Spectrometry Spectrometry can be defined as:
The study of molecular or atomic structure of a substance by observation of its interaction with electromagnetic radiation.
Two ways of studying this: Quantitatively: For determining the amount of material in a sample. Qualitatively : For identifying the chemical structure of a sample.
The Electromagnetic Spectrum
The "electromagnetic spectrum" of an object is the characteristic distribution of electromagnetic radiation emitted or absorbed by that particular object.
The difference between these sources of radiation is the amount of energy they radiate.
radiation is transmitted in waveform
Long wavelength: low radiation energy. Short wavelength: high radiation energy
Wavelength: time or distance to complete one cycle of wave form
Frequency: number of cycles in a given time
Laws of Specphotometry
There are two very important basic laws and a third one which is a
combination of the two.
- LAMBERTS LAW - ABSORBANCE (A) proportional to the PATHLENGTH (l) of the absorbing medium.
- BEERS LAW - -ABSORBANCE (A) proportional to the CONCENTRATION (c) of the sample.
- BEER- LAMBERT LAW - ABSORBANCE (A) proportional to c x l
Molar extinction coefficient
should be for pure samples and with known molecular weights. If an E value has been calculated for a sample then the % purity of the sample can be calculated as follows:
E sample / E standard x100
There are instances when either the MW or a pure sample is not available and there are no literature E values.
In these cases solutions are prepared at a concentration of 1% and absorbance measured in a 1cm pathlength cuvette to determine.
- Use of glass or plastic cuvettes for Visible Use of quartz or special plastic cuvettes for UV measurements
- Glass cuvettes absorb UV radiation but not visible radiation whilst quartz cuvettes do not absorb any radiation at all
UV or Visible spectra can both be obtained form the same instrument There are two basic designs the DISPERSION system and the more modern DIODE ARRAY system.
This design permits ALL wavelengths of light from source to pass sample in cuvette before reaching the diffraction grating and more sensitive.
- WHAT IS COLOUR? Colour is a sensation which occurs when light enters the eye and focuses on the retina at the back of the eye.
- CONES - Give colour and three types which pick up red, blue and green RODS - Give grey/black and also used for night vision.
Sunlight is white light and covers a wavelength range of 380-750nm
- If the material completely REFLECTS all light it appears WHITE
- If the material completely ABSORBS all light it appears BLACK
- If the material absorbs a constant fraction of the light across the spectrum it appears GREY.
Why Are Dye Molecules Coloured?
Why Are Dye Molecules Coloured?
Starting with ethanol, this is colourless because it only absorbs UV radiation.
The parts of a molecule which contain absorption features are called Chromophores and if a sufficient number are present then the molecule will be coloured.
Increasing the number of chromphores means that the compound will absorb light at a longer wavelength and will therefore increase its v max value.
Auxochromes play major role in dye chemistry as apart from changing the intensity depth of a colour they can have a profound influence on solubility and hence its ability to dye an object.
forensic application :
fluorescence-Fingerprints with use of dfo for maximum contrast on paper types
uv-visible -One of the current techniques for the analysis of ink samples is microspectrophotometry
calculations for this topic:
- beer lambert law