Spectroscopy

Question 1

What is light?

Answer 1

An electromagnetic wave that is made up of an electric field and a magnetic field that oscillate perpendicular to each other

Question 2

What equation involves wavelength?

Answer 2

Energy (E) = hc/λ

h = Planck’s constant
c = speed of light
λ - wavelength

OR

λ = c/v

v = frequency (s-1 or Hz)

Question 3

Define the types of energy radiation?

Answer 3

High energy radiation -
short wavelength and high frequency

Low energy radiation -
long wavelength and low frequency

Question 4

What is amplitude?

Answer 4

The maximum value the electric or magnetic vector can have

Question 5

What happens when two waves are superimposed on each other?

Answer 5

The resultant wave = the sum of the waves

180 degree shifted - cancel out
90 degree shifted - the wave will form in the middle of the peaks

Question 6

How does light interact with molecules?

Answer 6

Scattered - the direction of propagation changes

Absorbed - energy is transferred to the molecule

Question 7

What happens when energy is absorbed?

Answer 7

When the electromagnetic radiation is absorbed and the molecule becomes excited

The area that absorbs the energy is the chromophore

The energy acquired moves and electron to another orbital - this is called a transition
Ground state -> first excited state

Question 8

What do excited molecules possess?

Answer 8

Discrete amounts of energy = quanta

Amounts - electron energy levels

Vibrational energy levels - superimposed on electron energy levels

Question 9

What can we measure as a graph?

Answer 9

Probability of photon absorption vs wavelength

This is called the absorption spectrum

Question 10

What is the beer lambert law?

Answer 10

A = εcl

A - absorbance
ε - molar extinction coefficient
c - concentration
l - length

Question 11

What is significant about the beer lambert law?

Answer 11

Where the absorbance appears proportional to concentration the beer lambert law is obeyed

If concentration is increased it can lead to: oligomerisation, aggregation and denaturation

Question 12

How do we measure absorbance?

Answer 12

Using a spectrophotometer

Light source
Monochromator - provides selectivity of wavelength 
Sample holder
Detector - photocell
Recorder

The spectrophotometer needs to be zeroed

Question 13

What factors affect the absorption of a chromophore?

Answer 13

Polarity
pH
Orientation

Question 14

How does polarity affect absorption of a chromophore?

Answer 14

A polar chromophore placed in a polar solvent can lead to:

Hypsochromic shift - shorter wavelengths (more blue)
Non bonding orbital -> pi anti-bonding orbital

bathochromic shift - longer wavelength (more red)
pi bonding orbital -> pi antibonding orbital

Question 15

How does pH affect the absorption of a chromophore?

Answer 15

pH can determine the ionisation state of a chromophore that is ionisable
This can shift the equilibrium to the right sue to: more delocalised electrons and increased molar extinction co-efficient (bathochromic shift)

Question 16

How does orientation affect the absorption of a chromophore?

Answer 16

The geometric features of nucleic acids can lead to:

Hypochromism - a materials decreasing ability to absorb light

Hyperchromism - a materials increasing ability to absorb light

Question 17

How do we correct scattering?

Answer 17

This is needed if particles are in suspension and not solution
- as it leads to an artificially high absorbance

To correct: measure points away from the max wavelength in order to extrapolate the linear region

Question 18

What is a reporter?

Answer 18

A ‘non-natural’ molecule added if the molecule has no chromophore

It has to bind at a single site and can’t affect the function or interaction

Question 19

What information can be obtained from UV-visible spectroscopy?

Answer 19

The concentration of a substance
An assay of certain chemical reactions
Identification of materials
Solvent perturbation

Question 20

What is solvent perturbation?

Answer 20

If you change the solvent of nucleic acids from H2O to 50% of D2O
It can change the mono-nucleotides but not the base pairs

This is due to N-D having a greater reduced mass, altering the vibrational levels and disallowing the ability to hydrogen bond

Question 21

What can happen after absorption of a photon?

Answer 21

The energy can be reemitted as heat
OR
Emit a lower energy photon seen as fluorescence

Question 22

How does fluorescence work?

Answer 22

A photon is absorbed by a fluorophore and the excited electrons move to a higher energy level (the first excited state) in a transition
When the excited molecule relaxes a new low energy photon is emitted

The emission spectrum is shifted to a longer wavelength compared with the excitation spectrum (Stokes shift)

Question 23

How is probability of fluorescence described?

Answer 23

The quantum yeild (Q)
Q = ratio of emitted photons to absorbed photons

Q is determined by:
The properties of the molecule itself
The local environment of the molecule

Question 24

How else can energy be transferred is not heat/fluorescence?

Answer 24

If the vibrational levels of the excited and ground states overlap, energy can be transferred from the higher energy level to the lower – without re-emission of a photon, therefore no net transfer of energy
= nonradiative transfer

Question 25

How is fluorescence measured?

Answer 25

Using a fluorimeter

Commonly measured at 90º or as backscatter because fluorescence is emitted in all directions

Question 26

What type of fluorescence is measured?

Answer 26

Intrinsic fluorescence: an intrinsic fluorophore is one contained within the macromolecule
an amino acid (W, Y or F) – (Mainly tryptophan as it has the highest yield – most commonly studied)

Extrinsic fluorescence: an extrinsic fluorophore is one added to the macromolecule
It should be attached at a single site and not change its properties

Each fluorophore can be distinguished by its excitation and emission wavelengths

Question 27

What environmental impacts can we use to alter fluorescence readings?

Answer 27

Polarity
Quenchers
Temperature
pH
Ligand binding

Question 28

What are quenchers?

Answer 28

Quenching a process which decreases the fluorescence intensity of a given substance

Tryptophan quenchers: iodine, nitrate, cesium and acrylamide

A quencher only works if the fluorophore is on the surface not buried

Question 29

What is FRET?

Answer 29

FRET - Förster Resonance Energy Transfer
If the emission of one fluorophore overlaps with the absorption peak of a second fluorophore the energy can be transferred between them in a nonradiative process