2 Types of Infrared Molecular Vibrations
1) Stretching Vibrations: continuous change in interatomic distance along the axis of the bond between two atoms.
2) Bending Vibrations: change in angle between two bonds. Types of bending vibrations include: rocking, scissoring, wagging, and twisting.
What is Surface-Enhanced Ramen Spectroscopy?
- Involves very large enhancements of Raman spectra under the following conditions:
- 1) Adsorption of the Raman scattering molecule onto surface of noble metal nanoparticles (gold, silver, and copper)
- 2) Adsorption of the Raman scattering molecule onto roughened metal substrates (gold, silver, and copper)
What are 2 Enhancement Mechanisms for SERS?
1) Electromagnetic Enhancement Mechanism:
2) Chemical Enhancement Mechanism:
- a) Dominant source of enhancement
- b) Highly dependent on size, shape, and composition of metal nanoparticles or roughness features on metal substrates.
- c) Raman active molecules confined within large electromagnetic fields generated.
- a) Much smaller contribution to overall enhancement.
- b) Involves the creation of a charge transfer state between Raman scattering molecules and the nanoparticles/substrate.
Give 2 Examples of Sample Handling for SERS.
Colloidal gold and silver nanoparticles suspended in dilute solution of the sample
- 1) Thin film of the metal nanoparticles deposited on a glass slide and drop or two of sample spotted on the film.
- 2) Deposition of sample electrolytically on a roughened metal electrode
What is Matrix-Assisted Laser Desorption Ionization?
Ionization method used to obtain accurate molecular mass information from a few thousand to several hundred thousand Da.
Describe the process of MALDI.
- 1) Low concentration of the analyte is uniformly dispersed in solid or liquid matrix deposited on end of stainless steel probe or on a metal plate
- 2) Plate then placed in vacuum chamber and laser beam focused onto the sample. MALDI matrix must strongly absorb the laser radiation
- 3) Matrix and analyte are then desorbed and ionized, creating an ion plume.
Describe the mechanism of the MALDI ion plume formation.
Mechanism not completely understood, but current understanding is as follows:
- 1) Absorption of the laser beam by the matrix
- 2) Then transfer of energy from the matrix to the analyte
- 3) Next, desorption of analyte and matrix occurs
- 4) Analyte desorbs as a neutral molecule and then is ionized by proton-transfer reactions with protonated matrix ions in dense phase over the surface containing the matrix
- 5) Series of photochemical reactions produces the protonated matrix ions.
What are the 3 methods used to improve the resolution of HPLC separations?
- 1) Increasing the number of plates in the column
- -Requires more time to complete the separation.
- 2) Reduction of the plate height
- -Can be done without any sacrifice in the amount of time for separation.
- 3) Manipulating the retention factor
- -Results in increased elution time so separations take longer time
- -Usually easiest way to improve resolution
What is Cyclic Voltammetry?
Consists of scanning the potential of a stationary working electrode (unstirred solution) linearly by using a triangular potential waveform.
During the potential sweep, the potentiostat measures the current resulting from the applied potential
- Cyclic voltammogram—resulting current-potential plot
- Forward scan—O reduced to R
- Reverse scan—R reoxidized back to O
Describe Peak Current in Cyclic Voltammograms.
- Two peak currents: cathodic and anodic
- Two peak potentials: cathodic and anodic
Current is directly proportional to concentration and increases with square root of the scan rate—indicative of mass transportcontrolled reactions
Current peaks measured by extrapolating the preceding baseline current.
- Peak position-related to formal potential of the redox process
- Peak separation-used to determine number of electrons transferred as criterion for Nernstian behavior
Describe Irreversible and Quasi-Reversible Electron Transfer Processes.
- Irreversible processes: individual peaks are reduced in size and widely separated
- -Shift in peak potential with scan rate
- -Peak current still proportional to bulk concentration, but lower in height
- Quasi-reversible processes: current controlled by charge transfer and mass transport
- -Voltammograms more drawn out and larger separation in peak potentials compared to reversible system
What are some applications of Cyclic Voltammetry?
Applied for the study of oxidation-reduction reactions
Detection of reaction intermediates
Observation of follow-up reactions of products formed at electrodes