Biospectroscopy Flashcards
1
Q
What state of matter has rotational energy?
A
Gas only
2
Q
What are the challenges of Raman spectroscopy?
A
Can be highly effected by fluorescence
Laser can destroy sample if power is too high
May require several modes of operation and different laser wavelengths to cover all applications
3
Q
A
4
Q
Define
Symmetric stretching
A
Simultaneous vibration of two bonds, in which the bonds elongate together and contract together
5
Q
What happens when PCA is applied to spectroscopic data?
A
When applied to spectroscopic data, scores proved a map of the sample similarities/dissimilarities and the loadings represent the spectral features that cause the samples to group the way they do
6
Q
Definition
Map of samples: Projected locations of objects onto the principal components
A
Scores (T)
7
Q
Draw a FT/Raman Spectrometer
A
8
Q
Define
Vibrational energy
A
The energy in a vibrating system, otherwise at rest; especially that in a molecule due to the vibrations of its atoms
9
Q
What is the equation for Beer’s law?
A
A = ελ c l
Where:
A = Absorbance
- c* = concentration
- l* = path length
ελ = Extinction coefficient
10
Q
Draw a Michelson Interferometer
A
11
Q
Definition
a measure of the degree to which electrons are displaced relative to the nuclei (i.e. how much to the electrons slosh about)
A
Polarizability
12
Q
Which has a higher frequency (and wavenumber)?
Stretching or Bending modes
A
Stretching modes
13
Q
What are the two types of light scattering?
A
Rayleigh scattering: elastic - same frequency - weak
Raman scattering: inelastic - changed frequency - weaker
14
Q
Definition
One of the possible Raman interactions that leads to the material losing energy and the emitted photon has a higher energy than the absorbed photon
A
Anti-Stokes Raman scattering
15
Q
Definition
finds a data value by averaging the values within a segment of data points
A
Moving average
16
Q
Why are the spectra for the same molecule different?
A
Different chemophores are enhancing different excitation wavelengths
17
Q
Define
De-trending
A
Removing unwanted baseline effects by estimating a polynomial baseline
18
Q
Which mode has the strongest absorbance?
A
Asymmetric stretching
19
Q
What is the relationship between number of double bonds and the position of v(C=C)?
A
↑ congugation
=
↓ frequency v(C=C)
because
↑ electron delocalisation
=
↓ strength of double bonds
=
↓ force constant
↓ wavenumber
20
Q
What two components make up molecular energy levels?
A
Electronic
Nuclear motion
21
Q
Do all CO2 modes give rise to IR transitions?
A
No. The electric component of the electro-magnetic radiation must interact with an oscillating dipole of the same frequency. Asymmetric stretching and bending modes do but symmetric stretching does not.
22
Q
- The fundamental transition of a diatomic molecule that is a harmonic oscillator is found at 3000 cm-1. Where would you expect to observe the 2nd overtone? (1 mark)
A
9000 cm-1
23
Q
Which of the following statements about DNA is correct?
- DNA has a strong bands assigned to the symmetric phosphodiester stretching and the asymmetric stretching vibration at 1080 cm-1 and 1220 cm-1, respectively.
- In the hydrated state the B-DNA asymmetric stretch appears at 1240 cm -1 whereas in A-DNA the band appears at 1220 cm -1
- The A-DNA conformation is adopted by bacteria under hydrating conditions
- The B-DNA form is a more narrow and disordered form compared to A-DNA
- The A-DNA conformation is observed when DNA is hydrated
A
Which of the following statements about DNA is correct?
- DNA has a strong bands assigned to the symmetric phosphodiester stretching and the asymmetric stretching vibration at 1080 cm-1 and 1220 cm-1, respectively.
- In the hydrated state the B-DNA asymmetric stretch appears at 1240 cm -1 whereas in A-DNA the band appears at 1220 cm -1
- The A-DNA conformation is adopted by bacteria under hydrating conditions
- The B-DNA form is a more narrow and disordered form compared to A-DNA
- The A-DNA conformation is observed when DNA is hydrated
24
Q
How can fluorescence be prevented?
A
Choosing an appropriate wavelength
25
What is chemimetrics?
Chemometrics is the science of relating measurements made on a chemical system or process to the state of the system via application of mathematical or statistical methods
26
What does a molecules eletronic energy level depend on?
The arrangement of electrons in molecular orbitals
27
Why does the loadings of a PCA model matter?
The loading plot is useful to understand the correlations between the variables
28
What is the Boltzman distribution?
The number of molecules in the ground state compared to the number of molecules in the excited state
29
What does a high residual indicate?
That it is an outlier
30
What are the four types of smoothing?
Moving average
Gaussian Filter
Median Filter
Savitzky-Golay
31
# Definition
a row-oriented transformation used to get all data in approximately the same scaling
Normalisation
32
# Define
Rotational energy
kinetic energy due to the rotation of an object and is part of its total kinetic energy
33
# Define
Anti-Stokes Raman scattering
One of the possible Raman interactions that leads to the material losing energy and the emitted photon has a higher energy than the absorbed photon
34
The amide I mode is a complex mode made up of 3 modes and appears at ~1650 cm-1 depending on the protein secondary structure.
What are the main modes in order of importance that make up the Amide I mode?
