2. Molecular Spectroscopy and Structure Flashcards
1
Q
What are the two perpendicular components of electromagnetic radiation?
A
Electric field and magnetic field (these are perpendicular to each other)
B (magnetic field) and E (electric field) are in phase with each other
2
Q
What are the three key properties of a wave?
A
Wavelength, frequency, speed
C = v.lambda
3
Q
What is wavenumber?
A
The reciprocal of the wavelength in cm^-1
4
Q
What equation allows you to find the energy of a photon?
A
E = hv = hc/lambda
5
Q
What is the equation for the refractive index n(lambda)?
A
n(lambda) = c/speed of light in material
6
Q
What does strength refer to in terms of waves?
A
Amplitude of wave - same for B and E
7
Q
Which component of the electromagnetic wave dominates? By how much?
A
The electric field component, 10^5 times stronger than the magnetic field. All light-matter interactions thus stem from electric field interactions.
8
Q
What axis is the Z axis in an electromagnetic wave?
A
The direction of travel
9
Q
What equation represent the electric component of the electromagnetic radiation?
A
E(t,z) = E0.cos(omega.t - kz.Z)
E(t,z) = electric field vector at position z and time t E0 = amplitude of the wave (electric field strength) omega = angular frequency - 2pi.v t = time kz = wave vector - 2pi/lambda Z = position
10
Q
What is the units of the electric field strength?
A
N/C or V/m
11
Q
How is the energy carried by a classical light wave usually expressed?
A
As energy density U
12
Q
What are the equations for energy density and intensity/irradiance of light waves?
A
Energy Density U (J/m^3) = 0.5(epsilon0)|E0|^2
Intensity I (W/m^2) = 0.5c(epsilon0)|E0|^2
Epsilon0 = permittivity of a vacuum - 8.854x10^-12 F/m E0 = amplitude of the wave c = speed of light
13
Q
What was Planck’s postulate?
A
Light energy is quantised into discrete packets called photons with Energy hv.
14
Q
What is the equation for finding the force of an electrically charged particle?
A
F = qE
F = force of particle q = electric charge of particle E = electric field
15
Q
How does a radio antenna work?
A
Movement of an electron in a transmitter generates an electric field. The filed may then move through space as an EM wave, this hits a receiver and causes an electron to feel the oscillating electric field. This causes the 2nd electron to start moving (electrons raised up an energy level).
16
Q
What is the ground state of an electron?
A
A point at which the electron is simultaneously in it’s lowest vibrational, rotational and electronic energy levels. Molecules in levels higher than this are said to be in the excited state.
17
Q
Can an electron be excited differently and simultaneously?
A
Yes, a molecule may absorb a photon that excites it both electronically and vibrationally (as long as photons energy = electronic + vibrational excitation energy)
18
Q
Can the energy of a photon be partially absorbed?
A
No, once a molecule absorbs a photon all the energy of that photon must be absorbed.
19
Q
What equation determines the electronic states of hydrogen atoms?
A
Rydberg equation:
En = -h.c.R/n^2
En = energy of level n n = energy levels - 1, 2, 3 etc
20
Q
What is the resonance condition?
A
The ability of an atom/molecule to absorb a photon ONLY when the energy of the photon corresponds precisely to the energy separation between 2 quantum states.
21
Q
How may an excited molecule lower it’s energy?
A
- Spontaneous emission of a photon
- stimulated photon emission by absorption of a second photon (LASER)
22
Q
For a system at equilibrium at room temperature, are all molecules in their ground rotational, vibrational and electronic states?
A
Nope, at equilibrium the molecules int he system will be spread between different energy levels depending on the amount of thermal energy available to them. Here the lower energy levels will be more populated than the higher energy levels though.
23
Q
What is the boltzmann distribution equation?
