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Biochemistry > Spectroscopy and spectrometry! > Flashcards

Spectroscopy and spectrometry! Flashcards

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1
Q

What is interference?

A

When waves meet

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2
Q

What are the types of interference?

A
  1. Constructive: Crests and troughs of waves align in phase
  2. Destructive: crests and troughs align 180 degrees out of phase (opposite eachother - essentially cancel eachother out into a straight line)
  3. Intermediate: inbetween constructive and deconstructive waveforms
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3
Q

What is the energy of a wave directly proportional to?

A

Proportional to amplitude (A) squared

e.g. louder sounds carry more energy

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4
Q

What are the 4 ways energy can be released?

A
  1. Translation
  2. Rotation
  3. Oscillation
  4. Electronic excitation
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5
Q

What is translation of energy?

A

Movement of a molecule through space

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6
Q

What is rotation of energy?

A

Molecule spinning around its centre of mass

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7
Q

What is oscillation of energy?

A

Atoms within a molecule moving back and forth like springs

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8
Q

What is electronic excitation?

A

Where electrons are promoted to higher energy levels, causing fluorescence - this requires more energy than other methods of energy release

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9
Q

What is the order of relative energies within molecules?

A

Translation > Rotation > Oscillation > Electronic excitation

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10
Q

What is the equation for calculating energy?

A

E = hf
* h = Planck’s constant
* f = frequency in Hz
* E = energy in J

frequency can also be written as v!!

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11
Q

How do you calculate transmittance (T) of radiation through an object?

A

T = transmitted radiation / incident radiation
OR
T = 10^-A

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12
Q

How do you calculate absorbance (A) from T?

A

A = log 1/T

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13
Q

What is Beers law?

A

A=εcl
* A = Absorbance
* ε = molar absorptivity (L/mol/cm)
* c = concentration of the solution (mol/L)
* l = path length (cm)

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14
Q

Order the molecular orbitals from highest to lowest in terms of energy

A

Sigma > Pi > non bonding orbitals (n) > Pi(star) > sigma(star)

wont let me use asterisks here!!

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15
Q

Where does an electron move to when it has been excited (e.g. when it absorbs light)?

A

HOMO > LUMO

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16
Q

What are HOMO and LUMO?

A
  • HOMO = Highest Occupied Molecular Orbital e.g. σ, π
  • LUMO = Lowest Unoccupied Molecular Orbital e.g. σ(star) and π(star)
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17
Q

What transition has the highest ΔE: σ to σ(star) OR π to π(star)?

A

σ to σ(star) has the highest ΔE (as it is literally moving from the lowest energy level to the highest)

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18
Q

Which is bigger: the wavelength of excitation, or the wavelength of emission?

A
  • The wavelength of emission has a higher wavelength
  • This is because some energy is lost when the e- is in its excited state, and therefore has less energy to emit
  • Less energy = larger wavelength
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19
Q

What is fluorescence spectroscopy?

A

Fluorescence spectroscopy is a technique used to study substances that absorb light at one wavelength and then emit light at a longer wavelength (lower energy)

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20
Q

What does fluorescence spectroscopy tell us?

A
  • What substances are present
  • How much is there
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21
Q

What is the excitation spectrum in fluorescence spectroscopy? What is measured?

A
  • The input excitation wavelength is varied
  • Emission intensity is recorded - this tells you what wavelength excites the molecule best
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22
Q

What is the emission spectrum in fluorescence spectroscopy? What is measured?

A
  • Excitation wavelength is fixed
  • Emission wavelength is varied
  • Emitted light is measured across varying wavelengths - this tells you the colour of the wavelength
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23
Q

What is emission intensity proportional to?

A

Concentration

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24
Q

What is UV-vis spectroscopy used for?

