UV-VIS Flashcards
What are the different species of matter that can be analyzed with spectroscopy? [2]
Molecular
Atomic
How does wavelength impact frequency and energy?
Wavelength is inversely proportional to energy and frequency.
What are the energy levels in atoms and molecules? [3]
- Electronic, vibrational, and rotational
- Atoms have different electronic states
- Molecules have electronic, vibrational, and rotational states
What is the difference between absorption and fluorescence?
Absorption is the process where a molecule absorbs light energy, causing an electron to move from a lower to a higher energy state.
Fluorescence occurs when the excited electron relaxes back to a lower energy state, emitting light of a longer wavelength (lower energy) than the absorbed light.
What is vibrational relaxation?
Dissipation of the energy within one
excited state; dissipated as heat to neighboring molecules. Vibrational relaxation is a non-radiative transition.
What is fluorescence?
Dissipation of energy by emitting a photon. The emitted photon will be of lower energy and thus of longer wavelength.
What is the accessible UV range in UV-Vis spectroscopy?
200-350 nm for UV
350-700 nm for VIS
Quantitative absorption spectroscopy is most common within UV-Vis spectroscopy.
True or False?
True.
Qualitative absorption spectroscopy is most common within UV-Vis spectroscopy.
True or False?
False.
Quantitative absorption spectroscopy is most common within UV-Vis spectroscopy.
What is the purpose of quantitative absorption spectroscopy?
To determine the concentration of an analyte in a given sample solution.
What are the two scenarios of quantitative absorption spectroscopy?
UV-VIS
- Analytes naturally absorb radiation in the UV-Vis range → no chemical modification of the analyte required
- Analytes do not absorb radiation in the UV-Vis range → chemical modification of the analyte required to convert into a species that absorbs radiation
In quantitative absorption spectroscopy, what does absorbance tell you?
- Absorbance is a measure of the amount of light absorbed
- The higher the value, the more of a particular wavelength is being absorbed
What is transmittance?
The fraction of light that passes through a sample, expressed as a ratio of transmitted light intensity.
What is absorbance?
A measure of how much light is absorbed by a sample, related to transmittance by the logarithmic relationship.
Absorbance is […] to the concentration of the absorbing species in the solution.
directly proportional
Absorbance is inversely proportional to the concentration of the absorbing species in the solution.
True or False?
False.
Absorbance is directly proportional to the concentration of the absorbing species in the solution.
Absorbance is directly proportional to the concentration of the absorbing species in the solution.
True or False?
True.
The fraction of the incident beam that is not transmitted does not equal the solution’s absorbance.
True or False?
In quantitative spectroscopy
True.
The fraction of the incident beam that is not transmitted equals the solution’s absorbance.
True or False?
In quantitative spectroscopy.
False.
The fraction of the incident beam that is not transmitted does not equal the solution’s absorbance.
What is Beer’s Law?
- The law relates the attenuation of light in a sample to the material properties of the sample itself.
- From the absorbance, the concentration of an analyte can be directly measured
- Only works when measuring a single, known compound.
- Only applicable at limited concentrations.
Beer’s law strictly applies to situations in which the radiation passing through the sample is monochromatic
Beer’s law only works when measuring a single, known compound.
True or False?
True.
Beer’s law strictly applies to situations in which the radiation passing through the sample is monochromatic
Beer’s law works when measuring multiple known compounds.
True or False?
False.
It only works when measuring a single, known compound.
Beer’s law strictly applies to situations in which the radiation passing through the sample is monochromatic
Why is Beer’s Law only applicable at limited concentrations?
- If too concentrated, some molecules are blocked from exposure to incident radation.
- High concentrations can lead to aggregation or changes in distribution of the analytes in solution.
- Beer’s Law applicable in dilute solutions, up to approximately 10 mM.
The optimal range for absorbance using Beer’s Law using simple, less expensive spectrophotometers is approximately […]
- 0.2-0.8 absorbance units
- 15-65% transmittance
What is the molar attenuation coefficient (ε)?
- Measurement of how strongly a chemical species attenuates light at a given wavelength
- Intrinsic property of the chemical species which can vary based on:
- Solvent (polar/nonpolar)
- pH
- Ionic strength
This is why standard curves are more common than Beer’s Law.
attenuate = ‘reduce’
The molar attenuation coefficient (ε) can vary based on: [3]
- Solvent (polar/non-polar)
- pH
- Ionic strength
ε is an intrinsic property of the chemical species.
When will a sample exhibit deviations from Beer’s Law? [5]
- High concentrations
- Analyte and solvent react chemically
- Turbidity
- Analytes
- Ionization due to pH change
Why are chemical reactions not ideal if intending to use Beer’s Law?
- If the analyte and solvent react chemically, there will be changes to the chemical structure of the absorbing species that will interfere with accurate results.
Why is turbidity a concern when applying Beer’s Law?
- Decreases intensity of the light beam that passes through the solution, which can lead to light scattering, reflectance, and some absorption.
What are concerns with the analyte when applying Beer’s Law? [2]
Analytes may:
- Polymerize in solution
- Undergo reversible association-dissociation of analyte molecules.
How can you determine the unknown concentration of a smple without using Beer’s Law?
A) Identify the maximum absorbance wavelength of the item of interest
B) Determine absorbance of a set of standards of the same compound at different concentrations
C) Determine equation of line
D) Solve for x (concentration)
What types of molecules absorb light? [2]
- Chromophores
- Auxochromes
What is a chromophore?
- Part of a molecule responsible for colour/absorbance of light
- A single functional group, or a combination of several
What are auxochromes?