A
ni/n0 = (gi/g0)exp(-deltaE/Kb.T)
ni = number of molecules in energy level i n0 = number of molecules in the ground state gi = degeneracy of level i g0 = degeneracy of the ground state exp = e to the power ... deltaE = difference in energy between level i and ground state Kb = boltzmann constant (1.38x10^-23 J/K) T = temperature in K
24
Q
How are the four states populated at varying temperatures?
A
NMR: almost equally populated
Rotational: excited are well populated
Vibrational: excited state only populated at high temperatures
Electronic: will remain in ground state unless you excited them with something other than heat (although there’ll be a few in excited state as you increase temp)
25
How does the process of UV/vis absorption spectroscopy work? Why are their broad peaks in the spectra?
Shining a light source through a sample and recording the signal attenuation (how much light drops) as a function of wavelength.
Photons will only be absorbed in their energy is precisely equal to the energy gap between two quantum states (resonance) so peaks occur, defining a footprint.
Saying this, broadening of absorption/emission peaks occurs due to quantum uncertainty, collisions between molecules and instrumentation
26
How does emission spectroscopy vary form absorbance spectroscopy?
Sample first excited by illumination with short electromagnetic pulse. Then any subsequent luminescence is recorded.
The incident light is monochromatic
27
How does the incident light of absorbance and emission spectroscopy vary?
Absorbance spectroscopy emits light of multiple wavelengths whereas the incident light of emission spectroscopy is monochromatic.
28
What is the benefit of emission spectroscopy?
A lack of background signal makes it more accurate
29
How are absorption and emission spectrum related in simple systems? In what situation does this occur?
They are usually mirror images of each other
molecular must have: similar geometries in ground and excited state, and emission must be from lowest vibrational level
30
What is the beer-lamber law?
I/I0 = exp(-e.c.l)
OR... log10(I0/I) = A = e.c.l
e = natural molar absorptivity
31
What observations did Mr. Beer and Mr. Lambert make?
- fraction of light transmitted decreases exponentially w/ increasing distance it travels through the sample
- fraction of light transmitted decreases exponentially w/ increasing concentration of solute
32
What do we assume for the beer-lambert law?
Thinking of sample in cuvette as series of thin slices width dl.
1. All photons travelling perpendicular to large face of 'slice'
2. If a molecule within this slice lies in the path of a photon then it will absorb the photon
3. Each molecule has an effective cross section: rho
4. No cooperative absorption between molecules
5. Absorbing species forms single homogenous phase and doesn't scatter light
33
What fraction of the area of the 'slice' is opaque to photons?
Equal to the ratio of the total cross sectional area of all the absorbing molecules compared to the total area of the large face of the slice
--> number of photons absorbed in slice is proportional to the concentration
34
How are the many very small slices combined together?
An integral is used,
this is because the slices are very thin and the intensity of the light entering the slice depends on the light leaving the previous slice
35
What is another name for the decade molar absorptivity?
The extinction coefficient
36
What is the transmittance T?
The ratio I/I0 (%)
37
What is the relationship between T and A?
A = -log10(T)
38
What is the units of the extinction coefficient?
dm3.mol-1.cm-1
39
Describe a typical beer-lambert plot of A against c
The gradient of the line would be linear initially, but at higher concentrations the gradient would become less steep
40
Why does the deviation from linear behaviour occur in the beer-lambert plot?
- Refractive index changes with conc.
- Absorbance saturates
- Association/Dissociation of the solute
- Solute may fluoresce, contributing to I
41
How do we use absorption spectroscopy to determine the concentrations of two distinct solute species in a solution? What assumptions are made? When is this method most effective?
The measured absorbance at a given wavelength is the sum of the contributions from each solute. So, measure the absorbance of the solution at two separate wavelengths (lambda 1 and 2), knowing the two molar absorptivity's, we can put solve this using simultaneous equations.
Assume that: solutes do not react and both obey the Beer-Lambert law
Most effective when one solution absorbs more strongly at one wavelength and vice versa
42
How may you calculate concentrations of two solutes which REACT using absorption spectroscopy (e.g. a weak acid)?
