UV, Fluoro And IR W7 Flashcards
Spectrum
Image
Issac newton 17th century
Describes the dispersion of white light into colours by diffraction
Spectroscopy
The study of the interaction between matter and electromagnetic radiation
The longer the wavelength the …
Lower the energy the radiation carries
Red light
750nm
Lower energy
Blue light
400 nm
Higher energy
Visible light wavelength
10 to the power of -6 to 10 to the power of -7 m
Infrared
10^13 - 10^14 Hz
10^-5 - 10^-6 m
UV
10^15 - 10^17 Hz
10^-7 - 10^-9 m
When is radiation absorbed by matter
When the energy matches that required for transition between one state and another
High frequency =
Short wavelength = high energy = potentially harmful
Electromagnetic radiation contains …
Energy which is inversely proportional to the wavelength
An example of DNA damage by UV irradiation
Cross-linking of thymine residues
Molecular energy levels
Excited electronic state
Ground electric state
What does the typical UV visible transition involve?
Promotion of an electron from the highest occupied molecular orbital HOMO to the lowest occupied molecular orbital LUMO
Strongest bond in a molecule
Sigma bond
Where are UV excitations usually?
Between HOMOs that contain N or P electrons
The more conjugated the P system…
The lower the energy required for an electronic transition
Chromophore
The functional group responsible for UV visible absorption
Increasing conjugation will increase y and E
Cis double bonds give longer y but lower E
Can be affected by solvents and pH
y = wavelength
E = absorbtion coefficient
Beer lambert law
A = log10 (i0/it) = ECL
A= Absorbance
i0 = Intensity of incident radiation
it = Intensity of transmitted
radiation ε = absorption
coefficient c = concentration of
sample l = path length
Auxochrome
functional group attached to the chromophore which modifies the ability of the chromophore to absorb light
– eg lone pair
– Can change with pH eg if deprotonation increases conjugation
Bathochromic
shift to longer wavelength (red shift)
“Hypsochromic
shift to shorter wavelength (blue shift)
Hyperchroism
increase in
absorbance
Aromatics
Simple aromatic compounds absorb light in the UV region, and will be
colourless.
Alkenes
simple alkenes absorb in the UV end of the spectrum.
They will be colourless.
highly conjugated natural products absorb light in the visible region, and will be coloured as a result. Ie carrot and tomato
UV-visible Absorption in Biomolecules
• Proteins absorb at 280 nm due to the amino acids tryptophan and tyrosine.
• Coloured proteins exist e.g. haemoglobin due to complexation with iron containing heme.
• Nucleic acids absorb at 260 nm due to the nitrogen heterocycles.
• Many natural products are coloured, absorbing in the visible region.
Monitoring reactions
Design of coloured dyes by modulation of the chromophore
• HPLC usually uses a UV trace
– eg monitor at 254 nm
• TLC can use UV to visualise
Fluorescence: decay of the excited electron
Sometimes when EM energy is absorbed it decays by emitting light.
This can be by the slow process of phosphorescence or by the rapid process of fluorescence.
Ff = photons emitted / photons absorbed
Max value of Ff is 1
Green Fluorescent Protein
• GFP naturally produced by jellyfish is fluorescent due to a spontaneous chemical reaction within its protein structure.
• GFP can be tagged to other proteins.
• This allows the tagged protein to be visualised by fluorescent imaging.
Fluorescence Based High-throughput Screening
• The rate of enzyme reaction is measured by a probe that reacts with the cofactor NADH to produce red fluorescence.
• If the sample contains an inhibitor of the enzyme, less NADH will be consumed leading to increased fluorescence
• Fluorescence can be detected at lower levels than UV-visible absorption,
Kcal to kj
X 4.18
Vibrational Transitions : equilibrium
No vibrations
No dipole change
No IR absorption
Vibrational Transitions : symmetric stretch
Vibration side to side
No dipole change
No IR absorption
Vibrational Transitions : bending mode
Movement up and down
Dipole change
IR absorption
Asymmetric stretch
One side stretching more than the other
Change in dipole
IR absorption
Infrared Spectroscopy and Functional Groups
By measuring the IR abrorptio of a molecule we can identify the FGs
Each bond have different frequency for a vibrational transition
Wave number
Inverse of wave length
1/y = n
The stronger the Bon …
The higher the IR absorption wave number
C=C higher than C-C
Wave number is proportional to…..
Spring constant which is proportional to bond strength
Reduced mass is inversely proportional to…
IR absorb frequency
C-H = 3000cm-1
C-Cl = 750cm-1
Heavier the atoms in the bond the…
Lower the frequency
Characteristic IR Frequencies
N-H stretch / O-H = >3000
C-H stretch = 3000
N-CH3 = 2800
CtripleN = 2100
CtripleC = 2100
C=O stretch stretch = 1700
Fingerprint = <1500
What affects C=O frequency
E.withdrawing group increases K ad increases wave no.
Conjugation reduce C=O frequency, decrease bond strength and decrease wave no.
E.donating group decreases spring constant and decreases wave number