1. C=O stretch, C-N stretch and NH2 in plane bend
2. C-N stretch, C=O stretch, NH2 in plane bend
3. NH2 in plane bend, C=O stretch, C-N stretch
4. C-C stretch, C=O stretch, NH2 in plane bend
The amide I mode is a complex mode made up of 3 modes and appears at ~1650 cm-1 depending on the protein secondary structure.
What are the main modes in order of importance that make up the Amide I mode?
1. **C=O stretch, C-N stretch and NH2 in plane bend**
2. C-N stretch, C=O stretch, NH2 in plane bend
3. NH2 in plane bend, C=O stretch, C-N stretch
4. C-C stretch, C=O stretch, NH2 in plane bend
35
What is a score plot (T)?
A 2D plot of the projected objects using PC1 and PC2 as a new coordinate system
36
What does Principal Component Analysis (PCA) do?
* Extract information and remove noise
* Reduce dimensionality/compression
* Exploratory data analysis
* Classification/identification
* Variable reduction
* Process monitoring
* Visual Analysis of Variance
37
# Definition
used to reduce the nise in the data without reducing the number of variables. It is a row-oriented transformation, that is to say the contents of a cell are likely to be influenced by its horizontal neighbours
Smoothing
38
# Definition
uses a diffraction grating spectrometer (essentially operating like a prism) to disperse the light scattered from a sample and then will detect this upon a multi-channel detector such as a CCD. The wavelengths of light detected are the Raman spectrum
Dispersive Raman spectrometer
39
# Define
Fluorescence
Signal noise on a Raman spectrometer that occurs due to real electronic transitions
40
The force constant increases with what?
Increased bond strength
41
What does the intensity of overtone and combination bands depend on?
The intensity of fundamentals
The anharmonicity of the molecule
Resonance with strong fundamentals
42
What is the difference between A-DNA and B-DNA?
43
What is the difference between a molecules' IR and Raman wavenumers?
They occur at the same frequency but at different intensities
44
Low frequencies (wavenumber) correlate with what?
Vibrational motion of heavy masses and low force constants
45
# Definition
the scattering of light by particles in a medium, without change in wavelength. It accounts, for example, for the blue colour of the sky, since blue light is scattered slightly more efficiently than red
Rayleigh scattering
46
What happens is you increase the number of smoothing points when using Savitzky-Golay Derivatives?
Increasing the number of smoothing points reduces the amount of noise, but can also eliminate important chemical information through reduced resolution
47
What are category variables?
Strictly qualitative variables without order and non-overlapping
48
What are the disadvantages of FT/Raman?
High laser power required
Low sensitivity
Not very useful for microspectroscopy
49
Below a Scores Plot and Loadings Plot for FTIR transflection spectra of cancer versus normal liver tissue
1. How much variance is explained by PC1 and PC2? (1 mark)
94%
50
\_\_\_\_\_\_\_\_\_\_: For each sample, the value of the lowest point in the spectrum is subtracted from all the variables
\_\_\_\_\_\_\_\_\_\_\_\_\_: Transforms a sloped baseline into a horizontal baseline. The rechnique is to point out two variables, both defined as o, which should define the new baseline
\_\_\_\_\_\_\_\_\_\_\_\_: Removes unwanted baseline effects by estimating a polynomial baseline
**Baseline offset**: For each sample, the value of the lowest point in the spectrum is subtracted from all the variables
**Linear Baseline Correction**: Transforms a sloped baseline into a horizontal baseline. The rechnique is to point out two variables, both defined as o, which should define the new baseline
**De-trending**: Removes unwanted baseline effects by estimating a polynomial baseline
51
# Define
B-DNA
the most common double helical structure found in nature, the double helix is right-handed with about 10–10.5 base pairs per turn
52
# Define
Frequency
the number of occurrences of a repeating event per unit of time
53
A fundamental frequency of 5Hz would seen overtones at what frequencies?
10Hz, 15Hz, 20Hz etc.
54
# Define
Virtual states
a very short-lived, unobservable quantum state
55
Define Residual variance
Variance remaining in E
56
# Definition
the most common double helical structure found in nature, the double helix is right-handed with about 10–10.5 base pairs per turn
B-DNA
57
# Definition
an alcohol and ether attached to the same carbon
Hemiacetal
58
What are the advantages of Dispersive micro-Raman spectrometers?
Multiplex advantage as for FT/Raman
High sensitivity detector
Low power OK
Choice of laser frequency
59
How many modes does CO2 have?
3x3-5 = 4 modes
| (CO2 is linear)
60
1. Why are symmetric stretches generally lower in wavenumber value than asymmetric stretches? (2 marks)
In **symmetric stretching**, two or **more** bonds vibrate in and out together. In **asymmetric stretching**, some bonds are getting shorter at the same time as others are getting **longer**. Longer bonds mean lower wavenumber value because the force constant is smaller. Therefore symmetric stretches are lower in wavenumber value.
61
When should you use a second derivative?
When the spectra is not noisy and you want to minimise broad features and highlight inflection points
62
# Define
Boltzman distribution
a probability distribution or probability measure that gives the probability that a system will be in a certain state as a function of that state's energy and the temperature of the system
63
What does Multiplicative scatter correction do?
Removes offset and divides by slope to normalise
64
What is the loading (p) of a variable?
The cosine of the angle that the PC makes with the original variables
65
# Definition
an oscillating system is a pattern of motion in which all parts of the system move sinusoidally with the same frequency and with a fixed phase relation
Normal modes
66
What happens is you use a higher degree polynomial when using Savitzky-Golay Derivatives?
