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3.8.7 UV-visible spectroscopy

General principles

Absorption of incident radiation by bonding/non-bonding electrons represents a high energy (^~100 kCal/ mole) transition. This corresponds to a high frequency, i.e. low wavelength, absorption band which is observed at 200 ^~ 800 nm in the UV and visible range of detection. In solution, electronic absorption spectra are found with broad, generally unresolved bands. These contrast with the vibration fine structure in the vapour phase and with a series of sharp peaks within a continuum in non-polar solvents.

For a solution of an absorbing substance, an absorptivity ratio at a monochromatic wavelength is defined as: {(incident light, Io)/(transmitted, I)} and this is logarithmically related to concentration and optical path-length by the Beer Lambert law: Absorbance (A) = log₁₀(Io/I) = k.c.l., where c mg/ml is the concentration of solute and 1 cm is the distance travelled between parallel optical faces of a suitable cell, and k is a proportionality constant. It is frequently convenient to normalise to a concentration c = 10 mg/ml [i.e. 1%] and l = 1 cm, which is expressed as the specific absorbance [A^1%_1cm]. Molar absorptivity is defined by the coefficient ε = M_r.(A/cl), and is related to the relative molecular mass, M_r. This coefficient is computed for each wavelength maximum, and also at minima if this is of diagnostic value. It may be useful in showing relationships within an homologous series.

For light source emissivity, the common radiation source is a deuterium lamp covering the operating range 180^~ 350 nm and supplemented by a tungsten filament lamp in the near UV, through the visible, into the near-IR, i.e. over the range 320^~1000 nm. Standardisation of equipment and monochromators is necessary to ensure the acceptability of data. The wavelength scale is calibrated with a Holmium perchlorate solution, within a tolerance of ±1 nm below 400 nm and ± 3 nm in the 400^~600 range (see British Pharmacopoeia, 1993). The absorbance may be checked with NPL calibrated neutral density filters; or should agree within defined corresponding ‘windows’ with absorbances obtained with a potassium dichromate solution of specified strength, at wavelengths 235, 257, 313 and 350 nm. Stray light is usually checked with a 1.2% potassium chloride solution, where the absorbance for 1 cm path length should exceed 2.0 at 200 nm against a water reference. This solution can be replaced by 1% NaBr or NaI at the more accessible wavelengths of 215 or 240 nm respectively. Glass optics absorb UV light below about 300 nm and quartz systems are used to extend the working range down to 200 nm, and even to 185 nm if there are high quality optics and stray light control. At lower wavelengths, absorption of UV-radiation by air requires the use of vacuum systems in research instruments. For practical UV-vis spectrophotometry, the effective working range is 200^~800 nm.

Operating conditions

Selection of a suitable solvent is influenced by the wavelength expected to be studied. Water and the lower (polar) alcohols, through diethyl ether and dioxan to nonpolar cyclohexane and light petroleum (‘aromatic free’ in a spectroscopic grade) can be used above 190 nm, whereas chloroform absorbs below ~245 nm. The table below provides a list of cut-off wavelengths. Measured absorbances should be less than 0.4 relative to air using prism monochromators; but higher absorptivity ratios are favoured with modern instruments.

The choice of cells depends on the target range. Silica is essential for measurements at UV wavelengths but glass is acceptable in the visible region; air must be evacuated below ^~200 nm. The matched pair required for test solution and solvent reference path should demonstrate effectively identical absorbance when filled with the same solvent. The optical faces of the cells should be parallel; the absorbance of a matched pair of cells containing the same solvent should not differ by more than 0.005 units. In quantitative work all solutions should be at the same temperature; conveniently, they are transferred from a waterbath at, say, 20° and the absorbance measured immediately. Sensitivity of the solution to laboratory and natural lighting should be established in a pilot experiment. One test (British Pharmacopoeia, 1993) of Resolution Power is the discrimination of adjacent maximum (269 nm) and minimum (266 nm) light absorption of toluene with a ratio not less than 1.5.

Quantitative procedures

UV photometry is a frequently used assay technique. Provided that proper calibration checks are maintained, the UV-vis technique is particularly useful for assay of formulations after extraction or separation of the active substance by suitable chromatography. In the assay calibration, there may be some deviation from Beer’s Law. This may be attributable to association in solution or an effect of slit width. The latter should be large enough to gain a reasonable I-value but remain small compared with the (half-) bandwidth for the absorption measured. If in doubt, reduce the slit width slightly and check if the apparent absorbance increases. UV photometric data can also be of value in determining the kinetics of a process, or in following a reaction sequence, such as the disappearance of an absorption peak representing starting material.

