Spectrophotometry Flashcards
What is electromagnetic radiation?
- Electromagnetic radiation comprises radiant energy extending from cosmic rays (λ 10-9 nm) to radio waves (λ km).
- ‘Visible Light’ – describes radiant energy in the visible portion of spectrum where our eyes work.
- All forms of electromagnetic radiation travel at the speed of light.
speed of light in a vacuum (c): 299,792,458 metres per second.
186,000 miles per second.
What is the equation of wavelegnth?
Wavelength is inversely proportional to a given frequency:
v = c / λ
v = frequency of light (cycles per second)
c = speed of light in a vacuum (3 x 1010 cm/s)
λ = wavelength in cm
What is energy proportional to?
Energy is proportional to frequency
- The relationship between the energy of photons and their frequency is proportional.
- It makes sense that the more times a wave wobbles in a second, then the more energy required to make that happen:
E = hv
E = energy (ergs)
h = Plank’s constant (6.62 x 1027 erg s)
v = frequency of light (cycles per second [Hz])
What is energy inversely proprotional to?
Therefore, it stands that wavelength (λ) is inversely proportional to E
E = hc / λ
For example: UV radiation at 200 nm possesses greater energy than IR radiation at 750 nm.
What happens when light interacts with matter?
- Be absorbed
- Be transmitted
- Be scattered
- Be reflected
What happens when absorbed light interacts with electrons?
- Electrons in the “ground state” promoted to higher energy orbitals
- Energy absorbed corresponds to difference between orbitals
- Energy is then lost either vibrationally (non-radiative) or radiatively e.g. fluorescence
Why are organic molecules useful in spectrophotemetry?
- Organic molecules often have conjugated double bond systems, so easy for electrons to move around (delocalise).
- Conjugated double bond systems have fairly low energies for absorption.
- Since colour occurs with absorption, many organic conjugatedcompounds are coloured
Why are some chemical coloured?
- lThe wavelength of light absorbed will depend on the chemical structure of the compound in solution.
- Since “white” light is a spectrum, only certain parts of the spectrum will be absorbed. The remainder produces the colour observed.
What is absorbance and how is it calculated?
- Absorbance is calculated from intensity
- Incident light with intensity (I0). Light reaching the detector with intensity (I)
- Transmittance (the fraction of incident light reaching the detector):
T = I /I0
%T = I x 100 / I0
- Relationship between concentration and transmittance is non-linear:
- From the transmittance, one can calculate the absorbance (A)
- Convert to absorbance which is the logarithm of the reciprocal
A = log10 1/T
A = 2 – log10 %T
What is the Beer-Lambert Law?
- Light is absorbed when a photon collides with a molecule.
- Therefore, it is not surprising that the amount of light absorbed depends on the concentration of the compound in solution.
- Beer-Lambert Law: Concentration of a substance is directly proportional to the amount of light absorbed.
What is the equation for Beer-Lambert Law?
A = εcl
Where:
- ε = molar absorptivity (molar extinction coefficient)
- c = concentration (mol/L)
- l = path length (cm).
How can molar absoptivity be used to calculate concentration?
- A proportionality constant for any given compound at any given wavelength of light.
ε = A / cl
- Since most cuvettes have a path length of 1 cm, the above equation can be simplified to:
ε = A / c
- We can then rearrange to calculate concentration:
c = A / ε
Where:
ε = molar absorptivity (molar extinction coefficient)
c = concentration (mol/L)
- So that concentration can be calculated from a absorbance reading if the molar absorptivity is known.
Why is absorbance an ideal analysis?
- Measurement is quick and easy
- (relatively) cheap
- Readily integrated into automation
- Reproducible
What is Spectrophotometry?
- The quantitative measurement of the reflection or transmission properties of a material as a function of wavelength.
- The light passing through the solution is detected by the photo-detector, generating an electrical current proportional to the intensity of the light, which is then converted into a reading.
What are the main types light sources of spectroscopy?
Need to produce light at wavelength where absorbance is measured
Tungsten
- Covers the visible spectral range reasonably well
- Tends to have higher intensity in the red region of the spectrum
- Cheap
Deuterium
- Deuterium: isotopic hydrogen (abundance ~ 1 in 6000 H atoms)
- Deuterium arc light produces mainly UV light (so invisible to the eye)
- Expensive
- Relatively short lifetime
What is needed for optimal analytical performance in Spectrophotometry?
- Incident light beam is parallel and of a constant wavelength (monochromatic)
- Incident light beam is of the wavelength which gives the maximum absorption (minimum transmission) of the light.