Vary the degree of dissociation by varying pH of the solution. Carry out 3 experiments, at low, med and high pH. At low pH can assume all HA, at high pH all A- and in the middle it'll be a mixture of both.
Low pH: Eha = A1/c'
High pH: Ea- = A2/c'
Med pH: A3 = Eha[HA] + Ea-[A-]
Rearrange to find [A-] or [HA]
[HA] = c'.((A2-A3)/(A2-A1))
If you choose med pH as the pH of the solution you are testing then can find out the concentration of HA and A- in it.
pKa = pH - log({A3-A1}/{A2-A1})
43
What is the Henderson-Hasselbach equation?
pH = pKa + log([A-]/[HA])
44
What wavelength should a spectroscopy involved solutes which react be undertaken at?
A wavelength at which the absorbance of the products are very different form one another
45
What is the isobestic point?
A point on a (absorbance/ wavelength) plot where all absorbance curves converge - the absorbance is independent of pH At this point. So literally the worst for testing but shows there is true equilibrium between the solutes
46
What broad categories can we separate molecular orbitals into (2)?
Valence - bonding orbitals of the atoms - change significantly in reactions/ionisation etc
Core - core orbitals of the atoms - don't change
47
What 6 categories can molecular orbitals be split into? Describe each category briefly
1. sigma bonding orbitals - constructive overlapping along line joining centre of atoms - no modal planes between nuclei - single bonds - form molecular backbone but not often involved in chemical reactions
2. sigma* anti-bonding orbitals - destructive overlapping - one perpendicular modal plane - much higher energy than sigma
3. pi bonding orbitals - constructive p atomic orbital overlap above & below atom bonding centres - double bonds - one nodal plane in plane of nuclei
4. pi* anti-bonding orbitals - two nodal planes, in plane and perpendicular to plane of nuclei - pi-pi* orbitals have much less energy separation than sigma (UV vis)
5. n non-bonding (not bonding/anti-bonding) - no restriction on nodal planes - lone pairs - least tightly bound
6. d electrons - colourful
48
What is a chromophore? How do they fluoresce?
The chromophore is a region in the molecule where the energy difference between two different molecular orbitals falls within the range of the visible spectrum.
Visible light that hits the chromophore can thus be absorbed by exciting an electron from its ground state into an excited state
Two categories
49
How would you label the molecular orbitals of methanal?
1sO
50
What is the shorthand for the n to pi* transition?
pi*
51
What is the electronic state of a molecule?
The electronic configuration and arrangement of electrons' spins in a molecule
52
What would the ground state of a closed shell molecule look like?
All electrons paired up in molecular orbitals so their spins cancel to give a total angular spin angular momentum of 0.
e.g. 2 Electrons in the sigma MO (one spin up and one spin down)
53
If you excite a closed shell species, what two possible spin states may occur?
Singlet excite state - electrons in ground and excited states have opposite spin (S = 0)
Triplet excited state - electrons in ground and excited state have the same spin (S = 1)
54
What does S stand for in electronic configuration stuff?
Total spin angular momentum
55
What is the name of states with S=0 and S=1? How does the energy of these states vary?
```
S = 0 = Singlet = S
S = 1 = triplet = T
```
```
S0 = ground state
S1/T1...S3/T3 = excited states
```
There is no T0 state - at the ground state all stuff is singlet as most molecules have closed shell ground state configurations
56
If they have the same electronic configuration, would the triplet or singlet state typically be lower in energy?
Triplet state is typically lower in energy than the singlet state
57
What is the electronic configurations notation including electronic states?
If: (pi up)(n)(pi* down) then 1(pi, pi*)
58
Why does electronic excitation not occur with thermal energy?
Because the electronic states are separate day large energy gaps - too large for thermal excitation to broach. So electronic excitation occurs with absorption of photon of correct frequency.
59
What is the equation for excitation due to photon absorption?
S1 = S0 + hv
60
What is Iabs?