A higher degree polynomial will fit better to the data, giving a more precise derivative. However, noise sensitivity increases
67
What are the benefits of Near-IR spectroscopy?
Non-destructive
Minimal sample preparation
Can be easily integrated into processes
Chemical and physcial information
68
# Definition
Error of the data
Residuals (E)
69
# Define
Michelson Interferometer
a device that produces interference between two beams of light
70
Consider the following diagram
Why does the position of ν(C=C) shift to lower wavenumber as the number of double bonds (0-12) increases?
Delocalisation
71
What factors determine the intensity of an IR band in a spectrum?
∂μ/∂x that is the change in electric field over the change in bond length
72
1. Sketch the main fundamental modes of CO2 (2 marks)
2. Which of these are infrared and Raman active? (2 marks)
The asymmetric stretching mode and the bending modes ae IR active while the totally symmetric mode is Raman active
73
Why are bending modes generally lower in wavenumber value than stretching modes?
It is easier to bend a bond than to stretch it because the nuclei are not moving against the attraction of the bonding electrons.
74
1. How many fundamental modes of vibration will CO2 have? (1 mark)
3N-5 = 4 modes of vibration
75
Where are beta-sheet bands found?
1635 cm-1
76
# Define
FT Raman spectrometer
A type of Raman designed to eliminate the fluorescence problem encountered in conventional Raman spectroscopy
77
# Definition
the spatial frequency of a wave, measured in cycles per unit distance or radians per unit distance
Wavenumber
78
How can the size of a data table be reduced?
Averaging samples or variables
79
What are the advantages of FT/Raman?
Highly fluorescent samples usually not a problem
80
What are second derivatives used for?
To correct for both linear and quadratic baseline effects such as scatter dues to particle size
81
What are ordinal variables?
Qualitative variables with an inherent rank (e.g. small, large)
82
What happens at each end of the data after Savitzky-Golay smoothing? What does it depend on?
After smoothing, the data will be slightly truncated at both ends. Truncation depends on segment size
83
Define Residuals, E
Error. The data can be divided into structure (Xstruct) and residual (E) : X = Xstruct + E
84
Which PC line accounts for the most variation?
The lowest (i.e. PC1)
85
# Definition
the number of occurrences of a repeating event per unit of time
Frequency
86
# Define
Dispersive Raman spectrometer
uses a diffraction grating spectrometer (essentially operating like a prism) to disperse the light scattered from a sample and then will detect this upon a multi-channel detector such as a CCD. The wavelengths of light detected are the Raman spectrum
87
Define Principal components
Main data variations, also known as ”latent variables”, ”factors” and ”eigenvectors”. The first PC accounts for the majority of the varaince with each successive PC accounting for less varaince in the data set.
88
# Define
Smoothing
used to reduce the nise in the data without reducing the number of variables. It is a row-oriented transformation, that is to say the contents of a cell are likely to be influenced by its horizontal neighbours
89
List in order from highest to lowest wavenumber value the following functional groups
C-Cl, C-O, C=O, C-F, C-H and Fe-O
C-H, C=O, C-O, C-F, C-Cl and Fe-O
90
Convert 3100 cm-1 into wavelength (micron mm), frequency (s-1) and energy (KJ).
![]()
v= c/λ = 3 x108 ms-1/3.226 10-6 m = 9.299 x 1013 Hz
*E* = *hv* = 6.62 x 10-34 Js x 9.299 x 1013 s-1 = 6.16 x 10-20 J = 6.16 x 10-23 KJ
91
# Define
Multivariate analysis
a subdivision of statistics encompassing the simultaneous observation and analysis of more than one outcome variable
92
What does the intensity of hot bands depend on?
Boltzman distribution
Temperature
93
What is residual variance?
Variance remaining in the residuals
94
What are the benefits of Raman spectroscopy?
Non-destructuve
Minimal sample preparation
Can be easily integrated into processes
Can provide enhanced chemical information over NIR
95
What is usually the first preprocessing step?
Mean centering of data
96
How many modes does a linear molecule have?
3N-5 (where N=number of atoms)
97
What is a disadvantage of using an FT-Raman system? (2 marks)
Less energy means more power is required to produce a spectrum because the scattered intensity of the light is smaller at longer wavelenghts
98
# Definition
a non-destructive identification method that measures the vibrational energy in a compound
Vibrational spectroscopy
99
# Definition
The energy in a vibrating system, otherwise at rest; especially that in a molecule due to the vibrations of its atoms
Vibrational energy
100
# Define
Hot band
a band centred on a hot transition, which is a transition between two excited vibrational states
101
Which of the following functional would have the **lowest** wavenumber value in the mid-infrared spectrum?
C-H
C-C
C-O
C-Cl
C-Br
Which of the following functional would have the **lowest** wavenumber value in the mid-infrared spectrum?
C-H
C-C
C-O
C-Cl
**C-Br**
102
# Define
Scores (T)
Map of samples: Projected locations of objects onto the principal components
103
# Definition
define the curvature of a data set and are best applied to data with quadratic (curved) baselines
Second derivative
104
What does a bond require to be IR active?
