Ultraviolet–visible spectroscopy
Ultraviolet–visible spectrophotometry refers to absorption spectroscopy or reflectance spectroscopy in part of the ultraviolet and the full, adjacent visible regions of the electromagnetic spectrum. Being relatively inexpensive and easily implemented, this methodology is widely used in diverse applied and fundamental applications. The only requirement is that the sample absorb in the UV–Vis region, i.e. be a chromophore. Absorption spectroscopy is complementary to fluorescence spectroscopy. Parameters of interest, besides the wavelength of measurement, are absorbance or transmittance or reflectance, and its change with time.
A UV–Vis spectrophotometer is an analytical instrument that measures the amount of ultraviolet and visible light that is absorbed by a sample. It is a widely used technique in chemistry, biochemistry, and other fields, to identify and quantify compounds in a variety of samples.
UV–Vis spectrophotometers work by passing a beam of light through the sample and measuring the amount of light that is absorbed at each wavelength. The amount of light absorbed is proportional to the concentration of the absorbing compound in the sample.
Optical transitions
Most molecules and ions absorb energy in the ultraviolet or visible range, i.e., they are chromophores. The absorbed photon excites an electron in the chromophore to higher energy molecular orbitals, giving rise to an excited state. For organic chromophores, four possible types of transitions are assumed: π–π*, n–π*, σ–σ*, and n–σ*. Transition metal complexes are often colored owing to the presence of multiple electronic states associated with incompletely filled d orbitals.Applications
UV–Vis can be used to monitor structural changes in DNA.UV–Vis spectroscopy is routinely used in analytical chemistry for the quantitative determination of diverse analytes or sample, such as transition metal ions, highly conjugated organic compounds, and biological macromolecules. Spectroscopic analysis is commonly carried out in solutions but solids and gases may also be studied.
- 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. 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.
A UV–Vis spectrophotometer may be used as a detector for HPLC. The presence of an analyte gives a response assumed to be proportional to the concentration. For accurate results, the instrument's response to the analyte in the unknown should be compared with the response to a standard; this is very similar to the use of calibration curves. The response for a particular concentration is known as the response factor.
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 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.
The method is most often used in a quantitative way to determine concentrations of an absorbing species in solution, using the Beer–Lambert law:
where A is the measured absorbance, is the intensity of the incident light at a given wavelength, is the transmitted intensity, L the path length through the sample, and c the concentration of the absorbing species. For each species and wavelength, ε is a constant known as the molar absorptivity or extinction coefficient. This constant is a fundamental molecular property in a given solvent, at a particular temperature and pressure, and has units of.
The absorbance and extinction ε are sometimes defined in terms of the natural logarithm instead of the base-10 logarithm.
The Beer–Lambert law is useful for characterizing many compounds but does not hold as a universal relationship for the concentration and absorption of all substances. A 2nd order polynomial relationship between absorption and concentration is sometimes encountered for very large, complex molecules such as organic dyes.
UV–Vis spectroscopy is also used in the semiconductor industry to measure the thickness and optical properties of thin films on a wafer. UV–Vis spectrometers are used to measure the reflectance of light, and can be analyzed via the Forouhi–Bloomer dispersion equations to determine the index of refraction and the extinction coefficient of a given film across the measured spectral range.
Practical considerations
The Beer–Lambert law has implicit assumptions that must be met experimentally for it to apply; otherwise there is a possibility of deviations from the law. For instance, the chemical makeup and physical environment of the sample can alter its extinction coefficient. The chemical and physical conditions of a test sample therefore must match reference measurements for conclusions to be valid. Worldwide, pharmacopoeias such as the American and European pharmacopeias demand that spectrophotometers perform according to strict regulatory requirements encompassing factors such as [|stray light] and wavelength accuracy.Spectral bandwidth
Spectral bandwidth of a spectrophotometer is the range of wavelengths that the instrument transmits through a sample at a given time. It is determined by the light source, the monochromator, its physical slit-width and optical dispersion and the detector of the spectrophotometer. The spectral bandwidth affects the resolution and accuracy of the measurement. A narrower spectral bandwidth provides higher resolution and accuracy, but also requires more time and energy to scan the entire spectrum. A wider spectral bandwidth allows for faster and easier scanning, but may result in lower resolution and accuracy, especially for samples with overlapping absorption peaks. Therefore, choosing an appropriate spectral bandwidth is important for obtaining reliable and precise results.It is important to have a monochromatic source of radiation for the light incident on the sample cell to enhance the linearity of the response. The closer the bandwidth is to be monochromatic the more linear will be the response. The spectral bandwidth is measured as the number of wavelengths transmitted at half the maximum intensity of the light leaving the monochromator.
The best spectral bandwidth achievable is a specification of the UV spectrophotometer, and it characterizes how monochromatic the incident light can be. If this bandwidth is comparable to the width of the absorption peak of the sample component, then the measured extinction coefficient will not be accurate. In reference measurements, the instrument bandwidth is kept below the width of the spectral peaks. When a test material is being measured, the bandwidth of the incident light should also be sufficiently narrow. Reducing the spectral bandwidth reduces the energy passed to the detector and will, therefore, require a longer measurement time to achieve the same signal to noise ratio.
Wavelength error
The extinction coefficient of an analyte in solution changes gradually with wavelength. A peak in the absorbance curve vs wavelength, i.e. the UV–VIS spectrum, is where the rate of change of absorbance with wavelength is the lowest. Therefore, quantitative measurements of a solute are usually conducted, using a wavelength around the absorbance peak, to minimize inaccuracies produced by errors in wavelength, due to the change of extinction coefficient with wavelength.Stray light
Stray light in a UV spectrophotometer is any light that reaches its detector that is not of the wavelength selected by the monochromator. This can be caused, for instance, by scattering of light within the instrument, or by reflections from optical surfaces.Stray light can cause significant errors in absorbance measurements, especially at high absorbances, because the stray light will be added to the signal detected by the detector, even though it is not part of the actually selected wavelength. The result is that the measured and reported absorbance will be lower than the actual absorbance of the sample.
The stray light is an important factor, as it determines the purity of the light used for the analysis. The most important factor affecting it is the stray light level of the monochromator.
Typically a detector used in a UV–VIS spectrophotometer is broadband; it responds to all the light that reaches it. If a significant amount of the light passed through the sample contains wavelengths that have much lower extinction coefficients than the nominal one, the instrument will report an incorrectly low absorbance. Any instrument will reach a point where an increase in sample concentration will not result in an increase in the reported absorbance, because the detector is simply responding to the stray light. In practice the concentration of the sample or the optical path length must be adjusted to place the unknown absorbance within a range that is valid for the instrument. Sometimes an empirical calibration function is developed, using known concentrations of the sample, to allow measurements into the region where the instrument is becoming non-linear.
As a rough guide, an instrument with a single monochromator would typically have a stray light level corresponding to about 3 Absorbance Units, which would make measurements above about 2 AU problematic. A more complex instrument with a double monochromator would have a stray light level corresponding to about 6 AU, which would therefore allow measuring a much wider absorbance range.