BIOSPECTROSCOPY Flashcards
Hierarchy of energy from nuclear motion
electronic energy (highest) vibrational energy rotational energy (lowest)
Equations for calculating energy
E = hv = (hc)/lambda = hc x wavenumber
what is planks constant
6.626 x 10^-34 J s
what is the speed of light
3.0 x 10^8 m/s
What is the beer lambert law
A = epsilon x concentration x path length = -log(I0/I)
what does epsilon in the beer lambert law represent
related to inherent transition strength B, is a property of molecules measuring how strongly a substance absorbs light at a specific wavelength.
When is a molecule IR active
if it has a dipole moment that changes with bending or stretching of the molecule. A dipole changing isn’t affecting the actual electrons and therefore isn’t changing polarizability like in raman.
what is the wavenumber force constant equation
wavenumber = (1/2pi)x √(k/v)
k= force constant, stronger bond is higher k
what is the equation of reduced mass
mu = (m1m2)/(m1+m2)
m= mass of atom, greater mass = greater mu
what is a fundamental energy change
from ground state to v=1 and also downwards
what is an overtone energy change
where is goes from ground state, skips v=1 to another v.
1st overtone is ground to v=2
2nd is ground to v=3 etc
what is a vibrational mode
a specific way a molecule can vibrate.
How to calculate number of vibrational modes
3N - 6 where N=no. of atoms
3N - 5 for linear molecules
What are the modes of CO2
v1 = symmetrical stretching
v2 = 2 equivalent bending modes
v3 = asymmetrical stretching.
These would from highest appear on a spectrum:
v3, v1, v2 however v1 would not have a signal as it has no change in dipole moment meaning not IR active.
which gives higher readings, stretching or bending
stretching is higher than bending as it affects the electron density more and is therefore in a higher energy state.
What are the spectral trends observed in IR
cm-1 is inversely proportional to reduced mass
cm-1 is proportional to strength of bond, force constant
strength of bond, force constant, is inversely proportional bond length
cm-1 is inversely proportional to bond length
higher cm-1 = lighter mu and larger k
What are the differences between A and B forms of DNA
Lower humidity = A form
More hydrated = B form = more h bonds, less vibration, smaller cm-1
Base pair carbonyl: B(1717) > A(1712)
Antisymmetric phosphate: A(1238) > B(1225)
Intensity of scattered light equation
Is proportional to a^2/lambda^4
a = polarisability
What are the 3 different types of scattering
Stokes: leaving photon has less energy
Anti stokes: leaving photon has more energy
Rayleigh: leaving photon has same energy
What is polarisability
polarisability is a measure of electrons being displaced relative to the nuclei.
Molecules with high polarisability
show strong raman scattering
When is a mode raman active
modes that cause a change in polarizability and therefore a change in the ability to form instantaneous dipoles OR a change in the electrons being displaced relative to their nuclei. Inactive IR modes are active Raman modes
is asymmetric or symmetric higher in cm-1
asymmetric is higher due to the due to the change in bond length.
What causes band broadening
If the bond in question can hydrogen bond it will have a broader peak
Baseline offset
represents number of signals generated when no light present due to noise.
corrected by subtracting the lowest point in the spectrum from all the variables to bring the spectrum down to the baseline or fitting a linear/polynomial baseline to data
Average spectra
reduces uncertainty in measurements and the effect of noise. allows comparison between 2 spectral tables collected at different resolutions (different number of raw spectra).
done by using a reducing factor to create any smaller number of spectra from the original number. Each value corresponding to the wavelength is taken and averaged to give a value.
Mean centre
subtracts the mean from each variable producing a new set of axes centered around the mean.
Savitsky-Golay smoothing
Smoothing is used to reduce noise without reducing the number of variables.
fits a polynomial to data. polynomial order = actual shape of peaks (parabola, cubic etc.).
Normalisation
used to get all data in approximately the same scaling for good comparison.
done by dividing variables by a specific value to give relative values.
Standard normal variate
Normalises the spectra by subtracting the mean and dividing by the standard deviation on each variable. Corrects path length variation
Multiplicative Scatter Correction
Corrects for both baseline slope and offset. Normalises for variations in pathlength and uses a linear model of the whole data to bring each sample as close to the average
What does MVA allow us to do
analyse chemical nature/composition
provide information on physical characteristics specific to samples
quantify and predict unknown things.
What does PCA allow us to do
allows spectra to be represented as one point for large comparisons from variation
replace expensive and or destructive testing,
perform rapid analysis,
reveal true structure and classification of data + spectral features
Model equation of PCA
X = (TPT )mode + (E) residuals
What is an object
An object is an observation of the spectra, in the case of the project each observation is one spectrum collected from the sample.
What is a variable
A variable is the things changing in the object, in the case of the project the variables are the absorbance values at each wavenumber of a spectrum.
What is PC
A principal component is a main variation in the collect data. It can be used to contrast objects and determine significant factors affecting their data.
What is a score
A score is a projected locatios of an object with reference to the principal components. A map of scores is a scores plot used to compare different objects with reference to the variation between them.
What is a loading
A loading is a correlation between a variable important in association and comparison
What is a residual
A residual refers to the error associated between a score and variance
What is a dummy variable
A dummy variable is a variable that takes the form of a 0 or a 1 to represent the presence or absence of a specific variable.
allow us to use a single regression equation for both groups instead of having separate equations.
The ‘Switch’ to turn parameters on and off.
What is leverage
High leverage refers to the ability for a score to greatly affect the variance between the data.
What is an indirect observation
Collect the desired data without actually directly measuring something. Gives a correlation instead.