A dipole moment
105
# Definition
a change of wavelength exhibited by some of the radiation scattered in a medium. The effect is specific to the molecules which cause it, and so can be used in spectroscopic analysis
Raman effect
106
# Definition
used to remove linear baseline effects in continuous spectral data
First derivative
107
Consider resonance from of amide I mode. Why does the amide I mode appear at ~1650-1630 cm-1 And not ~1730 cm-1
Resonance structures for an amide. Remember that the molecule does not actually switch between these structures. Instead, the actual structure is somewhere in between the structures shown. It can be thought of as some average of these structures.
108
The nitrile group is another reliable functional group that generally is easy to identify. There is a significant dipole moment associated with the C ≡N bond which leads to a significant change when it interacts with infrared radiation usually leading to an intense sharp peak at 2200-2280 cm-1 Very few other groups absorb at this region with this intensity.
Why is the C ≡N band more intense than the C ≡C band?
The C ≡N bond has a larger dipole moment compared to the C ≡C bond due to the electronegativity of the nitrogen atom
109
1. List the molecules HC≡CH, H2C=CH2 and H3CCH3 in descending order of their C–C vibrational stretching frequencies. (2 marks)
**HC**≡**CH\> H****2C=CH****2\>H****3CCH****3**
110
What is Dark DNA?
The phenomenon that describes the nucleosomes of an inactive nucleus appearing as "black" strings or dots that yield no spectral information from the DNA
111
How many normal modes of vibration are there in the molecule CH4 ? (2 marks)
**3N – 6 =9 modes of vibration**
112
# Definition
A type of Raman designed to eliminate the fluorescence problem encountered in conventional Raman spectroscopy
FT Raman spectrometer
113
Why does the scores of a PCA model matter?
The score plot shows how the data are distributed, Sample patterns, groupings, similarities and differences can be studied
114
# Define
Hemiacetal
an alcohol and ether attached to the same carbon
115
What are the yellow and green lines?
Fluorescence
116
What are the challenges of Near-IR spectroscopy?
Spectra features are broad and undefined
Suffers highly from scatter effects
Requires multivariate analysis for complete understanding
117
Raman spectroscopy involves the scattering of light.
1. Explain the difference between Stokes, anti-Stokes and Rayleigh scattering using an energy diagram and list in order from **highest to lowest** the intensity of the scattering from these three processes. (6 marks)
Stokes scattering is totally elastic scattering where the incoming photon leaves at the same energy as the scattered photon. It is the most intense form of scattering.
Anti-Stokes scattering results when the incident photon is scattered from a molecule in an excited vibrational state. The scattered photon leaves at a higher energy than the initial photon and is the weakest type of scattering.
Stokes scattering results when the incident photon interacts with a molecule in the ground vibrational state and the photon scatters at a lower energy than the initial photon. The intensity of the scattered light is less than Rayleigh scattering but more than anti-Stokes scattering.
In both Stokes and anti-Stokes the energy difference between the incident and scattered photon is equal to wavenumber of the vibrational mode.
118
Why doesn't symmetric stretching result in a IR transition?
There is no change in dipole
119
What must occur in the molecule for a Raman spectrum to be generated?
Application of an electric field (laser) can induce a dipole moment. If this induced dipole moment varies during a vibrational motion, a Raman spectrum will be produced
i.e. the "mean" polarizability (α) must vary during the vibrational motion
120
# Define
Linear baseline correction
A transformation of a sloped baseline into a horizontal baseline. The technique is to point out two variables, both defined as o, which should define the new baseline
121
# Definition
The first preprocessing step that involves subtraction of the mean for each variable
Mean centering
122
What caused the peaks between 3400 and 3900?
Overtones
123
1. List the molecules HF, HI, HCl and HBr in descending order of their vibrational frequencies. (2 marks)
HF\>HCl\>HBr\>HI
124
1. Why are bending modes lower in wavenumber value than stretching modes? (2 marks)
It is easier to bend a bond than to stretch it because the nuclei are not moving against the
attraction of the bonding electrons.
125
# Definition
a normalisation of spectra to remove effect of variations between samples due to differences in pathlength. Corrects for both baseline slope and offset
Multiplicative scatter correction
126
# Definition
one of the possible double helical structures which DNA can adopt. It is thought to be one of three biologically active double helical structures. It is a right-handed double helix fairly similar to the more common form, but with a shorter, more compact helical structure whose base pairs are not perpendicular to the helix-axis as in the common form
A-DNA
127
Below a Scores Plot and Loadings Plot for FTIR transflection spectra of cancer versus normal liver tissue
1. Along which PC is the separation between cancerous and normal spectra of cells? (1mark)
PC1
128
What does Raman measure?
Scattering of UV, vis or near IR laser light (or X-ray etc.)
129
# Definition
finds a data value by making a polynomial fit to the data points using a number of data points on each side
Savizky-Golay smoothing
130
Draw a diagram illustrating the difference between Rayleigh, Stoke and Anti-Stokes scattering
131
What does the wavenumber of a bond depend on? State the equation
The wavenumber depends on the force constant and the mass of the atoms. An increase in bond strength and/or a decrease in mass causes an increase in wavenumber
*v* = (1/2π)\*sqrt(k/μ)
Where:
*v* = wavenumber
k = force constant
μ = mass
132
What allows Raman microprobes to rapidly line map?
Line of laser diodes the give high laser energy output
133
How many modes does a non linear molecule have?