The intensity/rate of absorption of photons by electrons
61
What does the selection rule state?
Transitions between electronic states as the result of the absorption/emission of a photon must occur with no change in spin multiplicity
Most molecules have singlet ground states so absorption is singlet->singlet
62
Remembering the selection rule, how is possible for triplet states to be populated directly from the ground state (3)?
- Bombarding molecule with electrons
- Collisions with other electrically excited molecules
- Chemical reactions that leave a product in an excited state
63
What are the 7 major relaxation pathways?
1. Fluorescence
2. Phosphorescence
3. Vibrational relaxation
4. Internal conversion
5. Intersystem crossing
6. Physical quenching
7. Chemical reaction (with another species)
64
Describe the process of fluorescence and state it's related equation. What timescales does this process occur on?
-Radiative: Emission of a photon with no change in spin (allowed by selection rule)
- S1 --> S0 + hv
- Rate Kf[S1] = 10^-5 - 10^-9 seconds
65
Describe the process of phosphorescence and state it's related equation. What timescales does this process occur on?
- Radiative: Emission of a photon with a change in spin (forbidden by selection rule)
- T1 --> S0 + hv
- rate lp[T1] = 10^3 - 10^2 S
66
Describe the process of vibrational relaxation and state it's related equation. What timescales does this process occur on?
- Non-radiative: Change in vibrational level but no change in electronic state
Vibrational energy from one species is transferred to another during collision - energy can go into rotational. vibrational or translational modes of the collided particle. VR is efficient as there is almost always a state with the correct energy to excite collided particle.
- S1 --> S1 + heat
- Rate Kvr[S1] = 10^-12 S
67
Describe the process of internal conversion and state it's related equation. What timescales does this process occur on?
Non-radiative: change in electronic state with no change in spin
- S1--> S0 + delta (heat)
- rate Kic[S1] = SLOW
Electronic energy converted into vibrational energy on ground electronic surface, vibrationally relaxes producing heat
68
Describe the process of intersystem crossing and state it's related equation. What timescales does this process occur on?
Non-radiative: change in electronic state with a change in spin
- T1--> S0 + delta (heat)
- rate Kisc[T1] = quite slow
Electronic energy in upper state converted to vibrational energy on lower state, rapidly decays to lowest vibrational level of lower electronic state by vibrational relaxation
69
Describe the process of physical quenching and state it's related equation. What timescales does this process occur on?
Loss of electronic excitation via collision with another molecules
- S1 + Q --> S0 + Q + delta
- speed depends on pressure
Electronic excitation becomes translational motion in the quenching species (heat)
70
Describe the process of chemical reaction and state it's related equation. What timescales does this process occur on?
Chemical reaction with another species (e.g. isomerisation, dimerisation)
- R*(S1) --> R1(S0) + R2(S0)
OR --> R1*(S1) + R2*(S1)
- time depends on reactant/physical conditions
71
How do relaxation processes compete with each other?
Depends on the specific molecule, basically whichever pathway occurs fastest will preclude ability of molecule to travel down other pathways
72
What is the name of the diagrams drawn to represent the transitions between different electronic states?
Jablonski diagrams
73
How does the electronic energy vary as bond length changes?
Goes down then up again :) - look at page29
74
What does a Jablonski diagram look like?
Electronic states are listed horizontally and show the vibrational levels vertically (page 30)
75
How and when do you simplify a jablonski diagram to an energy state diagram?
For systems with efficient vibrational relaxation (like gases and liquids) we don't need to account for vibrational relaxation so can put all the singlet states in one column and all the triplet states in a another column
76
What equation shows the rate of change of concentration S1 in quenching?
The rate of change of concentration of S1 given by:
d[S1]/dt = Iabs - Kf[S1] - Kq[S1][Q] = 0
=0 is because S1 is considered as a reactive intermediate and so assumed that after a short period of initiation its concentration approaches a constant value --> Steady-state approximation (SSA) - linear relationship can then be derived for the Stern-Volmer plot
77
What is the equation of the Stern-Volmer plot?
