Spectroscopy

Question 1

What do the following types of energy involve?

1. Nuclear
2. electronic
3. vibrational
4. rotational
5. translational

Answer 1

1. Nuclear: protons and neurtrons within the nucleus
2. Electronic: Electrons between a few atoms
3. Vibrational: Few atoms
4. Rotational: Whole molecule
5. Translational: Whole molecule moving large distance

Question 2

Whats the formula for the Boltzmann distribution of molecules over energy levels?

What makes molecules move up into a higher energy level?

Answer 2

As temperature increases, the moelcules inhibit higher energy levels

Question 3

What is electromagnetic radiation?

What is the energy for a given frequency described as?

Answer 3

Electromagnetic radiation has an electric and magnetic component. where the electric component is oscillating and the magnetic component is rotating from north and south

Energy (E) for a given frequency (u) is

E= hu

where h is the Planks constant (6.626x10^-34 J s)

Question 4

Whats the order of the electromagnetic spectrum from the smallest frequency to the largest?

Answer 4

Question 5

What are the conditions in order for the electromagnetic radiation to be absorbed?

Answer 5

• must be two or more energy levels
• Charge displacement must occur:

a. Linear displacement: electric transition dipole
b. Rotation of charge: magnetic transition dipole
* ∆E= hu (must be between the ground and excited state)

Question 6

Once the incident radiation, I₀, is absorbed what are the types of radiation you can get from this?

Answer 6

1. Transmitted radiation, I_t
2. Emitted radiation, I_e

Question 7

What are the characteristics of a spectrum?

Answer 7

Question 8

What does the intensity depend on?

Answer 8

The number of moelcules that can be excited from ground state

Question 9

What determines if population different is small and therefore sensitivity to be low?

Answer 9

∆E is roughly kT

Question 10

Name 3 types of spectroscopy and which one sees more absorption of the electromagnetic radiation?

Answer 10

1. NMR
2. rotational
3. electronic (sees more absorption)

Question 11

What is absorption dependent on?

What equation describes this?

What does each part of the equation represent? Units?

Answer 11

The concentration of absorbing species

The Beer Lambert law:

Log₁₀(I₀/ I_t) = OD = ecl

e= the extinction coefficient (absorbing power of a species at a particular wavelength)

C= the concentration of absorbing species (mol dm^-3)

l= length of solution of light passes through (cm)

Question 12

In the Beer Lambert law, when OD >1 what does this mean?

Answer 12

It is no longer linear–> not accurate

Question 13

What is the Beer Lambert law?

Answer 13

Its combined of 2 laws and each are correlates which state that, the absorbance of light is proportional to the thickness of the sample or absorbance is proportional to the concentration of the sample

Question 14

When is maximum absorbance seen in intensity?

Answer 14

When the transition dipole is aligned with the polarised light

Question 15

The orientations of light leads to differential…

Answer 15

absorption

Question 16

What is natural linewidth determine by?

Answer 16

Lifetime of excited state (t)

Question 17

Whats the Heissenberg uncertainty principle?

Answer 17

it states that its impossible to know simultaneously the exact position and momentum of a particle

That is, the more exactly the position is determines, the less known the momentum and vice versa

Question 18

How does decay from an excited state occur?

Answer 18

• stimulated emission: through application of external field
• conversion to other excited state
• non-radiative loss to environment
• resonance energy transferred to nearby molecule
• chemical reactions

Question 19

Why is spectroscopy important?

Answer 19

1. Study conformation of molecules

• Structure of molecules
• Reports on their environment

2. Quantitation

• Protein/ DNA concentration determination
• Biochemical assays

3. Detection

• DNA sequencing
• Microscopy
• Cytometry

Question 20

Learn absorbance/ emittence

Question 21

Coupling to vibrational states

Answer 21

Question 22

What are the types of electronic transitions?

Answer 22

1. Transition involving p, s and n electrons

Organic compounds:

• Saturated (σ –> σ (< 150 nm)*, n –> σ*(<250 nm))

leads to displacement of charge when σ –> σ*

• unsaturated (π- π*, n–> π* (200-700nm))

2. Transitions involving charge transfer

• electrons move from ligand donor to metal acceptor

3. Transitions involving d and f electrons

• transitions between d orbitals split by the presence of a ligand

Question 23

Whats determines

Answer 23

1. Dependent on molecular structure
2. No easy rule for assignment, however:

• Presence of resonance >300 nm suggests presence of conjugated polyene chain/ polycyclic aromatic
• Conjugated systems typically have larger extinction coefficients

3. Refer to databases (as above doesn’t give much detail)

Question 24

What are the changes that can occur in a UV/ visible spectra?

Answer 24

Question 25

Name the chromophores found in biology?

Answer 25

1. **Proteins** 2. **Nucleic acids** 3. **​Metal ions**

Question 26

Whats the most common protein chromophore?

Answer 26

Peptide bonds

Question 27

How many transition states are associated with this molecule?

Answer 27

2

Question 28

In proteins sidechains are there transition dipoles and conjugates bonds present? What determines a higher extinction coeffient in a sidechain?

Answer 28

yes The number of delocalised electrons

Question 29

What can influence energy levels? and how?

Answer 29

Solvent polarity by stabilising or destabilising the excited state

Question 30

How does changing the exposure to light influence the wavelength in prosthetic groups?

Answer 30

When in dakr conditions and then exposed to light, the wavelength can decrease

Question 31

Do nucleic acids have more transition dipoles then protens?

Answer 31

Yes

Question 32

How does nucleic acid transition coupling effect the excited state? What does coupling depend on?

Answer 32

When two transition dipoles are coupled, they interact, splitting the energy levels (excitation coupling) The coupling depends on relative orientation

Question 33

1. When the transition dipole is parallel to the induced dipole what occurs? 2. When the transition dipole os collinearl to the induced dipole what occurs?

Answer 33

1. Hypochromism 2. Hyperchromism

Question 34

1. What effect does structured DNA cause 2. What effect does unstructured DNA cause

Answer 34

1. Structured DNA --\> parallel dipoles **Hypochomism** 2. Unstructured DNA --\> Dipole unaligned **hyperchromic** shift