- A group of atoms attached to a chromophore which modifies the ability of that chromophore to absorb light
- Nonbonded electrons which alters both the wavelength and intensity of absorption of the
chromophore - Examples: Amine, hydroxyl, carboxyl, aldehyde, hydrogen sulfite
What are conjugated chromophores?
- Alternating single (sigma) and double (pi) bonds
- Can involve C=C, C=N, C=O, N=N
- Typically, the more conjugated (longer) the pi-system is, the longer the wavelength of photon can be captured
Why do leaves change colour in the fall?
- Their chromophores (chlorophyll molecules) break down and stop absorbing red and blue light.
Why are healthy plants perceived as green?
- Because chlorophyll absorbs mainly the blue and red wavelengths but green light, reflected by plant structures like cell walls, is less absorbed.
Define: auxochrome.
A group of atoms that can get attached to a chromophore, thereby increasing the colourfulness of the chromophore.
Define: chromophore.
The part of a molecule that is responsible for the colour of that molecule.
Describe how chromophores and auxochromes relate to the intensity of the colour.
- Auxochromes increase the colour intensity of the chromophore.
- Chromophores are responsible for the colour of a colourless compound.
Give an example of an auxochrome in action.
Pale yellow coloured nitrobenzene becomes dark yellow coloured when a hydroxyl group is attached to the molecule.
Give an example of a chromophore in action.
Colourless benzene gets a pale yellow colour when a nitro group is added to the benzene molecule.
Name some common functional groups.
What are the electronic absorption bands for nucleic acids?
What are the electronic absorption bands for proteins?
What does the maximum wavelength depend on?
The local environment of the group
What does the local environment mean?
- The chemistry surrounding the chromophore
- The type of solvent
- How much of the solvent is around the chromophore
- Other ions/salts in solution and/or ‘touching’ the chromophore
Impacts both absorbance and fluorescence!
What is the basic design and set-up of a spectrophotometer?
- Two basic designs: single-beam or double-beam
Describe a single-beam spectrophotometer.
What is the purpose of a monochromator?
- To isolate the specific, narrow, continuous group of wavelengths to be used in the spectroscopic assay.
- NOT single wavelength
Polychromatic radiation from the source enters the monochromator and is dispersed according to wavelength, and monochromatic radiation of a selected wavelength exits the monochromator
Describe the dispersing device of a spectrophotometer.
-
Diffraction grating
- Equally spaced grooves on a highly polished surface
- Layer of aluminum deposited under vacuum
- Second layer of quartz or glass
-
β, blaze angle
- Incident light angle determines diffracted light wavelength
Describe a double beam spectrophotometer.
Simultaneously measure and compare the relative absorbance of a sample and a reference cell.
Compare the basic design of a single beam and a double beam spectrophotometer.
What is the advantange of a double-beam spectrophotometer.
More accurate: will compensate for deviations in the radiant output of the source
What is the disadvantage of a double-beam spectrophotometer?
The radiant power of the incident beam is diminished because the beam is split
What is a diode array spectrophotometer?
- Rapid: λ range 200-800 nm can be obtained in usually under 1 second
-
Array detector can contain 256 to 2048 diodes
- One diode gives a spectral coverage of ≤ 1.25 nm
- E.g.: ProStar 335 Diode Array Detector
- Two 1024 diode arrays, 190-950 nm range, resolution < 1 nm
-
Can be made of different materials
- Silicon (190-1100 nm)
- Germanium (400-1700 nm)
Diode array instruments are ideal for collecting complete spectral data on rapidly changing samples in disciplines such as kinetics, dissolution, liquid chromatography, and multicomponent analysis.
Diode array instruments are ideal for […]
Collecting complete spectral data on rapidly changing samples in disciplines such as kinetics, dissolution, liquid chromatography, and multicomponent analysis.
Use Beer’s Law to measure the change in NADPH.
- NADPH and glucose determination
- ε = 6300 M-1cm-1
- Specific to measurement at 340 nm in a specific buffer
- Glucose is converted to glucose-6-phosphate by enzymes in the presence of ATP
- Glucose-6-phosphate is oxidized, which reduces NAPD+ to NADPH
- Change in NADPH is measured directly from absorbance using Beer’s law
Describe the use of Beer’s Law in the TBARS assay for measuring lipid oxidation.
- ε = 1.56x10^6 M^-1cm^-1
- Specific to measurement at 532 nm in a specific solvent system
- Malondialdehyde (MDA) is a product of lipid oxidation
- Can react MDA with thiobarbituric acid (TBA) under acidic conditions to form adducts
- Determine concentration directly from the absorbance
Describe the use of spectrophotometry to study chemical reactions.
- Can be used to follow enzymatic (or
chemical) reactions- Can get reaction rate
- Only useful if substrate and product have different absorption spectra
- Example: Studying the reaction rate of polyphenol oxidase on different substrates
Describe the use of spectrophotometry in the bicinchoninic acid assay for measuring protein.
- Peptide backbone will reduce Cu2+ (cupric) to Cu+ (cuprous)
- Cu+ forms complexes with BCA
- Compare absorbance of a sample to the absorbance of a standard curve of known concentrations (mg/mL) of a protein
What is derivative spectrophotometry?
- Method to enhance the detectability of overlapping spectral bands/increase assay resolution
- In first derivative spectra, peaks become inflection points
- In second derivative spectra, peaks (relative to normal spectra) become troughs
Describe how derivative spectrophotometry can uncover hidden peaks.
Hidden peaks are revealed in 2nd derivative.
Describe how derivative spectrophotometry can be used for background elimination.
After the 1st derivative, the shift is no longer relevant
Typically, the more conjugated (longer) the pi-system is, the […]
longer the wavelength of photon can be captured
What does the collimating mirror do?
Change the radiation from a point source into a parallel beam.