Look on page 31
1/If (Y) is plotted against [Q]
Stern Volmer plots are used assuming that the only deactivation pathways are quenching and fluorescence
78
Page 32 kill yourself
:)
79
What is the equation for the primary quantum yield?
(number of excited molecules, R*, undergoing process i)/(total number of photons absorbed by R)
Where we are assuming that the initial R* state is formed only by absorption of a photon and not by any secondary processed.
80
What does Nf, Nic, Np, Nisc and N stand for? What are their related Singlet/Triplet equations?
Nf: number of excited molecules that fluoresce back to S0 (S1--> S0 + hv)
Nic: number of excited molecules that internally convert back to S0 (S1--> S0 + delta)
Np: number of excited molecules that phosphoresce back to S0 (T1 --> S0 + hv)
Nisc: number of excited molecules that intersystem cross back to S0 (T1 --> S0 + delta)
N: total number of excited molecules
N = Nf + Nic + Np + Nisc
81
What is the equations for the quantum yield of fluorescence and phosphorescence?
(Phi) f = Nf/N
(Phi) p = Np/N
82
What does the sum of all the quantum yields add up to?
1
(assuming that the only way R* is produced is by photon absorption, and that all R* ultimately relax form excited R* state to lower energy states)
83
How do we demonstrate that all the primary processes are competing with each other to reduce fluorescence?
Assuming that:
Nisc (S1-->T1)= Np + Nisc (T1-->S0)
Therefore:
(Phi)isc (S1-->T1) = (phi)p + (phi)isc (T1-->S0)
You can show that all the primary processes are competing with each other. the primary quantum yield gives a useful indication of the efficiency of each process and therefore an indication as to the relative likelihood that a given process will win for a given excite molecule.
84
What do biological studies use to measure emission?
Fluorophores - have high quantum fluorescent yield (bright) and long tau (time)
85
How does the tryptophan emission vary depending on it's position on the protein?
If the Trp is on the surface of the protein then the emission will be at a longer wavelength than if it was in the proteins interior
86
How do we probe membranes?
Attach fluorophores to lipids. DPH fluoresces in membrane but is quenched by water.
87
What are fluorophores typically bound to?
DNA - to enhance its emission
88
What plot would we use to determine the rate of quenching?
A Stern-Volmer plot. Quenching is the process of other non-radiative processes competing with radiative (fluorescence and phosphorescence) to de-excite electrons
89
How may tyrosine fluorescence become quenched?
By amino, carboxyl groups or tryptophan.
90
How do bromide ions quench?
Br- have states at similar energies to excited states of organic molecules. So energy is transferred from excited organic molecule to the Br- when the species collide.
91
How is oxygen a peculiar quencher?
Exists in a triplet state in its ground state and is also able to promote non-spin-conserving transitions (e.g. ISC)
92
What is the general concept of FRET?
In physical quenching if the acceptor molecule (A) itself fluoresces. AKA An excited molecule (D) collides with a quencher which is in itself capable of fluorescing.
Can therefore monitor the energy transfer by observing fluorescence of acceptor molecule
93
What is the reaction scheme for FRET?
D(S0) + hv --> D*(S1)
D*(S1) + A(S0) --> D(S0) + A*(S1)
A*(S1) --> A(S0) + hv'
94
What is the difference between FRET and physical quenching?
In physical quenching the quenching molecule tends to remain in the ground state (and release heat energy). In FRET the electron transfer excited the acceptor molecule (A) to an excited electron state.
95
How do we envisage electronic excitation? HINT: coupling
Electronic excitation is the result of coupling between the oscillating electric field vector (in light waves) and the induced oscillating dipole in the molecule (D).
96
How does FRET work?
Use D as the donor molecule and A as acceptor.
