UV, fluorescence, and IR spectroscopy

what is spectroscopy

• the study of the interaction between matter and electromagnetic radiation

• measuring how light is split

if a wavelength is longer what does this mean for energy

the longer the wavelength the lower energy the radiation carries

is red light lower or higher energy

• lower

• 750 nm

is blue light lower or higher energy 

• higher

• 400 nm 

what is the equation for energy

• E = h x c/gamma 
  where:

• E= energy

• h= planks constant (6.626 × 10^-34 Js)

• c= speed of light (2.998 × 10^8 ms-1)

• v= frequency 

• gamma = wavelength 

what does electromagnetic radiation contain 

energy 

what is electromagnetic radiation inversely proportional to 

wavelength 

what will radiation be absorbed by and when will it do so 

• absorbed by matter

• when the energy matches that required for transition between one state and another 

what is non-ionising radiation

doesnt have harmful effects

what is ionising radiation

when electromagnetic energy is super high it can cause ionisation 

what does high frequency mean for wavelength and energy 

• high frequency = shorter wavelength = high energy 

• potentially harmful 

what does ionising radiation provide

provides enough energy to make or break chemical bonds 

give examples of non-ionising radiation

• radio

• microwaves

• infrared

• visible light

• ultraviolet 

give examples of ionising radiation

• x rays 

• gamma

• cosmic 

what are the different applications of spectroscopy

• determining the structure of an unknown molecule

• confirming the structure of a known molecule 

• examples of spectroscopic application to drug discovery and pharmaceuticals 

what are the experimentally determined spectroscopic properties used for predict the presence of functional groups and molecular structure 

predict the presence of functional groups and molecular structure 

confirming the structure of a known molecule

• testing the purity of a molecule

what are experimental values compared to

a reference standard to confirm the identity of the sample

what are spectroscopic techniques used for

• can be very sensitive

• used to detect minute concs. of a material 

how can spectroscopy be applied

in a quantitive manner to determine the conc. of a material

what are different examples of spectroscopic application to drug discovery and pharmaceuticals

• real-time monitoring of chemical reactions

• imaging in vitro and in vivo

• trace and purity analysis

• determination of drug concs. 

• identification of drug metabolites in vivo 

what is the high energy of UV irradiation sufficient enough to cause

chemical reactions to take place 

what is an example of UV irradiation

DNA damage by UV irradiation is the cross linking of thymine residues 

what are the different molecular energy levels 

• rotational energy levels

• vibrational energy levels 

• ground and excited electronic state 

what does rotational transition promote

changes in the rotational state

what does vibrational transition promote

changes in binational energy states

what does electronic transition promote

enough energy to promote electrons to excited electronic state 

what are the several possible electronic transitions that can occur

• sigma —> sigma* - alkanes

• sigma —> pi* - carbonyl

• pi —> pi* - unsaturated cmpds. 

• n—> sigma* - O, N, S halogens

• n —> pi* - carbonyls 

why is sigma —> sigma* an unusual state

because it is a big jump

what does the typical UV-visible transition involve

promotion of an electron from the HOMO to the LUMO 

what are the strongest bonds in a molecule

• sigma bonds

• we normally dont see sigma electrons promoted

UV excitations are usually between what 

HOMOs that contain n or pi electrons 

what happens to energy the more conjugated the pi-system 

the lower the energy required for an electronic transition

what is a chromophore

a functional group responsible for UV-visibke absorption 

what is the equation fro beer-lambert law

A = log10 I0/It = ecl
where:

• A = absorbance

• I0 = intensity of incident radiation

• It = intensity of transmitted radiation

• e= absorption coefficient

• c= conc. of sample

• l = path length 

what are the different machines used to measure UV absorbance 

• UV spectrometer

• UV lamp

• nanodrop

what dies increasing conjugation increase

wavelength of absorption and absorptivity coefficient

what do Cis double bonds give

longer wavelength of absorption but lower absorptivity coefficient 

what can the chromophore be affected by

solvents

what is an auxochrome

functional group attached to the chromophore which modifies the ability of the chromophore to absorb light 

what can the auxoxchrome change with

pH e.g. deprotonation increases conjugation

what does bathochromic mean

shift to a longer wavelength (red shift)

what does hypsochromic mean

shift to shorter wavelength (blue shift)

