Ultraviolet-visible spectroscopy
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Ultraviolet-visible spectroscopy or ultraviolet-visible spectrophotometry (UV-Vis or UV/Vis) involves the spectroscopy of photons in the UV-visible region. This means it uses light in the visible and adjacent (near ultraviolet (UV) and near infrared (NIR)) ranges. The absorption in the visible ranges directly affects the color of the chemicals involved. In this region of the electromagnetic spectrum, molecules undergo electronic transitions. This technique is complementary to fluorescence spectroscopy, in that fluorescence deals with transitions from the excited state to the ground state, while absorption measures transitions from the ground state to the excited state.[1]
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[edit] Applications
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UV/Vis spectroscopy is routinely used in the quantitative determination of solutions of transition metal ions and highly conjugated organic compounds.
- Solutions of transition metal ions can be coloured (i.e., absorb visible light) because d electrons within the metal atoms can be excited from one electronic state to another. The colour of metal ion solutions is strongly affected by the presence of other species, such as certain anions or ligands. For instance, the colour of a dilute solution of copper sulfate is a very light blue; adding ammonia intensifies the colour 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 effect 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 colours, the colours 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.
A UV/Vis spectrophotometer may be used as a detector for HPLC. The presence of an analyte gives a response which can be 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 (e.g., peak height) for a particular concentration is known as the response factor.
[edit] Beer-Lambert law
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, I0 is the intensity of the incident light at a given wavelength, I is the transmitted intensity, L the pathlength 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 1 / M * cm or often AU / M * cm.
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 (Xylenol Orange or Neutral Red, for example).
[edit] Practical considerations
To actually make a valid measurement you must understand and be aware of the limitations of the particular instrument being used. This is especially important when making measurements using simple (and therefore relatively inexpensive) instruments, where a user is more likely to encounter an instrumental limitation, or when making measurements of materials that have not been well characterized yet.
The molar extinction coefficient, ε, is a function of the wavelength (that is, the color) of the light used. For the Beer-Lambert relation above to hold in a particular case, the light must be sufficiently monochromatic that the exctinction coefficient used is well defined.
There can also be limits imposed by the materials being measured, for instance if the material is not a simple solution.
Notice that the Beer-Lambert law implies that changes in concentration and path length should have equivalent effects. That is, for example, diluting a solution by a factor of 10 should have the same effect on absorbance as shortening the path length from the normal 10 mm to 1 mm. If cells of different path length are available, this is an alternative test to simply plotting absorption versus concentration in order to judge the validity of a measurement. Failure of such a test could indicate a concentration-dependent effect in the sample, such as absorption flattening.
[edit] Spectral bandwidth
For instance, the spectral bandwidth of the instrument (as FWHM), the portion of the spectrum selected for the measurement, must be much smaller than the width of the absorbance curve of the sample, so that the exctintion coefficient does not change significantly over the band. Some instruments allow selection of bandwidth. (The tradeoff is that reducing the bandwidth reduces the energy passed to the detector and will require a longer measurement time to achieve the same signal to noise ratio.)
[edit] Wavelength error
In liquids, the extinction coefficient usually changes slowly with wavelength. A peak of the absorbance curve (a wavelength where the absorbance reaches a maximum) is where the rate of change in absorbance with wavelength is smallest. Measurements are usually made at a peak in order to minimize errors produced by errors in wavelength in the instrument, that is errors due to having a different extinction coefficient than assumed.
[edit] Stray light
Another important factor is the purity of the light used. The most important factor affecting this is the stray light level of the monochromator [2] . The detector used 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 AU, 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.
[edit] Absorption flattening
The nature of the material being measured is also important. Solutions that are not homogeneous can show deviations from the Beer-Lambert law because of the phenomenon of absorption flattening. This can happen, for instance, where the absorbing substance is located within suspended particles. [3] The deviations will be most noticeable under conditions of low concentration and high absorbance. The reference describes a way to correct for this deviation.
