Ultraviolet–visible spectroscopy
The UV / Vis spectroscopy is a, the electromagnetic waves of ultraviolet ( UV) and visible (English visible, VIS ) light uses. The method is also known as UV / VIS spectrophotometry.
Principle
Molecules are irradiated with electromagnetic waves in the visible and ultraviolet light, thereby valence (for example, the p-and d- orbitals of the outer shells ) excited, that is, raised to a higher energy level.
To lift an electron, for example, of an occupied (HOMO ) to an unoccupied, higher orbital ( LUMO), the energy of the absorbed photon must exactly match the energy difference between the two energy levels. On the relation
Can be calculated, the wavelength of the absorbed light to the energy required, whereby the energy is Planck's constant, the speed of light, the frequency and the wavelength of the electromagnetic wave. This relationship is sometimes referred to as Einstein - drilling equation. As an approximation, this relationship can be represented in simplified form in the form of a tailored size equation:
Building a Zweistrahl-UV/Vis-Spektrometers
The light source radiates ultra-violet, visible and near infrared light in the wavelength range from about 200 nm to 1100 nm. In the monochromator, the wavelength selected for measurement is first selected, and then the light beam is incident on the mirror sector. The sector mirror can alternately fall through the test solution and the reference solution the light. Both solutions are in so-called cuvettes. The two light beams are received at the detector and comparing the intensities in the amplifier. The amplifier then adjusts the intensity of the light beam from the reference solution by inserting the comb diaphragm of the intensity of the light beam from the sample. This motion is transmitted to a recorder or passed the readings to a data processing.
Increasingly küvettenfreie UV / VIS spectrometer for determining the concentration of small sample volumes used (less than 2 microliters) of samples of higher concentrations. So-called NanoPhotometer work with layer thicknesses in areas of 0.04 mm to 2 mm. You do not need cuvettes, thinners and can samples with a volume of only 0.3 ul analyze ( at minimum layer thickness), but have due to the small layer thickness, a higher detection limit. A proven technology based on a compression of the sample, which thus is independent of the surface tension and the evaporation of the sample. This method finds use in the analysis of nucleic acids (DNA, RNA, oligonucleotides ) and proteins ( UV absorption at 280 nm). According to the Lambert - Beer's law, there is a relationship between absorption and thickness. The absorbance values for the different layer thicknesses (0.04 mm to 2 mm) can thus be calculated. Small layer thicknesses act as a virtual dilution of the sample, but can be used only with correspondingly higher concentrations. Often can therefore be dispensed with entirely on a manual dilution of the sample.
Chemical Examples
Useful are the π -to- π * transitions in unsaturated hydrocarbons (eg, alkenes ). They will take on longer-wavelength UV light and are easy to measure. One obtains information on the wavelength of the absorbing molecule, the structure and color. In this case, the light absorption occurs in the longer wavelength region, the greater is the number of conjugated double bonds. If the energy of the π -to- π * transitions in the visible light region, the molecule appears colored. It always assumes the complementary color of the absorbed light.
In the observed electron transitions, the following selection rules are always observed ( inter alia Laporte rule):
First spin rule: the total spin must be maintained
2nd banning of transitions of the same parity, eg:
- Prohibited is the transition 3s → 4s
- Allowed is the transition 3s → 3P/4P
- Prohibited is the transition from straight → straight ( orbital)
Attention: Forbidden does not mean that these transitions do not occur ex: observed due to the vibration coupling of the nuclei
3 overlap rule: only with similar symmetry and size