Fluorometric Determination of Sub-Nanogram Levels of Nitrite Using 5-Aminof luorescein Herman D. Axelrod and Nancy A. Engel’ National Center for Atmospheric Research, Boulder, CO 80303
Nitrite ion has recently attracted a great deal of attention because of its potential role in producing nitrosoamine, a carcinogenic material, in the human body. The concern stems from the fact that nitrites are commonly added to processed meats. The ability to measure nitrite a t extremely low levels is important to those studying nitrite reactions in our food system. A review of various ,techniques has been presented by Streuli and Averell (1). These techniques, in general, lack the sensitivity to reach the low nanogram range. Wiersma ( 2 ) and Dombrowski and Pratt (3) have developed sensitive fluorometric procedures but these are quite complicated relying on solvent extraction. A new procedure, presented here, is sensitive to 50 pg/ml (pg = g) and is also quite simple. Axelrod et al. ( 4 ) previously reported that 5-aminofluorescein is a suitable reagent for the analysis of sulfite. The 5-aminofluorescein is highly fluorescent, and, because it has a primary amino group, it was investigated and found to be useful for the trace determination of nitrite.
EXPERIMENTAL Instrumentation. A Perkin-Elmer Model 203 Spectrofluorometer equipped with a high-pressure xenon lamp (for continuous spectra) was used in the study. The excitation wavelength was 490 nm and the emission wavelength was 515 nm. All measurements were made in a 22 OC air conditioned room. Reagents. The 5-aminofluorescein (F.W. 347) was obtained from Eastman Kodak Co. (Rochester, N.Y.) and was used as received. All other chemicals were reagent grade; the water used was first passed through a deionizing column and then distilled. Procedure. A 1.5 X 10-3M 5-aminofluorescein (5AF) stock solution was prepared by dissolving the appropriate weight of the dye in 25 ml of absolute methanol in a 100-ml volumetric flask. After the 5AF has dissolved, 4 ml of concentrated HC1 (12.1M)was added and the solution was diluted to volume with distilled water. All further dilutions were made with distilled water. The analysis was performed in the following manner: 7 ml of an aqueous sample was placed in a 25-ml glass stoppered cylinder. Added to this was 1 ml of an appropriate dye concentration followed by 2 ml of 6M HC1. The sample was mixed and allowed to stand for an appropriate time period (up to 60 min-see later discussion). T o this solution, 5 ml of 5.4M NaOH was added. The solution was mixed and allowed to stand for 5 min prior to the fluorescence measurement. The product is stable for measurement up to 24 hr later. Nitrite standards of a suitable concentration range were analyzed in the same manner. In this particular analytical technique, the fluorescence increases with increasing nitrite concentration. Therefore, the standard blank (no nitrite) was used to set the 0 units fluorescence and the highest nitrite standard was used to set the 100 units fluorescence. This procedure was used to measure nitrite concentrations from iO-5M to 5 x I O - ~ Musing final dye concentrations of 10-5M to 3 X 10-8M, respectively.
RESULTS AND DISCUSSION Optimization of Acidity and Basicity. The acid and base concentrations were optimized by varying the acid or base over an appropriate range and noting a t a fixed nitrite concentration which acid or base concentration produced the best response. Present address, School of Pharmacy, University of Washington, Seattle, WA. 922
ANALYTICAL CHEMISTRY, VOL. 47, NO. 6, M A Y 1975
The analytical procedure was essentially independent of acid levels when the HC1 was 1-2M. Sulfuric and phosphoric acids were also used but they did not perform as well. The procedure was best and independent of NaOH levels when the NaOH was 1M final concentration. No other bases were investigated. The above acid and base concentrations were also independent of the 5AF and nitrite concentrations.
Reaction Time. The time for both the acid reaction and the base reaction to go to completion were studied. By varying the reaction times and comparing the emission of the respective products to a fluorescein standard, it was determined that the degree of completion of the acid reaction was a function of time and dye concentration, whereas that of the base reaction was not. At 10-5M 5AF, the acid reaction goes to completion within 5 min, but the fluorescence shows a slight decay with time, while a t lO-’M to 10-8M 5AF, the acid reaction product concentration rises sharply a t first and then levels off after 45 and 60 min, respectively. These reaction time curves are shown in Figure 1. Because of this time effect, the length of time allowed after the addition of acid but before the addition of base, should be held constant for each sample and for the standards. Unlike the acid reaction, the base reaction goes to completion within 5 min and the reaction product is stable for up to 24 hr. Variations in acid concentrations and solution temperature did not affect the reaction rate to any degree which would be useful in this analysis. Therefore, the 1.2M HCl was chosen to be an acceptable level for this procedure, Many times one can shorten reaction times by increasing the concentration of one of the components such as the 5AF. In this instance, however, this is not possible because the 5AF is itself fluorescent and thus one would not be able to observe the small increases in the solution fluorescence that would result from the presence of trace amounts of nitrite. Standardization Curve. The shape of the standardization or analysis curve was dependent upon the dye concentration and the nitrite working range. Figure 2 shows the various shapes. In general, as the 5AF concentration decreases, the standardization curve becomes curved and drawn out. The degree of curvature or the amount of nitrite required to consume all of the 5AF present was dependent upon the HC1 used. Different batches and brands of HC1 were used and, a t 10-7M 5AF, each acid produced its own unique standardization curve. The amount of nitrite required to consume all of the dye present varied between 1 and 10 times the dye concentration. In general, the best sensitivity was achieved with DuPont reagent grade HCl. The use of Ultrex HC1 (high purity, J. T. Baker Co.) did not show any sensitivity improvement over the DuPont acid. Although a study has not been made to determine why different lots of HCl have such an effect, one can prob-
I
I
Table I. Reproducibility at Various Nitrite Concentrations
1
a
Nitrite concn
Re1 std dev, % a
7.5 x IO-~.W (345 ng/ml) 1 .O x 10-7M(4.6 ng/ml) 1.O x 10-8.W (0.46 ng/ml)
5.7 5.2 6.7
Based upon 10 samples at each concentration.