"Electronic excitation is the result of coupling between the oscillating electric field vector in light waves and induced oscillating dipole in molecule D"
So the dipole is caused to oscillate by light waves.
If A is close to D and can absorb light of a similar wavelength then the oscillating dipole in D can stimulate an oscillating dipole in A. If the energy gap between the excited/ground states of A and B are similar then the resonance condition is satisfied and electronic excitation from D* may be converted into electronic excitation in A*
97
What does FRET stand for?
Fluorescence resonance energy transfer
OR
Forster resonance energy transfer
98
When can FRET only occur?
- When D and A are separated by less than 100angstroms
| - When the emission spectrum of D overlaps with the absorption spectrum of A
99
May the chromophores involved in FRET be part of the same molecule or not?
They may be part of the same molecule, and usually are... (BIOC2005-photosynthesis)
100
When is FRET analysis valid?
When donor and acceptor chromophores are connected via a covalent network
101
What is the symbol for the efficiency of energy transfer between D and A? What is the related equation involving R? What is the equation used to find (eta)T?
(Eta)T
1/(Eta)T = 1 + (R/R0)^6
```
R = distance between D and A
R0 = Forster distance at which half of the energy is transferred
```
Eta(T) = 1-((phi)f)/(phi)f0)
(phi) f0 = fluorescence quantum yield of D in absence of A
(phi) f = fluorescence quantum yield of D when A is present
102
What may FRET be used for? How (example)?
Probing changes in conformation of biomolecules
Add donor/acceptor dye genes to N/C-terminal of myosin - observe changes in conformation by observing changes in the FRET efficiency.
103
How may we visualise the FRET process?
Donor species emit a 'virtual photon' (we never see it) that is immediately absorbed by the acceptor
104
What are the uses of FRET in biochemistry (3)?
Hint: examples
1. Determining proximity of tryptophan to active site of carbonic anhydrase
2. Determining shape between hydrophobic groups of bovine serum albumin with ANS
3. Determining the shape of the visual pigment rhodopsin
105
What is the difference between unpolarised and plane polarised light?
Normal light has waves with the electric field pointing gin all directions. Plane polarised light has only waves with a single orientation of the electric field vector (and magnetic).
106
What is normally used to polarise light?
Nicol prisms: rhombohedral crystals of calcite (CaCO3) which are cut and glued back together. The refraction that takes place at the join between the two parts of the prism give rise to the plane polarisation.
107
What does optically active mean?
When plane polarised light passes through a sample containing these molecules, the plane of polarisation is rotated.
The chiral centres in the molecule are responsible for this.
Molecule and mirror image must NOT be superimposable.
108
What is a polarimeter?
An instrument used to measure the optical rotation of molecules in solution.
Consists of a monochromatic light source, fixed polariser, polarimeter tube (containing optically active substance) and a rotatable polariser (analyser).
109
What is the equation for the specific rotation [alpha]?
There is a little T superscript and a little lambda subscript after the ] - you just write these parts in
[alpha] = alpha/l.c
alpha = angle of rotation
l = path length of the sample cell
c =concentration of optically active species IN GRAMS PER 100cm^3
BE CAREFUL WITH UNITS HERE
110
If the medium causes the plane of polarisation to rotate to the right it is____. If it causes it to shift to eat left it is _____.
```
Right = dextrorotatory (+)
Left = levorotatory (-)
```
111
Why does the plane of polarisation rotate? What is the equation which determines the angle of rotation?
In an optically active medium the refractive index is different for left and right circularly polarised light.
The angle of rotation is determined by
alpha = (180/lambda)(nL-nR).l (degrees)
```
lambda = wavelength of incident light
nL = refractive index for left circularly polarised light
nR = refractive index for right circularly polarised light
l = path length of the sample
```
You can use this equation to work out the refractive index :)
112
Keep going you're doing well :)
:)
113
What does a spectropolarimeter do?
This measures how the optical rotation varies with wavelength, producing an optical rotatory dispersion curve.
