Medicine: EMR and UV-Vis Spectrophotometery Flashcards
What tends to shift absorption to longer wavelengths? What is this called?
Conjugated structure
This is called bathochromic shift
How to calculate the concentration of a drug in grams/100ml?
C= A/A1%,1cm
where A1%,1cm=extraction coefficient
What is Emission?
Electromagnetic radiation is produced when excited particles return to lower-energy levels or the ground state. This happens in florescence
What does EMR spectroscopy involve?
Absorption, emission, or scattering of EMR by substances
What are light waves made of?
Moving electric and magnetic fields vibrating 90 degrees to each other at the speed of light
The energy from these fields makes the wave move
Hence makes light waves electromagnetic
What is an Auxochrome?
a functional group containing lone pair(s)
e.g. OH, NH2 of electrons that does not absorb appreciable amount of UV/visible light on its own but shifts peaks of molecules that it is attached with to longer λ (bathochromic shift) and makes the peak higher intensity (hyperchromic shift)
What is a Chromophore?
A part of a molecule that absorbs UV or visible light (double bond, groups with lone pair atoms)
UV spectrum absorption for drug analysis
Wavelength range?
Molecule must contain what?
> 200nm absorption
Molecule must contain unsaturated pi bonds or atoms with non binding orbitals (oxygen, nitrogen, halogen)
In reality UV absorption is mostly restricted to drug molecules with conjugated bonds (pi to pi*)
Equation for the energy from each photon
E=hc/λ
E=energy
h= Plank’s constant
c= speed of light
λ= wavelength
Which part of the UV spectrum is..
a) not much use in analysis
b) most commonly used for molecular analysis
a) <200nm absorption
b) >200nm absorption
Using equations, if 100 photos enter a sample and only 10 leave, what is the absorbance value?
For 10% transmission
A=log(1/T)
1/T=10
thus A=log of 10=1
What do these stand for?
HOMO
LUMO
Highest occupied molecular orbital
Lowest occupied molecular orbital
where are n orbitals found?
n = non-bonding
e.g. in nitrogen, oxygen as lone pairs
Wave and energy equation
c=fλ
where c=speed of light
f=frequency
λ= wavelength
How is ε related to A1%, 1cm?
A1%,1cm = 10ε/Mr
where 1%=conc in g/100ml
1cm= path length in cm
How are sigma molecular orbitals formed?
by electrons in 2S orbitals
e.g. CH3-CH3 (ethene)
forming single bonds.
Electron must be close to nucleus
When photons hit something, EMR waves interact like…
other waves
Why is the UV-visible spectrum made of broad rather than sharp lines?
Photons with slight difference in energy can still cause electronic transitions by exciting electrons from the many vibrational state that corresponds to its energy (wavelength)
Total internal energy in a molecule=
sum of energy from
electrons
vibrations between the molecule’s own atoms
fluorescence emission - electrons returning back
How are pi orbitals formed?
by electrons in p orbitals forming double or triple bonds
Each energy level is discrete but has further vibrational and rotational levels which are also discrete, thus..
Excitation of a molecule can be electronic, vibration and rotational
Deviations from Beer-Lambert Law - about absorption in a homogeneous solution
May not be true for turbid samples, causing scatter of light
Classify the electromagnetic spectrum in order of decreasing wavelength
Radio waves, microwaves, infrared, visible light, ultraviolet, X-rays, Gamma rays
What are the “other factors” which are assumed if A is directly proportional to C?
Association, dissociation, photodegration, solvation, complexation or adsorption, or if sample emits fluorescence etc
Deviations from Beer-Lambert Law - about independent absorption
Each molecule does not act as an independent absorbing species in solution, especially at high concentration
Energy frequency equation
E=hf
E=energy
h= Plank’s constant
f= frequency
If A(1%,1cm) is for most drugs, what can you deduce?
Knowing absorbance of a solution its concentration can be easily calculated.