Light absorption’ measurements also provide a semi-quantitative test of identity. This relies on the specific absorbance (defined above as the A_1cm^1% value), or sometimes absorbance at a nominated concentration and path length, either exactly at a specified wavelength, or at the absorption maximum close to a named wavelength. If this test is used as the principal assay of a substance in a formulation, it is advisable to use an authenticated reference substance rather than rely on a published A₁¹. The absorption spectrum may be sensitive to control of pH. Chromophores involving an acidic or basic group will be affected by pH, e.g. the bathochromic shift (to longer wavelength) and hyperchromic peak (greater intensity) of phenates compared with their parent phenol. This is a useful test for a phenolic system.

In subtractive spectrophotometry, the difference between two (or more) spectra measures multicomponent mixtures and is especially useful in formulated product assays. This should be distinguished from the use of second derivative spectroscopy, in which there is computer differentiation of the algebraic function equivalent to the change of slope (i.e. second differential) of the digitalised spectrum (British Pharmacopoeia, 1993). This display sharpens separation of individual UV bands and thereby facilitates lower levels of control. In other applications of computer-aided spectroscopy, modern equipment will provide ‘smoothing’, deconvolution and regression (least squares) analysis.

General chromophores

Absorption bands are particularly evident for conjugated π-bond systems. Most single bond transitions are inaccessible, being derived from higher energy σ-orbitals, with wavelengths below 185 nm, i.e. in the ‘vacuum ultraviolet’. Many isolated triple bonds also absorb below 185 nm. The CC (^~185) and CN (^~190) double bonds exhibit strong π–π^* interactions but unless there is very good control of stray light, measurement is still unreliable in this region. At longer wavelengths there are rather weak n−π^* interactions, such as NO and keto CO in the range 280^~300 nm. Simple benzene compounds show medium intensity multiplets around 254 nm for non-conjugated derivatives, and shifted to longer wavelengths when substituents are conjugated to the aromatic system. In the table at the end of this section there are examples of commonly encountered chromophores, including conjugated alkene, carbonyl and aromatic systems which exhibit bathochromic (longer wavelength) and hyperchromic (enhanced absorptivity) changes. For very extensive catalogues of individual UV spectra, refer to DMS (1960–1971) (1160 substances), Hirayama (1967) (8500 selected values) and Sadtler (1979) (2000 spectra for 1600 compounds). Older spectra of specifically aromatic compounds were collated by Friedel and Orchin (1951). Schemes such as Woodward’s ‘Rules’ (1941, 1942), as further modified by L. and M. Fieser, for conjugated polyenes and en-ones, have considerable predictive power. (For a fuller discussion, see Scott (1964).)

References

British Pharmacopoeia (1993) Appendix IIB, A88-A89, HMSO, London.
DMS (1960–1971) Documentation of Molecular Spectroscopy: UV Atlas of organic compounds, Butterworth,
   London.
R. A. Friedel and M. Orchin (1951) Ultraviolet spectra of aromatic compounds, Wiley, New York.
K. Hirayama (1967) Handbook of UV & visible absorption spectra of organic compounds, Plenum Press.
Sadtler (1979) Handbook of UV spectra, Heyden, London.
A. J. Scott (1964) Interpretation of UV spectra of natural products, Pergamon Press, London.
R. B. Woodward (1941, 1942) J. Amer. Chem. Soc., 63, 1123 and 64, 72.

Solvent cut-off wavelengths

In this table, approximate wavelengths (nm) are specified below which the solvent absorbance may be unacceptable. For quantitative work, the cut-off may be set at a wavelength (L₀) where the absorbance for 10 mm pathlength of the solvent exceeds 0.05 absorbance unit (relative to water), i.e. A_{1 cm} > 0.05. For qualitative work, it may still be feasible to work at significantly lower wavelengths and most analysts accept a cut-off based on the wavelength (L₁) for A_{1 cm} > 1.0. However, if the UV absorption curve rises steeply, the accessible wavelength range may not be greatly extended.

UV absorption bands for typical chromophores

For nonconjugated π-systems, the bands may be inaccessible for conventional spectrometers. Conjugated alkene, carbonyl and aromatic systems are at longer wavelengths and more intense.

G.F. Phillips

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