what does hypochroism mean

decrease in absorbance

what does hyperchroism mean

increase in absorbance

where do the simple alkenes ethene, butadiene, and hexatriene absorb in the UV

at the end of the spectrum 

will ethene, butadiene, and hexatriene have colour or be colourless

colourless

what are some examples of UV-visible excitations- alkenes

• beta, beta-carotene

• lycopene 

what region do simple aromatic compounds absorb light

in the UV region

what colour will simple aromatic compounds be

colourless

what are examples of UV-visible excitations- aromatics 

• tumeric

• curcumin 

what does extended conjugation in cur cumin result in

absorption in the blue region, leading to an orange colour 

what is the characteristic absorbance of proteins and what is this due to

• 280 nm

• due to the amino acids tryptophan and tyrosine

what is an example of a coloured protein

haemoglobin die to complexation with iron containing heme

what is the characteristic absorbance of nucleic acids and what is this due to

• 260 nm

• due to nitrogen heterocyclic

how do you get hyperchroism in DNA 

denaturing the DNA with heat 

what is HPLC used for

separating mixtures and isolating particular compounds

what does HPLC usually use

a UV trace

what does using a combination of HPLC and a UV Trace allow for 

focus on a single absorbance 

what is stop-flow technology used for

measuring reactions after rapid mixing

what is fluorescence

the decay of the excited electron

what are the 2 processes in which EM energy can decay by emitting light

• phosphorescence

• rapid process of fluorescence

what can quantum yield for a fluorescent process be defined as

the ratio of photons emitted through fluoresce to the total number of photons originally absorbed

what is the maximum value for quantum yield 

• 1

• often much lower than this because of competing processes which deactivate the excited molecules 

what is the intensity of light emitted after the exciting light is turned off given by

I = I0 exp(-t/fancy looking t)
where:

• I0= the intensity at t=0

• I= intensity at time t

• T= time

• fancy looking t= mean lifetime of the fluorescent state 

what is difficult to predict

the structures that will be fluorescent 

what is fluoresce generally associated with

an extended chromophore/auxochrome system and a rigid structure 

what are some examples of fluorescent molecules with pharmacological importance 

• pentobarbitone

• adrenaline

• chlorpromazine

• riboflavin

• procaine

• noradrenaline

• quinine 

what is a green fluorescent protein naturally produced by

• a jellyfish

• due to a spontaneous chemical reaction within its protein structure

using recombinant techniques what can happen to GFP

can be tagged to other proteins 

what happens when a protein is tagged to a GFP

the tagged protein can be visualised by fluorescent imaging 

how have different fluorescent variants of proteins with different colours been produced

through changes in the amino acid sequence

what is an example of a 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, the less NADH will be consumed leading to increased fluorescence 

what are the levels that fluorescence can be detected at compared to UV-visible absorption and what does this mean 

• can be detected at lower levels

• leading to higher sensitivity for the assay 
  

what is UV-Vis spectroscopy comparable to

bond strength energies

what is IR spectroscopy comparable to

energies of conformations

what are the different vibrational transitions

• bending mode

• asymmetric stretch

which vibrations can be seen by IR

only those that result in a change in DIPOLE MOMENT 

what is dipole moment

a change in polarity in a molecular pair

as a first approximation in IR spec what can we treat chemical bonds like 

a spring 

what will each type of bond have

a different frequency for a vibrational transition

what is the wavenumber inversely proportional to 

wavelength 

how can IR wavenumber be calculated

using Hooke’s law 

what is force constant, K, proportional to 

bond strength 

what does a stronger bond mean for IR spec

the higher the IR absorption wavenumber

what is reduced mass, mui, inversely proportional to 

IR absorption frequency 

what does a heavier atom in the bond mean for IR spec 

lower frequency 

what is the exception peaks in the fingerprint region

• 600-860cm 

• can help determine ortho-meta-para substitution patterns for aromatics

what is the functional group in the wavenumber »3000cm. give an example and a characteristic of this peak  

• heteroatom- H, single bond stretch 

• O-H, N-H

• broad peaks

what is the functional group in the wavenumber - 3000cm. give an example and a characteristic of this peak  

• Carbon-H, single bond stretch 

• C-H

• present in all organics, hence not very useful 

what is the functional group in the wavenumber - 2800cm. give an example and a characteristic of this peak  

• amino-methyl stretch

• N-CH3

• above 2800 for aromatic N-methyl, below for aliphatic methyl 

what is the functional group in the wavenumber - 2100cm. give an example and a characteristic of this peak  

• triple bond stretch 

• C-C, C-N, N-N

• sharp peaks, no other interfering absorptions in triple bond region

what is the functional group in the wavenumber - 1700cm. give an example and a characteristic of this peak  

• carbon-oxygen double bond stretch

• C=O

• intense peaks, exact frequency dependent on nature of C=O

what is the functional group in the wavenumber - 1500cm. give an example and a characteristic of this peak  

• fingerprint region

• C-O, C-C

• unique to specific molecule, not very useful

what is the frequency of an aliphatic ketone

about 1715 cm

why does an ester have a wavenumber of around 1746 cm

the electron withdrawing group increases the C=O force constant

why does a ketone with an alkene have a reduced wavenumber of 1680 cm

conjugation reduces C=O frequency

why does an amide have a reduced wavenumber of around 1655 cm

electric donating groups reduced the C=O constant force