3N-6 (where N=number of atoms)
134
# Definition
an analytical technique to study the electronic structure and its dynamics in atoms and molecules
Electronic spectroscopy
135
# Define
Moving average
finds a data value by averaging the values within a segment of data points
136
# Definition
concerned with the measurement of the energies of transitions between quantized rotational states of molecules in the gas phase
Rotational spectroscopy
137
What are the max number of PC lines?
n - 1
Where:
n = number of spectra
138
List in order of magnitude:
Evib, Erot, Eelec
Eelec \>\> Evib \>\> Erot
139
What are first derivatives used for?
To remove linear baseline effects in continuous spectral data
140
How is FT/Raman different to FTIR?
* Scatter of Nd-YAG laser
* Requires Rayleight filter
* Requires different materials for:
* Beamsplitter
* Windows
* Detector
* Mirror coating
141
What is an issue with higher PC lines?
They pick up noise in the sample
142
Consider the following carboxylate group. Would you expect the carbonyl group to be at ~1730 cm-1 as seen in esters, aldehydes and ketones?
The carbonyl frequency for a bond and a half would be expected to fall halfway between 1735 and 1200 or at approximately 1465 cm-1. The carboxyl group has the same symmetry as the nitro and CH2 groups. Both a symmetric and asymmetric stretch should be observed. An asymmetric and symmetric stretch is observed at 1410 and 1560 cm-1 in sodium benzoate that averages to 1480 cm-1, in good agreement with the average frequency predicted for a carbon oxygen bond with a bond order of 1.5.
143
# Definition
kinetic energy due to the rotation of an object and is part of its total kinetic energy
Rotational energy
144
# Define
Rotational spectroscopy
concerned with the measurement of the energies of transitions between quantized rotational states of molecules in the gas phase
145
# Definition
corrects each spectrum by subtracting the mean and dividing by the standard deviation for that spectrum
Standard normal variate
146
# Definition
Signal noise on a Raman spectrometer that occurs due to real electronic transitions
Fluorescence
147
# Define
Loadings (P)
Map of variables: Correlation between variables (regression of X on 1)
148
What is a virtual state?
A continuum of vibrational states that occurs when there is not enough energy to be an electronic state change
149
# Definition
a probability distribution or probability measure that gives the probability that a system will be in a certain state as a function of that state's energy and the temperature of the system
Boltzman distribution
150
Spectroscopic transformations let you switch between \_\_\_\_\_\_\_\_\_and \_\_\_\_\_\_\_\_\_units and transform reflectance data into __________ units
Spectroscopic transformations let you switch between **absorbance** and **reflectance** units and transform reflectance data into **Kubelka-Munk** units
151
152
# Define
Second derivative
define the curvature of a data set and are best applied to data with quadratic (curved) baselines
153
What can happen to light that hits a sample?
Transmitted, absorbed or scattered
154
# Define
Resonance Raman
occurs when excitation laser frequency is chosen in such a way that it crosses the frequencies of electronic excited states and resonates with them. The intensity of Raman bands, which originate from electronic transitions between those states, are enhanced three-to-five orders of magnitude
155
Why are lipids good biomarkers?
* Lipids change in response to disease conditions
* e.g. cholesterol, which, has been used in risk calculations for heart disease
* Triglycerides are also used clinically for risk assessmet of heart disease and diabetes
* Lipids can be used as infrared biomarkers for malaria diagnosis
156
When should you not use a second derivative?
Don't use it when the spectra is noisy becauase it will amplify the noise
157
# Define
Overtone bands
the spectral band that occurs in a vibrational spectrum of a molecule when the molecule makes a transition from the ground state (v=0) to the second excited state (v=2), where v is the vibrational quantum number (a non-negative integer) obtained from solving the Schrödinger equation for the molecule
158
What does averaging samples and variables do?
Reduce the size of the data table
Reduces uncertainty in the measurements
Reduces the effects of noise
159
# Define
Biospectroscopy
The spectroscopic examination of specimens of living tissue or body fluids
160
Consider the following spectrum.
What type of molecule is this?
Aldehyde, ketone, carboxylate, organic halide, carboxylic acid, aliphatic, aromatic?
Hexanoic acid
The carbonyl stretch C=O of a carboxylic acid appears as an intense band from 1760-1690 cm-1. The exact position of this broad band depends on whether the carboxylic acid is saturated or unsaturated, dimerized, or has internal hydrogen bonding.
O–H stretch from 3300-2500 cm-1
C=O stretch from 1760-1690 cm-1
C–O stretch from 1320-1210 cm-1
C-O–H bend from 1440-1395 and 950-910 cm-1
161
# Define
Asymmetric stretching
Simultaneous vibration of two bonds, with opposite atomic motions. While one bond is elongating the other is contracting
162
# Define
Raman effect
a change of wavelength exhibited by some of the radiation scattered in a medium. The effect is specific to the molecules which cause it, and so can be used in spectroscopic analysis
163
Write down an expression for the PCA model equation. (1 mark)
164
Define PCA Loadings
Map of variables: Correlation between variables (regression of X on T). Explains what variables are causing the seaparation observed on the Scores Plot
165
Define Explained variance
The % variance explained by Xstruct
166
# Definition
the spectral band that occurs in a vibrational spectrum of a molecule when the molecule makes a transition from the ground state (v=0) to the second excited state (v=2), where v is the vibrational quantum number (a non-negative integer) obtained from solving the Schrödinger equation for the molecule
Overtone bands
167
What state of matter has vibrational energy?
All states (above 0K)
168
What are the disadvantages of Dispersive micro-Raman spectrometers?
Possible fluorescence
Requires calibration
169
How do you work out the score (t) of a sample?
