Brainscape's Knowledge GenomeTM

Browse over 1 million classes created by top students, professors, publishers, and experts.


See full index

1. BIOC 1301 S1 > Spectroscopy > Flashcards

Spectroscopy Flashcards

1
Q

What is light?

A

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

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
2
Q

What equation involves wavelength?

A

Energy (E) = hc/λ

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

OR

λ = c/v

v = frequency (s-1 or Hz)

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
3
Q

Define the types of energy radiation?

A

High energy radiation -
short wavelength and high frequency

Low energy radiation -
long wavelength and low frequency

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
4
Q

What is amplitude?

A

The maximum value the electric or magnetic vector can have

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
5
Q

What happens when two waves are superimposed on each other?

A

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

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
6
Q

How does light interact with molecules?

A

Scattered - the direction of propagation changes

Absorbed - energy is transferred to the molecule

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
7
Q

What happens when energy is absorbed?

A

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

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
8
Q

What do excited molecules possess?

A

Discrete amounts of energy = quanta

Amounts - electron energy levels

Vibrational energy levels - superimposed on electron energy levels

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
9
Q

What can we measure as a graph?

A

Probability of photon absorption vs wavelength

This is called the absorption spectrum

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
10
Q

What is the beer lambert law?

A

A = εcl

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

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
11
Q

What is significant about the beer lambert law?

A

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

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
12
Q

How do we measure absorbance?

A

Using a spectrophotometer

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

The spectrophotometer needs to be zeroed

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
13
Q

What factors affect the absorption of a chromophore?

A

Polarity
pH
Orientation

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
14
Q

How does polarity affect absorption of a chromophore?

A

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

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
15
Q

How does pH affect the absorption of a chromophore?

A

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)

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
16
Q

How does orientation affect the absorption of a chromophore?

A

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

17
Q

How do we correct scattering?

A

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

18
Q

What is a reporter?

A

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

19
Q

What information can be obtained from UV-visible spectroscopy?

A

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

20
Q

What is solvent perturbation?

A

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

21
Q

What can happen after absorption of a photon?

A

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

22
Q

How does fluorescence work?

A

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)

23
Q

How is probability of fluorescence described?

A

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

24
Q

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

A

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

25
Q

How is fluorescence measured?

A

Using a fluorimeter

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

26
Q

What type of fluorescence is measured?

A

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

27
Q

What environmental impacts can we use to alter fluorescence readings?

A 
Polarity
Quenchers
Temperature
pH
Ligand binding
28
Q

What are quenchers?

A

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

29
Q

What is FRET?

A

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

1. BIOC 1301 S1 (28 decks)

Brainscape helps you reach your goals faster, through stronger study habits.
© 2024 Bold Learning Solutions. Terms and Conditions