Table 11. Materials Investigated for Interferencea Compound
NH,'
L
I
0
20
I
I
40 60 TIME, minutes
cr3+
I
1
80
100
C ~ , O ~ ~ CU2'
Figure 1. Time study of acid reaction in 1.2MHCI ( A ) 10-5M NO*-, NOz-, 10-8M5AF
5AF; ( E ) lO-'M
I
I
NOn-, lO-'M
I
5AF; (C) 10-8M
Ni2* K+
I
I
Mg2* Ca2+ Mn2' Hg2'
-
AP+ ce3+ Fez*
a
% Error
Compound
%
-4 9 -22 0 0 0
0 0
Fe3' Pb2+
0 0 0 0 -40 0
NO3'
0
S042'
0 0 0 -88
Zn2+ Br' BrO,' ~ 0 ~ 3 -
Error
-100 0 0 0
SO3'-
-26
C2H302-
0 0 0
CH3COCH3 HCHO
10-7M NOz-; potential interferent at 100-fold mole excess.
* Fez+ at 10-fold mole excess. 5AF CONCENTRATION
0
05 NO,
1 x 5AF
IO
1.5
20
CONCENTRATION
Figure 2. Standardization curve at various 5AF concentrations
?.NOH
I 1
J
FINAL PRODUCT
Figure 3. Proposed reaction for the NO2- analysis procedure
ably attribute this observation to trace impurities present in the HC1 which can react with the nitrite. Reproducibility. The reproducibility of the method was determined over a range of concentrations and the data are shown in Table I. As the concentration approached the 10-sM nitrite range, instrumental factors such as lamp stability, photomultiplier response, etc., appeared to be a major factor in the reproducibility of the method. Sensitivity. The sensitivity of this procedure is about 5 X 10-9M, with the 5AF at 3 X 10-sM. As with the reproducibility, the sensitivity value depended upon instrument stability a t high signal amplification and the quality of re-
agents. Since the reaction was utilized at low concentrations, it is, therefore, possible to achieve measurements of even lower nitrite levels if better instrumentation and chemicals are available. Interferences. Various cations, anions, and organic compounds were added to an aqueous nitrite solution and the mixture was analyzed as in the previously described procedure. The results were then compared to the analysis of the same nitrite concentration with no potential interferent present. Table I1 shows the results of this experiment. As expected, oxidants and reductants tended to interfere. For the most part, the analysis is free of interferences. Proposed Reaction and Stoichiometry. The reaction stoichiometry between the dye and nitrite was determined by preparing a standard working curve and determining a t which point a further increase in nitrite concentration did not further increase the solution fluorescence. For and 10-6M dye levels, the stoichiometry was 1:1, nitrite-dye. However, a t 10-7M 5AF, the stoichiometry was found to vary from 1-1O:l depending upon which brand of reagent grade HC1 was used. Apparently, the variation is due to impurities in the acid so that it is imperative that standards be run when analyzing at very low levels. On the basis of the above observations and the literature (5),a reaction can be proposed for this analytical procedure (Figure 3). This reaction has not been studied, so the proposed mechanism is simply conjecture. It should be noted that there might be some additional reactions occurring, especially a t very dilute concentrations. If one examines the standardization curve for dilute solutions (Figure 2, lO-'M), the data show a fall-off in intensity as the nitrite concentration increases. One would normally expect that the fluorescence would increase linearly with increasing nitrite in a similar manner to the Beer-Lambert law for absorbance. Since this is not the case, it is conceivable that there is another reaction or other reactions present which affect the fluorescence intensity. Spectra and Optimum Wavelengths. The spectra for the 5AF and the reaction product were studied and found ANALYTICAL CHEMISTRY, VOL. 47, NO. 6, MAY 1975
923
I
$Jy\ ,I a 2 0
J LL
200
300
400 500 600 WAVELENGTH ( n m )
700
Figure 4. Spectra of 5AF-nitrite system. (A) Excitation spectra of 10-7M 5AF; ( B )Emission spectra of 10-7M 5AF; ( C ) Amplification of A; (0) Amplification of lO-'M 5AF, 10-7M NO*-
to be quite complicated. However, they both show excitation-emission at 490-515 nm. These two wavelengths (for the instrument and lamp used in this work) provide the best analytical sensitivity. Figure 4 shows the spectra for the 5AF-NO2- system. Lines A and B show that the 490515 nm combination is best. In addition, excitation at lower
wavelengths can be used but a t reduced analysis sensitivity. While the dye and the dye-nitrite spectra have large excitation maxima a t 490 nm, the spectra differ in the 250400 nm region. Lines C and D are amplifications of that lower nm region. It should be noted that excitation at wavelengths in the 250-400 nm region will produce emissions a t 365-385, 410-425, 690, and 750 nm in addition to 515 nm. However, only the 515-nm peak is suitable for analysis work, as the intensity of the other emission peaks is not altered by the reaction of nitrite with 5AF. It is also possible that some of the non-usable peaks are due to the presence of impurities in the 5AF dye.