The distance from the mean along th PC is the score
170
# Define
Mean centering
The first preprocessing step that involves subtraction of the mean for each variable
171
List these types of spectroscopy in order of increasing size of wavelengths detected:
Electronic, rotational and vibrational
Rotational \> vibrational \> electronic
172
Haemoglobin has 87% alpha helices while IgG has only 3%. Which would have a high Amide I band?
Haemoglobin (1651 cm-1; IgG 1632 cm-1)
173
# Define
Standard normal variate
corrects each spectrum by subtracting the mean and dividing by the standard deviation for that spectrum
174
# Definition
Simultaneous vibration of two bonds, with opposite atomic motions. While one bond is elongating the other is contracting
Asymmetric stretching
175
# Definition
the science of relating measurements made on a chemical system or process to the state of the system via application of mathematical or statistical methods
Chemometrics
176
# Definition
observed when more than two or more fundamental vibrations are excited simultaneously
Combination bands
177
1. What happens to maxima bands when a first derivative of the spectrum is calculated? (1 mark)
The maxima lie on the Y=0 axis
178
# Define
Residuals (E)
Error of the data
179
# Definition
occurs when excitation laser frequency is chosen in such a way that it crosses the frequencies of electronic excited states and resonates with them. The intensity of Raman bands, which originate from electronic transitions between those states, are enhanced three-to-five orders of magnitude
Resonance Raman
180
# Definition
One of the possible Raman interactions that leads to the material absorbing energy and the emitted photon has a lower energy than the incident photon
Stokes Raman scattering
181
# Define
Stokes Raman scattering
One of the possible Raman interactions that leads to the material absorbing energy and the emitted photon has a lower energy than the incident photon
182
What is the explained variance?
The % variance explained by Xstruct
183
What are the four modes of CO2?
Symmetric stretching
Asymmetric stretching
2 equivalent bending
184
What are the three types of spectroscopic transformations available in The Unscrambler?
1. Absorbance to reflectance or Absorbance to transmittance
2. Reflectance to absorbance or Transmittance to absorbance
3. Reflectance to Kubelka-Munk
185
\_\_\_\_\_\_\_\_\_\_: finds a data value by averaging the value within a segment of data points
\_\_\_\_\_\_\_\_\_\_\_\_: applies a normally distributed weighting function across the spectral region
\_\_\_\_\_\_\_\_\_\_: finds the median value within a segment of data points
\_\_\_\_\_\_\_\_\_\_: finds a data value by making a polynomial fit to the data points using a number of data points on each side
**Moving average**: finds a data value by averaging the value within a segment of data points
**Gaussian Filter**: applies a normally distributed weighting function across the spectral region
**Median Filter**: finds the median value within a segment of data points
**Savizky-Golay**: finds a data value by making a polynomial fit to the data points using a number of data points on each side
186
# Define
Combination bands
observed when more than two or more fundamental vibrations are excited simultaneously
187
What is the criteria for selecting the appropriate number of PCs?
* Explained variance/Residual variance
* Clear global maximum/minimum
* First local maximum/minimum
* Plateau
188
# Definition
a very short-lived, unobservable quantum state
Virtual states
189
# Define
Wavenumber
the spatial frequency of a wave, measured in cycles per unit distance or radians per unit distance
190
# Define
Rayleigh scattering
the scattering of light by particles in a medium, without change in wavelength. It accounts, for example, for the blue colour of the sky, since blue light is scattered slightly more efficiently than red
191
Draw a diagram illustrating the difference between Resonance Raman, Pre-Resonance Raman and Fluorescence scattering
192
What is the most common application of Beer-Lambert Law?
UV-vis determination of component concentrations in unknown solutions
193
Which of the following statements is **correct** in relation to Principle Component Analysis?
1. The first PC accounts for the lowest variance in the data set
2. Loadings plots show the projected samples onto the scores plot
3. The PCA matrix is decomposed into a scores and loadings matrix
4. The inclusion of outliers can improve the models predictive capacity
Which of the following statements is **correct** in relation to Principle Component Analysis?
1. The first PC accounts for the lowest variance in the data set
2. Loadings plots show the projected samples onto the scores plot
3. **The PCA matrix is decomposed into a scores and loadings matrix**
4. The inclusion of outliers can improve the models predictive capacity
194
Which region of the spectrum are fundamental, overtone and combination bads found?
**Fundamental:** IR region
**Overtones and Combination:** IR or near-IR region
195
# Definition
The spectroscopic examination of specimens of living tissue or body fluids
Biospectroscopy
196
# Define
Savizky-Golay smoothing
finds a data value by making a polynomial fit to the data points using a number of data points on each side
197
# Define
Multiplicative scatter correction
a normalisation of spectra to remove effect of variations between samples due to differences in pathlength. Corrects for both baseline slope and offset
198
# Define
Normalisation
a row-oriented transformation used to get all data in approximately the same scaling
199
A spectrum shows a band at 750 cm-1. What is the main functional group contributing to this vibrational mode? (1 mark)
1. ν(C=O)
2. δ(NH2)
3. ν(C-Cl)
4. ν(N-H)
A spectrum shows a band at 750 cm-1. What is the main functional group contributing to this vibrational mode? (1 mark)
1. ν(C=O)
2. δ(NH2)
3. **ν(C-Cl)**
4. ν(N-H)
200
A spectrum of an unknown compound shows a small distinct band at 3010 cm-1 plus strong bands between 3000-2800 cm-1 and a medium band at 1740 cm-1. The spectrum is likely to be from:
1. B-DNA
2. Protein
3. Unsaturated lipid
4. Saturated lipid
5. None of the above
A spectrum of an unknown compound shows a small distinct band at 3010 cm-1 plus strong bands between 3000-2800 cm-1 and a medium band at 1740 cm-1. The spectrum is likely to be from:
1. B-DNA
2. Protein
3. **Unsaturated lipid**
4. Saturated lipid
5. None of the above
201
# Definition
Removing unwanted baseline effects by estimating a polynomial baseline
De-trending
202
# Definition
a device that produces interference between two beams of light
Michelson Interferometer
203
What is PC1?
