Antonio H. Miguel
and Robert D. Braun University of Illinois Urbono, 61801
Fluorimetric Analysis of Nitrate in Real Samples
The fluorimetric analysisof "real" samples is often undesirable as a student experiment since prior separations or elaborate sample preparations are required. It is our purpose to report a fluorimetric, student experiment for the determination of trace nitrate in river water, soil, and fertilizer which requires no sample preparation except dissolution. Nitrate is a common pollutant resulting from the use of nitrogen fertilizers and their subsequent drainage into ground waters. Nitrate may he converted within the human body to nitrite which inhibits the oxygen carrying ability of blood hem~glohin.',~ The exoeriments listed in this . Daoer . have been oerformed by students in an undergraduate, instrumental analvsis course. All of the experiments were easily completed by the students within one laboratory pe&od of about three hours. Theory
These ex~erimentsare based on the method of Axelrod. Bonelli and Lodge.3 Upon excitation a t 435 nm, fluo: rescein exhibits a strong fluorescence emission hand a t 485 nm. The addition ornitrate to fluorescein in concentrated sulfuric acid results in the formation of a non-fluorescing compound which in all probability is the dye, Solvent Orange Nitrate is determined by the amount of quenching of the fluorescein fluorescence. The reaction of fluorescein to form Solvent Orange 16 is shown in Figure 1. Since the reaction between fluorescein and nitrate is slow. the actual fluorescence measurements on both the standard and the unknown solutions are made 45 min after the fluorescein and nitrate are mixed. Further reaction is wssihle hut is relativelv slow. For best results the reaction times of the standard and the unknown solutions should he equal within roughly 5 min.
SOLVENT OR4NbE 16
FLUORESCEIN
Figure 1. The reaction of fluorescein with nitrate to yield Solvent Orange 16. The reaction is carried out in concentrated sulfuric acid.
ty is obtained when the sulfuric acid concentration is 89% or greater.3 In each case the fluorescence measurement is made 45 min after the nitrate and fluorescein solutions are mixed. Fluorescence Spectra Fill the cell with the 5 x lo-' M disodium fluorescein in sulfuric acid solution and insert in the fluorimeter. To record the excitation spectrum, adjust the fluorimeter emission wavelength to 485 nm. Set the initial excitation wavelength at 350 nm and scan to about 5M) nm using the recorder to trace the spectrum. To record the emission spectrum, adjust the fluorimeter excitation wavelength to 435 nm, set the initial emission wavelength st 400 nm, and scan to about 600 nm. Typical excitation and emission spectra are shown in Figure 2. Working Curve Prepare a working curve by separately diluting 0.05, 0.10, 0.25, and 0.50 ml of a 2 x 10.' M nitrate solution to 50 ml with the 5 x 10-7 M disodium fluorescein in concentrated sulfuric acid. Set the fluorimeter excitation wavelength at 435 nm and the emission wavelength at 485 nm. After the nitrate and fluorescein solutions have reacted for 45 mi", separately fill the cell with each of the standard solutions and measure the fluorescence intensity. Mea-
Equipment
A Perkin-Elmer model 204 spectrofluorimeter and a Heath recorder were used in this experiment; however, any other comparable fluorimeter and recorder can he used. If complete excitation and emission fluorescence sDectra are not reouired.. i.e.., if the excitation and emission wavelengths are not scanned, the recorder is not needed and a ratio fluorimeter mav he suhstituted for the scanning fluorimeter. Fluorescence measurements were made in a 1.0-cm quartz cell. Experimental
In each case the nitrate solution is diluted in a 50-ml volumetric flask with a freshly prepared, 5 x lo-" M disodium fluorescein in concentrated sulfuric acid, solution. Warning: Extreme caution should be taken when working with concentrated sulfuric acid solutions. The use of safety glasses is imperative. In no case should more than 3 ml of a nitrate solution be added to the 50-ml volumetric flask since the greatest reaction sensitivi'Scientific American, 220.48 (1969). ZWolff,LA., and Wssserman, A. E., Science 177.15 (1972). 3Axelrod, H. D., Bonelli, .I. E., and Lodge, d . P., Anal. Chim. Aeto, 51,21 (1970). 682
I Journal ot Chemical Education
L
I
450 500 WAVELENGTH, nm
550
Figure 2. Fluorescence spectra of a 5 X t o - ' M disodium fluorescein in concentrated sulfuric acid solution. (A) Excitation spectrum observed at 485 nm: IB) emission spectrum with excitation at 435 nm.
fluorescein in sulfuric acid to hring the nitrate concentration within the range of the working curve. In our ease this was 0.2 ml for a high nitrate sail and 1.0 ml for a low nitrate sail, bath diluted to 50 ml. After 45 min measure the fluorescence intensity and determine the nitrate content of the soil sample by comparison with the working curve. Nitrate in Fertilizer Add 0.1 g of a commercial fertilizer to a 1-1 volumetric flask and dilute to the mark with distilled water. Dilute enough (about 0.05 ml) of this solution to 50 ml with disodium fluorescein solution to bring the nitrate concentration within the range of the ealihration curve. After 45 min measure the fluorescence intensity and determine the nitrate content of the fertilizer by comparison with the working curve. If the fertilizer contains ammonium, it is necessary to maintain the final ammonium ion concentration in the fluorescein solution at less than 10.' M. At higher concentrations, ammonium interferes with the determination. In most cases this problem should not be encountered since the final ammonium concentration will not normally exceed that of nitrate which is about 10.". Discussion and Conclusions Figure 3. A warking curve of
relative fluorescein fluorescence as a func-
tion of nitrate concentration, sure the fluorescence mtensity of fluorescein solution to which no nltrate has been added. Plot the fluorrsrence intensity a* a func. tiun of added nitrate. A typical working curve rs shuun in F l p r e 3.
Nitrate in River Water Dilute enough river water (in our case this was 0.2 mi) with the disodium fluorescein in sulfuric acid solution to hring the nitrate concentration within the range of the working curve (Fig. 3). After 45 min measure the fluorescence intensity as in the preceding section and by comparison with the working curve, determine the concentration of nitrate in the original river water sample. It is advisable for the student to do this determination in triplicate so that the precision of the method can he determined. Nitrate in Soil Pipet 10 ml of distilled water into 1 g of soil and shake for 10 min to dissolve the nitrate. Remove the solid particles by filtration and dilute enough of the soil-nitrate solution with disodium
Chloride concentrations on the order of 100 times the nitrate concentration and nitrite concentrations greater than 10 times the nitrate concentration interfere with this method.3 Except in those cases where interfering ions are present, the procedure is simple and rapid and can be used for "real" samples. Our students obtained results with this method which in each case agreed with those obtained by other laboratories which used different methods' of nitrate analysis. Acknowledgment We acknowledge t h e assistance of Mr. L. M. Henley of the Illinois State Water Survey for supplying analyzed river water samples and of Dr. L. F. Welch of the Agronom y Department of the University of Illinois for supplying the soil samples.
'West, P. W., and Ramachandran, T. P., Anal. Chim. Aeta, 51, 21 (1966).
Volume 51. Number 10. October 1974
/
683