This is quantified into the molar rotation using the following equation:
Mr.[alpha]/100
AA and NA can be expressed in per mole of repeating unit
114
What is the equation for the refractive index n(lambda)?
n(lambda) = c/(speed of light in medium)
115
What is the optical rotary dispersion?
The variation of optical rotation with wavelength, generate by a spectropolarimeter
116
What is the Cotton effect?
Characteristic change in optical rotatory dispersion in the vicinity of an absorption band of a substance.
In a wavelength region where the light is absorbed, the absolute magnitude of the optical rotation at first varies rapidly with wavelength, crosses 0 at absorption maxima and then again varies rapidly with wavelength in the opposite direction
Positive cotton effect - if optical rotation first increases as wavelength decreases
Negative cotton effect - vice versa
117
What type of protein structure shows a negative Cotton effect?
The beta sheet
118
At which wavelength does the main cotton effect for proteins occur? And nucleic acids?
200nm - absorption by the peptide bond
250-275nm - excitation of the nucleotide bases
119
What is circularly polarised light?
Plane polarised light where the plane of polarisation 'spins' as the light wave propagates. Magnitude of electric and magnetic field vectors stay constant but orientation varies - drawing a helix shape.
120
How is circularly polarised light generated?
The rotation of plane polarised light (clockwise or anti-)
121
What happens if you combine the left and right circularly polarised components?
Becomes plane polarised instead
122
How do you separate the left and right components of a circularly polarised light?
Using a Fresnel Prism
123
What is the basis of circular dichroism?
The fact that chiral species absorb left and right polarised light differently
124
How is the difference in absorptivity in circular dichroism quantised?
By epsilonR and epsilonL. Plots of delta(epsilon) = epsilonL - epsilonR
125
Which is better, ORD (optical rotatory dispersion) or CD (circular dichroism)?
CD, better resolving power
126
What are CD and ORD used for?
Studying conformations of biomolecules in solution (e.g. proteins and DNA). The optical activity is usually as a result of perturbations from other groups in the neighbourhood of the chromophore.
127
What provide the chirality in DNA and proteins?
DNA: the double helix is a good chiral environment
Proteins: the peptide bond has chirality
128
What protein structure is circular dichroism particularly sensitive to?
Secondary structure. Wavelengths of 260nm/180nm can be analysed to find: alpha helices, parallel/anti-parallel beta sheets, turns etc. All these structures have tell-tale CD spectral
129
When plotting a CD spectra what are on the X and Y axis?
Y axis: mean residue differential absorptivity (delta epsilon) in mol-1dm3cm-1
X axis: wavelength in nm
130
What is CD good at? What isn't it so good at?
CD spectra are not good at detecting absolute structural information, but they are good at detecting conformational changes and the magnitude of molecules which undergo a conformational change e.g. protein denaturation and folding, evidence for base stacking, RNA structure in ribosomes
131
What does LASER stand for?
Light Amplification by the Stimulate Emission of Radiation
132
Why are lasers often used in electronic spectroscopy methods? What improvements do they result in?
They produce light of high intensity, highly focussed, coherent and of very short pulses
Enable high resolution spectra to be produced in a short period of time with low detection limits
133
How does stimulated emission occur (in lasers)?
A photon is fired at an excited molecule to stimulate the release of 2 photons
This represents no advance on the intensity of normal luminescence
134
What is the process of population inversion?
Used by lasers, many molecules in an excited state waiting to be stimulated into emission in a cascade process
135
What are the components of a LASER (3)?
Active medium - luminescent material by which electromagnetic wave is amplified
Pump - supplies initial energy to active medium required to electronically activate first molecules, two types:
optical pump: high intensity light source (flash lamp/laser)
electrical pump: supplies energy by active discharge
optical resonator - contains active medium, box with highly reflecting mirror at each end
136
What are the two main types of laser?