LITERATURE CITED (1) C. A. Streuli and P. R. Averell, "The Analytical Chemistry of Nitrogen and Its Compounds", Wiley-lnterscience, New York. NY, 1970. (2) J. ti. Wiersma, Anal. Letts., 3, 123 (1970). (3) L. J. Dombrowski and E. J. Pratt, Anal. Chem., 44, 2268 (1972). (4) H. D. Axelrcd, J. E. Bonelli, and J. P. Lodge, Jr., Anal. Chem., 42, 512 (1970). (5) J. D. Roberts and M. C. Caserio, "Basic Principles of Organic Chemistry", W. A. Benjamin, New York. NY, 1965.
RECEIVEDfor review September 16, 1974. Accepted January 2,1975. The National Center for Atmospheric Research is sponsored by the National Science Foundation.
Analysis of Urine for Trace Elements by Energy Dispersive X-Ray Fluorescence Spectrometry with a Pre-Concentrating Chelating Resin Madhulika Agarwal,' Roy B. Bennett,2 1. G. Stump, and John M. D'Auria3 Department of Chemistry, Simon Fraser University, Burnaby, British Columbia, Canada
The role of trace elements in the body is an area which continues to receive a great deal of attention ( I ) , especially as newer analytical techniques are developed. Various studies have indicated correlations between particular elemental fluctuations and specific medical disorders (2, 3 ) . Analyses of human urine as a diagnostic tool has not been fully utilized, yet urine is easily obtainable, can be handled without complicated apparatus, and contains various trace elements indicative of medical disorder. Diagnostic elements of particular importance recently are copper ( 4 , 5 ) , zinc (5-7), and lead (8) with respect to a variety of medical disorders. Standard instrumental methods of analysis of trace elements include atomic absorption (9) and stripping voltametry (10). Wavelength dispersive X-ray fluorescence spectroscopy of nanogram amounts of chromium in urine has been performed by Beyermann et al. ( 1 1 ) . The advent of energy dispersive X-ray fluorescence spectroscopy (12, 13) does allow multielemental determinations, rapidly and accurately without destroying the samples. The areas of applicability of this new analytical technique are only now Present address, McMaster University Medical School, Hamilton, Ontario. * Present address, Bio-Tracers Services, 1908 Mahon Avenue, North Vancouver, B.C. Author to whom reprint requests should be sent. 924
ANALYTICAL CHEMISTRY, VOL. 47, NO. 6, MAY 1975
being fully appreciated and it is conceivable that it can provide valuable diagnostic information rapidly when used routinely on urine samples. It should be noted that matrix effects (14) must be taken into account when performing quantitative analysis; however, these could be avoided by using the sample material itself in the analysis. The purpose of the present study was to explore the applicability of an energy dispersive X-ray fluorescence spectroscopy (EDXS) system to determinations of trace elements, e.g., Cu, Zn, and P b in urine. The system available was a photon excitation, secondary target EDXS system (15) and two major factors had to be investigated: a sample preparation scheme which allows determination of the trace elements in the concentration levels normally present in urine; and a sample preparation scheme which is reasonably rapid and efficient but allows for reproducible and quantitative determinations. Various sampling procedures were studied including spotting onto filter paper, freeze-drying urine samples, and using an ion exchange chelating resin, Chelex 100 to provide pre-concentration prior to analysis. Earlier studies by Blount e t al. ( 1 6 ) have utilized similar chelating preparatory procedures for barium ions in an aqueous medium prior to wavelength dispersive X-ray spectroscopy. This resin shows a high preference for divalent and polyvalent metal ions, such as Cu, Hg, Zn, and Pb, over lighter metals corre-
