L6: Spectroscopy (UV) - Vis

Question 1

Define spectroscopy

Answer 1

• 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.

Question 2

Describe what happens when a beam of light is shone on a prism?

Answer 2

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

Question 3

What can spectroscopy tell us about proteins?

Answer 3

• 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

Question 4

What does the energy of the EM spectrum depend on?

Answer 4

Wavelength and frequency

Question 5

Describe the relationship between wavelength and energy on the EM spectrum

Answer 5

• 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

Question 6

What is the Planck-Einstein equation?

Answer 6

Question 7

What type of spectroscopy is mainly used in biology?

Answer 7

Ultraviolet-visible spectroscopy

Question 8

What does UV-Vis spectroscopy do to electrons?

Answer 8

• High energy
• Excites electrons and delocalises them

Question 9

Where can delocalised electrons be found?

Answer 9

• Metallic bonding as a sea of delocalised electrons around metal ions
• Benzene aromatic ring above and below the plane of the bonding atoms in

Question 10

Describe how absorbance works in spectroscopy

Answer 10

-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

Question 11

Between what wavelength are aromatic structures viewed on a spectrophotometer?

Answer 11

255-280 nm

Question 12

At what wavelength is a peptide bond observed at on a spectrophotometer?

Answer 12

200-230 nm

Question 13

Why does the amino acid tryptophan have a much larger absorbance than the other amino acids?

Answer 13

Tryptophan has a double aromatic ring - means greater excitation of the electrons in the molecule

Question 14

Why can some proteins show a low absorbance on a graph in the aromatic region?

Answer 14

An amalgamation of different peptides e.g.
serum albumin

Question 15

What is the Beer-Lambert equation?

Answer 15

Question 16

What do the different coefficients mean in the Beer-Lambert equation?

Answer 16

Question 17

Why is there a lambda symbol next to the A in Beer-Lambert equation?

Answer 17

• Absorbance needs to be at a particular wavelength

Question 18

What is molar absorption?

Answer 18

Describes how well a substance will absorb light

Question 19

Describe the relationship between absorbance and concentration?

Answer 19

Absorbance is proportional to concentration e.g. a high absorbance will mean a high concentration and a low absorbance will mean a low concentration

Question 20

How can the relationship between absorbance and concentration be shown?

Answer 20

• An a calibration curve on a graph
• Plot absorbance data for a concentration found using a colorimeter and cuvette

Question 21

What happens if there is no colour in the experimental solution?

Answer 21

• 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

Question 22

What is stoichiometry?

Answer 22

Ratios and relationships between different elements and compounds in biological systems

Question 23

What happens if there is no colour and molecules absorb at the same wavelength?

Answer 23

Can monitor another reaction that is taking place alongside the reaction, to understand what is happening in the first reaction - stoichiometry

Question 24

What is the rate determining step?

Answer 24

The slowest reaction which determines how the reaction progresses

Question 25

What other factors can affect absorption on a UV spectrum?

Answer 25

• Conformational and structural changes to the molecule being tested
• Thermal denaturation of the protein

Question 26

How does the solvent effect impact on the UV spectrum?

Answer 26

• 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