Name 3 uses of UV absorption
1) Identification of drugs - by comparing and interpreting spectra. Not absolute ID has overlapping of spectra can occur
2) Measuring reactions, ionisation (pKa determination., solubility, drug release - dependent on change in absorption spectrum λmax and/or intensity) can therefore determine the pka and the solubility
3) Quantification of drugs - most useful and widely used
(Beer Lambert Law)
Deviations from Beer Lambert - why is incident radiation not always monochromatic?
due to:
- instrument calibration errors,
- stray light (without passing through sample) going directly to the detectors
When a bonding orbital (HOMO) is created there is also created….
a corresponding anti-bonding orbital (LUMO) that is normally unoccupied but lies at higher (less stable) state
Deviations from Beer-Lambet Law - about standards such as potassium dichromate?
-Stray light can cause difference against standard
what happens when photons hit a molecule?
A molecule/atom changes its energy state by absorbing or emitting energy that is equal to the energy difference between ground E0 and excited E1 state
Absorption = change in energy is positive
emission = change in energy is negative
Explain the relationship between concentration of sample, absorbance and transmission.
A more concentrated sample would absorb more and transmit less. However transmittance is not linear with increase in concentration, instead exponentially. To make this relationship linear it is best reported as absorbance which is the logarithm (base 10) of the reciprocal of the transmittance:
A= log(1/T) = log(I0/I)
The more extensive the conjugated system… the 1 is the separation in energy levels and thus the 2 the wavelength of absorption
1= less
2=longer
the more delocalised and conjugated, the more easily excitable
What is absorption? Describe it
- Selective removal of certain frequencies by transfer of energy to atoms or molecules
- electrons promoted from lower-energy (ground) states to higher energy (excited) states
- Energy of exciting photon must exactly match the energy difference between the ground state and one of the excited states of the absorbing species
When does delocalisation (resonance stabilisation) extend?
- if benzene rings are present
- if alternating double and single bonds exist between C=C, C=O, C=N, N=N, N=O for example
- Lone pairs on substituent groups with nitrogen, oxygen or halogen become involved e.g. NO2 groups
BP monographs usually report what for most drugs?
A(1%,1cm)
Deviations from Beer-Lambert Law regarding florescence?
If sample emits fluorescence then positive or negative deviations of the Beer-Lambert law may occur
The amount transmitted (T) when light enters and leaves a sample is given by
T= I/I0
or
%T=100xT
Units of ε
units of molarity used, but in pharmaceutical analysis which units are preferred?
What is ε replaced by?
Grams
Here ε is replaced by the term A 1%, 1cm
Specific absorbance of a 1% (1g/100ml) solution using 1cm path length is at a given wavelength
Why does conjugation shift wavelength to red?
In conjugated systems electrons are easily delocalised making the energy gaps between the various orbitals smaller, thus lowering the energy to excite them. This gives batochromic shift
Describe Beer Lambert Law and give the equation
The amount of light absorbed (A) is dependent on the concentration of the sample
A=εbc
where ε= molar extinction coefficient (M-1cm-1) or molar absorpitivity (i.e. the absorbance value ofr a 1M solution)
It is constant for a particular compound at a given wavelength
b = path length of the cell in cm
c = concentration in moles/litre (molarity)
The Beer lambert equation implies linear relationship, but in practice this is only true for up to A <1.5
Why?
Molecules too close to each other
causing saturation of the detector and hence a non-linear relationship after A>1.5
BP standard set limits
Wavelength +/-1nm for standard at given wavelength
A269/A266 0.02% toluene resolution check - > or equal to 5
Stray light 1.2%KCL sol against water - A>2 at 198nm
Path length +/-0.005cm
What is a conjugated system?
Having double bonds with single bonds
ΔE=?
h x c
____
λ
Most drugs absorb
UV light
apart from a few coloured ones
Which parts of the electromagnetic spectrum are used in pharmaceutical analysis?
200-1000nm
For absorbance to be directly proportional to c which assumptions apply?
- Incident radiation must be monochromatic
- Absorbance checks can be made against standards such as potassium dichromate, the absorbance must lie within specified range in BP
- each molecule in solution acts as an independent absorbing species in solution
- absorption takes place in a homogeneous solution
- Other factors… see other flashcard