The line that points in the direction of the maximum data variation
204
Which one of the following statements is **incorrect** for infrared spectroscopy: (1 mark)
1. The absorbance is directly proportional to the concentration
2. The stronger the dipole moment the higher the wavenumber value
3. The larger the reduced mass the lower the wavenumber value
Hydrogen boning gives rise to broad OH stretching modes
Which one of the following statements is **incorrect** for infrared spectroscopy: (1 mark)
1. The absorbance is directly proportional to the concentration
2. **The stronger the dipole moment the higher the wavenumber value**
3. The larger the reduced mass the lower the wavenumber value
Hydrogen boning gives rise to broad OH stretching modes
205
What is PC2?
The line perpendicular to PC1 that explains the perpendicular variation
206
True or False:
The PCs are orthogonal to each other
True
This means that they are independent of one another
207
Why are the C=C bonds generally have a higher wavenumber value than C-C bonds?
C=C bonds are more electron rich and thus have a higher bond order than C-C bonds therefore they have a greater force constant and thus higher wavenumber value
208
True or False:
Water is a good Raman scatterer
False
Water doesn't scatter
209
Why does the number of components of a PCA model matter?
The number of components in the model characterises the structure of the data: the fewer PCs needed, the simpler the model
210
Which **one** of the following molecules is a **strong** Raman scatterer?
1. Asymmetric CO2
2. HCl
3. water
4. HF
5. benzene
Which **one** of the following molecules is a **strong** Raman scatterer?
1. Asymmetric CO2
2. HCl
3. water
4. HF
5. **benzene**
211
What are the two types of nuclear motions?
Rotational and vibrational energy
212
# Define
Polarizability
a measure of the degree to which electrons are displaced relative to the nuclei (i.e. how much to the electrons slosh about)
213
# Definition
a subdivision of statistics encompassing the simultaneous observation and analysis of more than one outcome variable
Multivariate analysis
214
# Definition
Map of variables: Correlation between variables (regression of X on 1)
Loadings (P)
215
Given this fundamental absorption frequency, what are the overtones?
216
How does Standard Normal Variate (SNV) correct each spectrum?
SNV corrects each spectrum by subtracting the mean and dividing by the standard deviation for that spectrum
217
# Define
Principal Component Analysis (PCA)
a method of analysis which involves finding the linear combination of a set of variables that has maximum variance and removing its effect, repeating this successively
218
What is the model equation of PCA?
X = TPT + E
Where:
X = data matrix
T = score matrix
P = matrix of loadings
219
What is smoothing used for?
Smoothing is used to reduce the noise in the data without reducing the number of variables
220
Where are alpha helix bands found?
1650 cm-1
221
What are the general guidelines for pre-processing spectral data?
* Baseline correction
* Scatter correction
* Derivatives
* Normalisation
222
# Definition
states that the quantity of light absorbed by a substance dissolved in a fully transmitting solvent is directly proportional to the concentration of the substance and the path length of the light through the solution
Beer-Lambert Law
223
# Definition
Simultaneous vibration of two bonds, in which the bonds elongate together and contract together
Symmetric stretching
224
# Define
First derivative
used to remove linear baseline effects in continuous spectral data
225
True or False:
Both C-C and C-F would be IR active
False
C-F would be but C-C would not since it does not have a dipole moment
226
Consider the following spectrum. Is this molecule aromatic, alkane, alkene, alkyne?
=C-H stretch between 3100-3000 cm-1
C–H stretch from 3000–2850 cm-1
CH2 bend or scissoring from 1470-1450 cm-1
C–H rock, methyl from 1380-1350 cm-1
C–H rock, methyl, seen only in long chain alkanes, from 725-720 cm-1
Alkene
227
1. What type of data would you avoid the use of a second derivative and why? (2 marks)
Data that is noisy because small spikes become large bands in the second derivative (large rise over small run).
228
Label the major bands in the spectrum of normal epithelial cell and cancer cell
229
Define PCA Scores
Map of samples: Projected locations of objects onto the principal components
230
# Define
Beer-Lambert Law
states that the quantity of light absorbed by a substance dissolved in a fully transmitting solvent is directly proportional to the concentration of the substance and the path length of the light through the solution
231
What does the force constant depend on?
The strength of the bond
232
Why is the O-H stretching mode at a higher wavenumber value than the C-H stretching mode?
Although the mass of oxygen is higher than carbon. The force constant for the O-H bond is stronger because the oxygen is more electronegative and forms a stronger bond than the C-H producing a larger force constant.
233
Consider the following spectrum of ethanol. Why is the OH so broad?
