4.4: Spectrophotometry
- Page ID
- 121644
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The concentration of a pure compound is to be determined by its absorbance at 290 nm and its known molar absorptivity at this wavelength. You have a choice of four different spectrophotometers available in the laboratory, employing either a tungsten, xenon, mercury or deuterium light source.
QUESTIONS
- Which lamp might be the best light source for this experiment?
- You subsequently learn that the spectrophotometer with the deuterium lamp has a glass cuvette, while the xenon spectrophotometer has a quartz cell. Does this affect your decision about which spectrophotometer to use? Why?
Questions to Consider
- Do all these light sources emit equal amounts of light energy throughout the ultraviolet and visible spectra?
- Does increased energy output of a light source have any advantage in a spectrophotometric assay?
- Do all optical materials transmit light with equal efficiency?
- Answer
-
- Usually the deuterium lamp is preferred because it gives uniform light of good intensity in the ultraviolet region. (see p. 90).
- Yes. The xenon spectrophotometer with a quartz cell will probably have overall better performance than will the instrument with regular glass. (Remember that sensitivity = L x M x R.) Thus, the instrument with the deuterium lamp would not be as useful as the spectrophotometer with the xenon light source.
Answers to Questions to Consider
- The energy emitted from a light source is the result of heating a material to incandescence. The energy released during the emission process depends upon the nature of the material being used. Thus, most of the energy of the tungsten lamp is emitted in the far-red (>700 nm) as heat, with very little light emitted at wavelengths less than 300 nm. Xenon lamps produce a fair amount of light throughout the UV and visible spectra, while most of the light emitted by deuterium lamps falls in the UV spectrum. Mercury lamps produce a discontinuous light spectrum, with light being emitted only at discrete wavelengths. This is shown in Figures 4-7 and 4-8 (p. 90). Thus, only the xenon or deuterium lamp would be useful for experiments at about 290 nm.
- As described on p. 92, the sensitivity of a spectrophotometric analysis is dependent upon the lamp output at a given wavelength. At a given photomultiplier tube response, the greater the light output, the higher the sensitivity. At a given concentration of material, the greater the input (incident) light, the greater the amount of light that will be transmitted (although the percent transmission remains constant). Thus, the more light transmitted, the greater the current output of the photomultiplier tube and the more precise and sensitive the assay.
- As depicted in Figure 4-10 (p. 91), the transmission characteristics of optical materials vary greatly. Quartz glass is much more efficient at transmitting light at 290 nm than regular glass or many plastics.