3 and 4 level
137
Describe how a three level laser works
1. PUMP: Ruby crystal (Active medium) illuminated by intense flash of radiation (correct frequency used to match energy gap between populated level |0> (energy E0) and initially unpopulated level |1> with energy E1, exciting some Cr3+ ions to |1>.
2. State |1> relaxes through ISC (non-radiative) to long lived |2> state with lower energy E2. Many Cr3+ ions in |2>. Note: pump is too high frequency to stimulate emission from |2> so metastable.
3. When an ion in |2> NATURALLY emits a photon emission is stimulated of another ion in |2>. 2 photons now moving in phase & same direction so these can both now stimulate emission from other |2> ions and emission is amplified = cascade
4. Emission takes place in a cavity with mirrors at either end (one totally reflecting, one partially), it occurs in all directions but only photons moving along axis between mirrors aid lasing. Each emitted photon travelling between mirrors can stimulate emission from many other ions as it is reflected back and forth.
5. This process causes a large build up of photons in the cavity, the partially non-reflecting mirror allows some coherent laser light to escape - beam is produced
Length of cavity must be carefully chosen to avoid destructive interference of light waves.
138
Why does the length of the optical resonator matter?
This must be an integer number of wavelengths of the laser radiation to avoid destructive interference.
139
What are the problems with 3-level lasers?
- Lasing relies on there being a |2> population which can be stimulated by the first natural emission photon - if there was a low |2> population then naturally emitted photon would only lead to emission of a few photons which wouldn't be enough for lasing
- As the photon emitted from |2> can be lost at non-reflective surfaces OR lead to stimulated emission of another photon from |2> OR be absorbed by molecules in |0> taking it to |2>, stimulated emission is therefore in direct competition with absorption so in order for stimulation to outweigh absorption |2> must have higher population than |0> --> this is the requirement for population inversion
For 3 level systems population inversion can only occur if more than 50% of molecules in active medium are excited to |2>
140
Give an example of an active species in a three level laser
Cr3+ - in synthetic sapphire (Al2O3) doped with 0.5% Cr2O3
141
Why are three level lasers a bit shit?
Very inefficient (large input = small output)
Require high population of excited ions
142
What is a bit less shit than the three level laser?
The 4 LEVEL LASER!!!!
level 4 fuck yeah
143
How does the level 4 laser work?
1. Molecules excited to |1> by pumping (w/ flash/ continuum lamp)
2. Ions ross to metastable state |2>
3. Stimulated emission occurs to intermediate state |3> (3 is lower than 2)
4. |3> then immediately depopulated non-radiatively to |0> (ground state) - happens fast to maintain population inversion
144
What are the advantages of a four level laser (4)?
- population inversion is between 2 excited state so easier to achieve and control then population inversion between excited state/ground state
- lower level |3> of lasing transition is rapidly depopulated is easy to maintain excess population in upper lasing level |2> --> less levels of pumping required = more efficient
- laser can be operated continuously (pumping transition provided by continuous light sourcE)
- Conversion efficiencies are much higher
145
Which organic species are good for use in 4 level lasers? Why?
Rhodamine 6G and Rhodamine B
These have electronic states ideally spaced for use in 4 level lasers
146
What is the advantage of using molecule chromophores?
In solution they possess many, very closely spaced, vibrational levels (including in the |3>) so by fine tuning the dimensions of the optical resonator (e.g. add diffraction grating) the laser can be tuned to produce light of a specific frequency.
If don't want specific frequency can also make a small range for spectroscopic sources.
147
What are high power dye lasers? What do they need? Why?
These are lasers involving molecular chromophores (organic dyes).
These require stirring and cooling to prevent heating of laser.
|3> is rapidly depopulated by collisions with solvent molecules which produce heat.
148
What are the 4 main properties of laser light?
1. Highly monochromatic
2. Coherent radiation - light wave oscillations are all in phase
3. Highly parallel radiation - allow v. good focussing
4. Pulsed lasers can give radiation in extremely small pulses - femtosecond spectroscopy