In any sample where hydrogen bonding occurs, the number and strength of intermolecular interactions varies greatly within the sample, causing the bands in these samples to be particularly broad.
234
What is polarizability?
A measure of the degree to which electrons are displaced relative to the nuclei
i.e. how much do the electrons slosh about
235
# Define
Chemometrics
the science of relating measurements made on a chemical system or process to the state of the system via application of mathematical or statistical methods
236
Which **one** of the following molecules **will show** an infrared spectrum?
1. oxygen
2. nitrogen
3. symmetric CO2 vibration
4. Br2
5. carbon monoxide
Which **one** of the following molecules **will show** an infrared spectrum?
1. oxygen
2. nitrogen
3. symmetric CO2 vibration
4. Br2
5. **carbon monoxide**
237
What does Multiplicative Scatter Correction (MSC) correct?
Corrects for both baseline slope and offset
238
Draw a Dispersive micro-Raman spectrometer
239
# Define
A-DNA
one of the possible double helical structures which DNA can adopt. It is thought to be one of three biologically active double helical structures. It is a right-handed double helix fairly similar to the more common form, but with a shorter, more compact helical structure whose base pairs are not perpendicular to the helix-axis as in the common form
240
High frequencies (wavenumber) correlate with what?
Vibrational motion of light masses and molecules with large force constants
241
# Definition
a band centred on a hot transition, which is a transition between two excited vibrational states
Hot band
242
# Define
Electronic spectroscopy
an analytical technique to study the electronic structure and its dynamics in atoms and molecules
243
# Definition
a technique, where for each sample, the value of the lowest point in the spectrum is subtracted from all the variables
Baseline offset
244
Why does a longer wavelength mean less fluorescence?
There is less energy so there is less scattering of light
245
In which region of the electromagnetic spectrum is radiation of wavenumber 10000 cm‑1?
Near-IR
Visible
far IR
microwave
infrared
UV
In which region of the electromagnetic spectrum is radiation of wavenumber 10000 cm‑1?
**Near-IR**
Visible
far IR
microwave
infrared
UV
246
# Define
Normal modes
an oscillating system is a pattern of motion in which all parts of the system move sinusoidally with the same frequency and with a fixed phase relation
247
# Definition
A transformation of a sloped baseline into a horizontal baseline. The technique is to point out two variables, both defined as o, which should define the new baseline
Linear baseline correction
248
# Define
Vibrational spectroscopy
a non-destructive identification method that measures the vibrational energy in a compound
249
The total energy of a molecule is made up of what components?
Electronic energy
Vibrational energy
Rotational energy
250
Why are asymmetric modes generally higher in wavenumber than symmetric modes?
In symmetric stretching, two or more bonds vibrate in and out together. In asymmetric stretching, some bonds are getting shorter at the same time as others are getting longer. Shorter bonds mean higher wavenumber value because the force constant is greater.
251
1. The Michelson Interferometer is an essential piece of apparatus in the FT Raman spectrometer. Sketch and label the basic components of an FT-Raman spectrometer including the Michelson Interferometer . (5 marks)
252
# Define
Baseline offset
a technique, where for each sample, the value of the lowest point in the spectrum is subtracted from all the variables
253
How do you choose an appropriate wavelength for Raman?
254
Below a Scores Plot and Loadings Plot for FTIR transflection spectra of cancer versus normal liver tissue
1. Assuming the spectra have been processed using a second derivative. What are the wavenumber values for the strong loadings (bands) associated with cancer cells? (2 marks)
2. What is the likely origin of these bands? (2 marks)
1153, 1084, 1026 and 995 cm-1
DNA
255
# Definition
a method of analysis which involves finding the linear combination of a set of variables that has maximum variance and removing its effect, repeating this successively
Principal Component Analysis (PCA)
256
1. What are some advantages of using FT-Raman spectrometer compared to conventional visible/UV dispersive Raman spectrometer ? (2 marks)
Using a longer wavelength can minimise fluorescence (not enough energy to cause electronic transitions). Damage from shorter wavelengths (high energy) is possibly reduced depending on the applied power.
257
Which nucleotide is the most acidic?
Cytosine
258
List the nucleotides in terms of nucleophilicity
G \> A \>\> T \> C
259
\_\_\_\_\_\_\_\_\_\_\_ between aromatic rings of base pairs stabilises DNA double helix
**π- stacking** between aromatic rings of base pairs stabilises DNA double helix
260
What happens to the melting point of DNA as the GC content increases?
Tm increases
261
What is the formula for the melting point of DNA?
Tm = 2(#ATbp)+4(#GCbp)
262
What are the three ways of targeting DNA with cytotoxic drugs?
* Thymine biosynthesis
* DNA replication
* Inhibition of mitosis (alkylating agents)
263
What are the steps of thymine biosynthesis?
1. Remove 2' OH
2. Remove 5' diphosphate
3. Add 5-CH3
264
Why do drugs targer thymine synthesis instead of other nucleotides?
Thymine does not form spontaneously; it requires enzymes
Targeting thymine prevents targeting of RNA
265
How are alkylating agents mutagenic?
They react as electrophiles to form stable covalent bonds, intercalating with the DNA causing mutation
266
Cyclophosphamide is the most commonly prescribed alkylating agent used for the treatment of cancer. It is converted to an active metabolite in the liver, which is capable of cross-linking DNA. Draw a curved arrow mechanism for the formation of the double stranded DNA adduct (Note: onlt the mechanism for the second step is required)
