This lab instrument is used in the analysis of compounds based on their absorbance in the UV (200-400 nm) and visible (400-800nm) spectra. Its history dates the back to early 1800s when the diffraction grating was invented, enabling researchers to separate a light beam into its wavelengths. The instrument is based on electronic transitions that occur when a compound is exposed to electromagnetic spectra. Electrons in a molecule would absorb light energy and move to a higher energy level. The instrument detects the absorption intensities at each wavelength and plots a graph of absorbance vs. wavelength (Fakhri et al., 2006).
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The instrument’s functioning is based on a simple principle. The light within the UV-visible spectra beamed to a monochrome is split into individual wavelengths. Each wavelength is further divided into a ‘sample’ beam (red light) and a ‘reference beam’ (blue light) by a half mirror (Fakhri et al., 2006). The red beam is directed through the test compound in a cuvette while the reference beam goes through a solvent.
Electronic sensors in the instrument then detect and compare the absorption intensities of the reference (I0) and the test compound (I). The wavelengths of a light beam that fall between 200nm and 800nm are scanned. The instrument then plots the absorbance (l-l0) against wavelength in a graph.
The maximum absorbance, denoted as λmax, varies between compounds and depends on molar concentration. Generally, for absorbing compounds, absorbance is more intense at a high molar concentration. Therefore, it is possible to quantify the organic content of a sample/compound based on its standard molar absorptivity.
Nitrate and Nitrate Compounds
Traces of nitrates (NO3–) and nitrites (NO2–) occur in most organic compounds. The determination of these two ions is usually challenging because of interferences from other materials. The common spectrophotometric methods depend on nitrate reduction. For example, the Griess-IIosvay method measures the absorbance of nitrite resulting from nitrate reduction reactions (Pasquali et al., 2007). NO3– in soil or water samples is first reduced to NO2– by acidic vanadium (III), which then is detected by the Griess test (Miranda et al., 2000).
The color intensity of the product peaks after 16 hours and can be measured at 540nm (Doane and Horwath, 2003). However, free NO3– ions in solution absorb at 220nm, which is also the wavelength where most organic ions absorb UV-visible light (Fakhri et al., 2006). A correction for the foreign ions associated with NO3– can be made by measuring absorptivity at 275nm, as NO3– ions do not absorb at this wavelength. Therefore, this approach can allow one to correct for the organic content or contaminants in the sample. Based on the absorbance readings, a calibration graph can be plotted to determine the quantity of NO3– in the substance.
The Indole Reagent
The absorbance is only measurable for compounds made into dilute solutions. The solvents used must be non-absorbing to reduce interferences. The indole reagent is used as a chromogen in an acidic medium to detect nitrates released from a nitrate reduction reaction (Fakhri et al., 2006). The reagent forms an intense yellow coloration with an optimal absorbance at 395nm. The intensity of the chromophore peaks at 5 minutes after dilution. Its stability lasts for 60 minutes, after which it gradually fades (Fakhri et al., 2006). The reagent has superior sensitivity and accuracy than 4,5-dihydroxy coumarin as a chromogenic reagent.
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Doane, T. A.; Horwath, W. R. Spectrophotometric Determination of Nitrate with a Single Reagent. Anal. Lett. 2003, 36, 2713-2722.
Fakhri, N. A.; Rahim, S. A.; Bashir, W. A. Indole as A Chromogenic Reagent for Traces of Nitrate in Aqueous Solution. Int. J. Environ. An. Ch. 2006, 16, 131-138.
Miranda, K. M.; Espey, M. G.; Wink, D. A. A Rapid, Simple Spectrophotometric Method for Simultaneous Detection of Nitrate and Nitrite. Nitric Oxide-Biol Ch. 2001, 5, 62-71.
Pasquali, C. E.; Hernando, P. F.; Alegria, J. S. Spectrophotometric Simultaneous Determination of Nitrite, Nitrate and Ammonium in Soils by Flow Injection Analysis. Anal. Chim. Acta. 2007, 600, 177-182.