UV/Vis spectroscopy is routinely used in analytical chemistry for the quantitative determination of different analytes, such as transition metal ions, highly conjugated organic compounds, organic compounds, and biological macromolecules. Determination is usually carried out in solutions.
- Solutions of transition metal ions can be colored (i.e., absorb visible light) because d electrons within the metal atoms can be excited from one electronic state to another. The color of metal ion solutions is strongly affected by the presence of other species, such as certain anions or ligands. For instance, the color of a dilute solution of copper sulfate is a very light blue; adding ammonia intensifies the color and changes the wavelength of maximum absorption (λmax).
- Organic compounds, especially those with a high degree of conjugation, also absorb light in the UV or visible regions of the electromagnetic spectrum. The solvents for these determinations are often water for water soluble compounds, or ethanol for organic-soluble compounds. (Organic solvents may have significant UV absorption; not all solvents are suitable for use in UV spectroscopy. Ethanol absorbs very weakly at most wavelengths.) Solvent polarity and pH can affect the absorption spectrum of an organic compound. Tyrosine, for example, increases in absorption maxima and molar extinction coefficient when pH increases from 6 to 13 or when solvent polarity decreases.
- While charge transfer complexes also give rise to colors, the colors are often too intense to be used for quantitative measurement.
The Beer-Lambert Law states that the absorbance of a solution is directly proportional to the concentration of the absorbing species in the solution and the path length. Thus, for a fixed path length, UV/Vis spectroscopy can be used to determine the concentration of the absorber in a solution. It is necessary to know how quickly the absorbance changes with concentration. This can be taken from references (tables of molar extinction coefficients), or more accurately, determined from a calibration curve.
The wavelengths of absorption peaks can be correlated with the types of bonds in a given molecule and are valuable in determining the functional groups within a molecule. The Woodward-Fieser rules, for instance, are a set of empirical observations used to predict λmax, the wavelength of the most intense UV/Vis absorption, for conjugated organic compounds such as dienes and ketones. The spectrum alone is not, however, a specific test for any given sample. The nature of the solvent, the pH of the solution, temperature, high electrolyte concentrations, and the presence of interfering substances can influence the absorption spectrum. Experimental variations such as the slit width (effective bandwidth) of the spectrophotometer will also alter the spectrum. To apply UV/Vis spectroscopy to analysis, these variables must be controlled or accounted for in order to identify the substances present. (Adapted from Wikipedia)
- Wavelength range: 190-1100 nm
- Bandwidth: 1 nm fixed
- Stray light: At 220 nm (NaI), < 0.01%T; At 340 nm (NaNO2), < 0.01%T; At 370 nm (NaNO2), < 0.01%T; At 200 nm (KCl) < 1%T
- Wavelength accuracy (At D2 peak (656.1 nm)): ±0.1 nm
- Wavelength reproducibility (10 measurements at 656.1 nm): ±0.05
- Photometric accuracy: At 1 A using NIST 930D filter), ±0.001 A; At 2 A using NIST 1930D filter, ±0.005 A
- Potassium dichromate: ±0.010 A
- Photometric reproducibility (Maximum deviation of 10 measurements at 1 A): < 0.001 A
- Photometric stability (Stability at 1 A, at 500 nm with 2-sec response time) < 0.00015 A/hour
- Photometric noise (Noise 500 nm/0 A RMS slit 1 nm): < 0.00005 A
- Baseline flatness (Slit 1 nm): ±0.001 A
- Construction: Solid CNC-machined aluminum chassis for thermal and vibration stability
- Optics: Double-beam, sealed, quartz-coated mirrors; lens-free system to reduce chromatic aberrations
- Monochromator: Seya Namioka
- Grating: Holographic, concave grating with 1053 lines per mm
- Source: Deuterium and Tungsten prealigned sources with automatic switch-over