Spectroscopy Flashcards
What is spectroscopy?
The study of the absorption and emission of electromagnetic radiation (light) by matter.
Why is spectroscopy useful?
Determines:
Whether the protein is bound or free
The size or charge of protein
The concentration of protein
Activity of the protein
What does the energy of EM depend on?
The energy carried by electromagnetic radiation depends on wavelength and frequency.
Low wavelength gives high energy.
What is the Planck Einstein equation?
E = hc / λ
E = energy
h = Planck’s constant
c = speed of light
λ = wavelength
What is ultraviolet visible spectroscopy?
UV-Vis radiation is high energy - it excites delocalised electrons.
Delocalised electrons are found in metallic bonding.
How are absorbance and spectroscopy linked?
Atoms will absorb light at a specific, unique wavelength.
Electrons in the atom move from ground state to excited state.
At a given wavelength, how much light is absorbed is used to estimate the concentration of the sample.
What is the absorption spectrum?
The absorption spectrum gives a fingerprint of a biological molecule.
Any molecule with conjugated double bonds will be able to absorb enough energy to become excited and produce an absorption spectrum.
What is the Beer-Lambert relationship?
Aλ = Eλcl
Absorbance (Aλ) is dependent on
Concentration (c) of solute
Light path length (l)
Molar absorption (Eλ)
Ey is the absorbance of 1M-1.cm-1 solution at wavelength (λ) of incident energy
What is the relationship between absorbance and concentration?
Absorbance is proportional to concentration.
High absorbance = high concentration
How can compounds be identified if there is no colour?
Every molecule has a specific wavelength.
Check the purity of the molecule using UV to see if there is still things in the mixture.
How are compounds distinguished when they are the same wavelength?
Use another reaction that relies on a compound from the first.
Look at the stoichiometry of the equation to see how much of one product was used up.
What is an example of using multiple reactions to distinguish compounds?
Phosphoenol to pyruvate, with ADP going to ATP.
There is no colour, so look at wavelength, but ADP and ATP absorb at the same wavelength.
So compare it to another reaction - pyruvate to lactate, where NADH goes to NAD+.
As ATP is synthesised NADH is oxidised.
Can use it to measure pyruvate, because NADH absorbs at 340nm but NAD+ does not.
What is the rate determing step?
The slower rate of reaction determines the overall rate of reaction.
How is UV spectroscopy used in thermal denaturation?
UV spectroscopy can check if DNA is denatured.
Single stranded DNA has a higher absorbance - so if the line at a higher temp has greater absorbance then DNA is denatured.
What is the importance of solvent in UV spectroscopy?
Some solvents interact with the solute molecules so affect absorption peaks.
In proteins, some solvents can disrupt hydrophobic interactions in the side chains of the amino acid residues.