A
  • Quantitative measurement of coloured and fluorescent substances
  • Tagging of targets
  • Histochemistry and imaging (seletive staining of cells/organelles)
  • Changes of molecules depending on their surroundings
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25
What is a chromophore?
A light-absorbing part of the molecule They have pi or sigma electrons
26
What is an auxochrome?
A group attached to a chromophore that changes: * How **strongly** the mol absorbs light * What **wavelength** of light it absorbs ## Footnote They do not produce light themselves
27
What are the four ways in which an auxochrome affects the absorption of light by a chromophore?
1. Bathocromic shift: to red region 2. Hypsochromic shift: to blue region 3. Hyperchromic shift: Increases absorbance intensity 4. Hypochromic shift: decreases absorbance intensity
28
What effect does the positioning of double bonds have on a molecule's absorbance?
- Isolated double bonds have little effect on absorbance - this is because the electrons are localised - Conjugated double bonds have delocalisation of e-, more stability, this lowers the HOMO-LUMO gap = bathochromic shift - Cumulated double bonds have less delocalisation = hypochromic shift
29
What is co-planarity?
Where different groups of a molecule lie in the same plane
30
How does co-planarity affect absorption?
- Co-planarity increases a molecule's absorbtion - This is because the electrons delocalise into an extended conjugation because the pi bonds are flat Absorption being stronger shifts the max wavelengths into longer wavelengths ## Footnote Essentially, the more declocalised the e- become, the lower the HOMO-LUMO gap, and the more bathocromic the shift is
31
If there is a small HOMO-LUMO gap, what region does absorption occur in?
- Red regions - bathochromic shift - This is because there is a smaller energy difference between the HOMO and LUMO, and the red side of the visible light spectrum is lower energy ## Footnote This can pass into infrared regions
32
What analogy can be used to describe delocalisation of e-
Think of delocalization like flattening a hill between two valleys. The more flattened the hill (i.e., the more delocalised the system), the less energy it takes to climb from one valley to the other ## Footnote the hills are HOMO and LUMO
33
What is mass spec based on? What is it used for?
- MS is based on the manipulation of ions in the gas phase in a vacuum - Used for identifying and characterising molecules by their m/z ratio
34
What are the 3 main steps in mass spectrometry?
1. Ionisation and vaporisation 2. Mass filtering 3. Detection, converted into a mass spectrum (graph of intensity vs. m/z)
35
What 3 methods are there of generating ions?
1. Electrospray ionisation 2. Electron impact ionisation 3. Matrix-assisted laser deportation ionisation ESI, EII, MALDI
36
What happens in electron impact (EI) ionisation? What compounds is it used for?
- High energy electrons bombard molecules, causing ionisation - leaves a highly unstable molecule - Used for small, volatile and thermally stable compounds and produces ion molecules
37
What happens in ESI ionisation? What compounds is it used for?
Produces ions by applying a high voltage to a liquid sample, creating a fine mist Operates under atomspheric pressure - Ideal for large, polar, or non-volatile molecules and creates multiply charged ions Little fragmentation occurs ## Footnote electro (electric) spray (creates a mist)
38
What happens in MALDI ionisation? What compounds is it used for?
Utilises a laser to ionise analytes embedded in a solid matrix - laser carries the sample into a gas phase - Effective for analysing large biomolecules like proteins and polymers which are fragile Produces singly charged ions Very little fragmentation
39
What happens in chemical ionisation (CI)? What compounds is it used for?
- Gaseous ions transfer their charge to the analyte - Produces larger fragments than EI - Used for small, volatile, and thermally stable compounds
40
How do you calculate the kinetic energy of the particle once it has been accelerated?
KE = 0.5 x m x v^2 * m = mass in kg * v = velocity in m/s
41
What is a mass analyser?
It is the engine that allows the instrument (the mass spectrometer) to measure different ions
42
What different types of mass analysers are there?
1. Sector field 2. Quadrupole 3. Ion trap 4. Time of Flight (ToF)
43
How does sector field mass analysis work?
- Uses a magnetic field to separate ions based on their momentum - Commonly used for high-resolution mass measurements
44
How does quadrupole mass analysis work?
- Composed of 4 rod that make an oscillating electric field - Filters ions by stabilising **specific** m/z ratios - Often used for targeted analysis
45
How does ion trap mass analysis work?
- Traps ions using electric fields in a 3D region with a ring electrode - Sequentially ejects ions for detection, allowing detailed fragmentation studies
46
How does Time of Flight (ToF) mass analysis work?
- Measures the time ions take to travel a fixed distance - Lighter ions reach the detector faster, enabling separation by m/z
47
What are the main uses of mass spectrometry?
- Identifying unknown compounds. - Determining molecular weights. - Studying molecular structures through fragmentation patterns. - Analyzing complex biological molecules.
48
What is spectroscopy?
Spectroscopy is the study of how matter interacts with electromagnetic radiation (light) — especially how it absorbs or emits energy
49
What is electromagnetic radiation made up of?
An electric field (E), and A magnetic field (H) These fields oscillate perpendicular to each other and to the direction of the wave.
50
What does Snell's law describe?
Refraction - bending of light as it passes through materials
51
What is diffraction?
Light bends around edges or through small openings
52
Define polarisation
Restricts the vibration of waves to one direction only
53
Easy absorption vs emission
54
What is Beer's law?
Describes how absorbance depends on: Concentration of substance c Path length d Molar absorptivity E A=ε⋅c⋅d
55
What is UV-Vis spectroscopy?
UV-Vis spectroscopy studies how molecules absorb ultraviolet and visible light. This absorption causes electrons to move from lower to higher energy molecular orbitals — this is called electronic excitation
56
This gives the electronic transitions - before looking, what jump requires the most energy?
sigma to sigma*
57
Easy way to remember HUMO LUMO
Electrons are tenants HOMO is the highest floor with tenants (electrons) LUMO is the next empty floor When a molecule absorbs light, an electron is promoted from the HOMO to the LUMO. HOMO→LUMO This jump = electronic excitation
58
When would an electron emit light?
When it relaxed down an energy level
59
Which is higher energy, fluorescence or phosphoresence?
Fluorescence
60
What are conjugated double bonds?
Conjugated double bonds are double bonds that alternate with single bonds within a molecule Causes electron delocalization → more stable → lower energy transition
61
What is the visible region?
400-700nm
62
What is a mass spectrum?
A mass spectrum is a graph: X-axis: m/z (mass-to-charge ratio) Y-axis: Intensity (relative abundance)
63
What is tandem mass spectrometry MS/MS used for?
Used to study fragmentation of selected ions

Biochemistry (14 decks)

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