[edit] Ultraviolet-visible spectrophotometer
The instrument used in ultraviolet-visible spectroscopy is called a UV/vis spectrophotometer. It measures the intensity of light passing through a sample (I), and compares it to the intensity of light before it passes through the sample (Io). The ratio I / Io is called the transmittance, and is usually expressed as a percentage (%T). The absorbance, A, is based on the transmittance:
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- A = − log(%T / 100%)
The basic parts of a spectrophotometer are a light source, a holder for the sample, a diffraction grating or monochromator to separate the different wavelengths of light, and a detector. The radiation source is often a Tungsten filament (300-2500 nm), a deuterium arc lamp which is continuous over the ultraviolet region (190-400 nm), and more recently light emitting diodes (LED) and Xenon Arc Lamps[4] for the visible wavelengths. The detector is typically a photodiode or a CCD. Photodiodes are used with monochromators, which filter the light so that only light of a single wavelength reaches the detector. Diffraction gratings are used with CCDs, which collects light of different wavelengths on different pixels.
A spectrophotometer can be either single beam or double beam. In a single beam instrument (such as the Spectronic 20), all of the light passes through the sample cell. Io must be measured by removing the sample. This was the earliest design, but is still in common use in both teaching and industrial labs.
In a double-beam instrument, the light is split into two beams before it reaches the sample. One beam is used as the reference; the other beam passes through the sample. Some double-beam instruments have two detectors (photodiodes), and the sample and reference beam are measured at the same time. In other instruments, the two beams pass through a beam chopper, which blocks one beam at a time. The detector alternates between measuring the sample beam and the reference beam.
Samples for UV/Vis spectrophotometry are most often liquids, although the absorbance of gases and even of solids can also be measured. Samples are typically placed in a transparent cell, known as a cuvette. Cuvettes are typically rectangular in shape, commonly with an internal width of 1 cm. (This width becomes the path length, L, in the Beer-Lambert law.) Test tubes can also be used as cuvettes in some instruments. The type of sample container used must allow radiation to pass over the spectral region of interest. The most widely applicable cuvettes are made of high quality fused silica or quartz glass because these are transparent throughout the UV, visible and near infrared regions. Glass and plastic cuvettes are also common, although glass and most plastics absorb in the UV, which limits their usefulness to visible wavelengths.[5]
[edit] Ultraviolet-visible spectrum
An ultraviolet-visible spectrum is essentially a graph of light absorbance versus wavelength in a range of ultraviolet or visible regions. Such a spectrum can often be produced directly by a more sophisticated spectrophotometer, or the data can be collected one wavelength at a time by simpler instruments. Wavelength is often represented by the symbol λ. Similarly, for a given substance, a standard graph of the extinction coefficient (ε) vs. wavelength (λ) may be made or used if one is already available. Such a standard graph would be effectively "concentration-corrected" and thus independent of concentration.
The Woodward-Fieser rules are a set of empirical observations which can be used to predict λmax, the wavelength of the most intense UV/Vis absorption, for conjugated organic compounds such as dienes and ketones.
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. UV/Vis absorption is not, however, a specific test for any given compound. The nature of the solvent, the pH of the solution, temperature, high electrolyte concentrations, and the presence of interfering substances can influence the absorption spectra of compounds, as can variations in slit width (effective bandwidth) in the spectrophotometer.
[edit] See also
- Infrared spectroscopy is another common spectroscopic technique, usually used to obtain free information about the structure of compounds.
- Spectroscopy
- Fourier transform spectroscopy
- Near infrared spectroscopy
- Vibrational spectroscopy
- Rotational spectroscopy
- Applied spectroscopy
[edit] Notes
- ^ Skoog, et al. Principles of Instrumental Analysis. 6th ed. Thomson Brooks/Cole. 2007, 169-173.
- ^ "Beer's Law - Alisdair Boraston". http://www.brewingtechniques.com/brewingtechniques/beerslaw/boraston.html. Retrieved 2009-02-06.
- ^ Wittung, Pernilla; Johan Kajanus, Mikael Kubista, Bo G. Malmström (8 August 1994). "Absorption flattening in the optical spectra of liposome-entrapped substances" (pdf). FEBS_Lett_352_37_1994.pdf (application/pdf Object). http://www.img.cas.cz/ge/FEBS_Lett_352_37_1994.pdf. Retrieved 2009-02-06.
- ^ Skoog, et al. Principles of Instrumental Analysis. 6th ed. Thomson Brooks/Cole. 2007, 349-351.
- ^ Skoog, et al. Principles of Instrumental Analysis. 6th ed. Thomson Brooks/Cole. 2007, 351.
