L6: Spectroscopy (UV) - Vis Flashcards
Define spectroscopy
- Light and matter always interact with each other in a specific way
- The study of the absorption and emission of electromagnetic radiation (light) by matter.
Describe what happens when a beam of light is shone on a prism?
A beam of light on a ‘prism’ can make the light separate into different components (a spectrum). Some of these components are visible while others are not
What can spectroscopy tell us about proteins?
- Where is the protein? - Is the protein bound or free
- Which protein is present and what is the size/charge
- How much of the protein are there? - concentration
- How effective is the protein? - activity
What does the energy of the EM spectrum depend on?
Wavelength and frequency
Describe the relationship between wavelength and energy on the EM spectrum
- At a low wavelength (gamma rays and blue/purple end of VL) there is a higher degree of energy
- At a high wavelength (radio waves and red end of VL) there is a lower degree of energy
- There is an inverse relationship
What is the Planck-Einstein equation?
What type of spectroscopy is mainly used in biology?
Ultraviolet-visible spectroscopy
What does UV-Vis spectroscopy do to electrons?
- High energy
- Excites electrons and delocalises them
Where can delocalised electrons be found?
- Metallic bonding as a sea of delocalised electrons around metal ions
- Benzene aromatic ring above and below the plane of the bonding atoms in
Describe how absorbance works in spectroscopy
-Biological macromolecules will absorb energy at a specific, unique wavelength depending on the energy needed to excite the electrons in the molecule
- Electrons in the atom move from ground state to excited state
- At a given wavelength, how much light is absorbed can be used to estimate the concentration of the sample
- The absorption spectrum gives a fingerprint of a biological molecule, by isolating a particular wavelength of light
Between what wavelength are aromatic structures viewed on a spectrophotometer?
255-280 nm
At what wavelength is a peptide bond observed at on a spectrophotometer?
200-230 nm
Why does the amino acid tryptophan have a much larger absorbance than the other amino acids?
Tryptophan has a double aromatic ring - means greater excitation of the electrons in the molecule
Why can some proteins show a low absorbance on a graph in the aromatic region?
An amalgamation of different peptides e.g.
serum albumin
What is the Beer-Lambert equation?
What do the different coefficients mean in the Beer-Lambert equation?
Why is there a lambda symbol next to the A in Beer-Lambert equation?
- Absorbance needs to be at a particular wavelength
What is molar absorption?
Describes how well a substance will absorb light
Describe the relationship between absorbance and concentration?
Absorbance is proportional to concentration e.g. a high absorbance will mean a high concentration and a low absorbance will mean a low concentration
How can the relationship between absorbance and concentration be shown?
- An a calibration curve on a graph
- Plot absorbance data for a concentration found using a colorimeter and cuvette
What happens if there is no colour in the experimental solution?
- Rely on having the wavelengths and running a UV spectroscopy
- Different molecules would absorb at a different wavelengths
- Absorption spectra useful for identifying different chromophores & their concentrations
What is stoichiometry?
Ratios and relationships between different elements and compounds in biological systems
What happens if there is no colour and molecules absorb at the same wavelength?
Can monitor another reaction that is taking place alongside the reaction, to understand what is happening in the first reaction - stoichiometry
What is the rate determining step?
The slowest reaction which determines how the reaction progresses
What other factors can affect absorption on a UV spectrum?
- Conformational and structural changes to the molecule being tested
- Thermal denaturation of the protein
How does the solvent effect impact on the UV spectrum?
- The choice of solvent is important in the use of UV-Vis
- Some solvents interact with the solute molecules thus affecting the absorption peaks
- In proteins, some solvents can disrupt hydrophobic interactions in the side chains of the amino acid residues, changing the resulting spectrum
