resolution available is 2 cm-l. Even with instruments capable of higher resolution, it is common to work with 2 to 4 cm-l resolution to decrease scanning and computational time. In solution spectra, bands with FWHH of 2 to 5 cm-' are not uncommon (8,11,24). For a 4 cm-' wide band measured with R = 2 cm-l, the results of this work indicate that the measured peak absorbance will be at least 16% low if triangular apodization is used. In this case, a considerable increase in accuracy is possible if the interferogram is not apodized. Triangular apodization is only one of many forms possible (12). Apodization somewhere between triangular apodization and no apodization at all would introduce smaller resolution errors than the former but not have the disadvantages of the latter.. Such a function might be an excellent compromise for analytical work.
LITERATURE CITED (1) D. M. Dennison, Phys. Rev., 31, 503 (1928). (2) D. A. Ramsay, J. Am. Chem. SOC.,74, 72 (1952). (3) J. R. Nielsen, V. Thornton, and E B. Dale, Rev. Mod. Phys., 16, 307 (1944). (4) H. J. Kostkowski and A. M. Bass, J. Opt. SOC.Am., 46, 1060 (1956). (5) S. Brodersen, J. Opt. SOC.Am., 44, 22 (1954) (6) R. N. Jones, D. A. Ramsay, D. S. Keir, and K. Dobriner, J. Am. Chem. SOC.,74, 80 (1952). (7) B. A. Russell and H. W. Thompson, Spectrochim. Acta, 9, 133 (1957). (8) H. J. Sloane, Appl. Spectrosc., 16, 5 (1962).
(9) J. Morcillo, J. Herranz, and M. J. de la Cruz. Spectrochim. Acta, 15, 497 (1959). (10) W. J. Potts. Jr. and A. L. Smith, Appl. Opt.. 6, 257 (1967). (11) K. S. Seshadri and R. N. Jones, Spectrochim. Acta, 19, 1013 (1963). (12) P. R. Griffiths, "Chemical infrared Fourier Transform Spectroscopy", Wiley Interscience, New York, 1975. (13) H. A. Lorentz, K. Ned. Akad. Wet. Proc., 8 , 591 (1906). (14) P. R. Griffiths, C. T. Foskett, and R. Curbelo, Appl. Spectrosc. Rev.. 6, 31 (1972). (15) D. C. Champeney, "Fourier Transforms and Their Applications." Academic Press, New York, 1973, p 20. (16) IBM Svsternl360 Scientific Subroutine Packaae. Version Ill. Subroutine DQG32. (17) G. Horlick, Anal. Chem., 43(8), 61A (1971). (18) G. Horlick, Anal. Chem., 44, 943 (1972). (19) R. L. Kirlin and A. M. Despain, Air Force Cambridge Research Laboratories. ReDort No. AFCRL-69-0039 11968). (20) W. J. Piice, in "Laboratory Methods in infrared Spectroscopy", 2nd ed., R . G. J. Miller and B. C. Stace, Ed., Heyden and Sons, Ltd., London, 1972, p 97. (21) P. R. Griffiths, Appi. Spectrosc., 29, 11 (1975). (22) L. Mertz. InfraredPhys., 7, 17 (1967). (23) T. Hirschfeld, Paper 307, Pittsburgh Conference on Analytical Chemistry and Applied Spectroscopy, Cleveland, Ohio, 1975. (24) L. W. Herscher, Spectrochim. Acta, 15, 901 (1959).
RECEIVEDfor review May 19,1975. Accepted September 5, 1975. The authors gratefully acknowledge the financial support provided by the National Science Foundation Grant GP-38728X. This work was presented in part at the 30th Symposium on Molecular Structure and Spectroscopy, Ohio State University, June 20, 1975.
Substituted Benzophenone as Fluorometric Reagent in Automated Determination of Nitrate B. K. Afghan and J. F . Ryan Analflical Methods Research Section, Canada Centre for Inland Waters, P. 0. Box 5050, 8 6 7 Lakeshore Road, Burlington, Ontario, Canada L7R 4 A 6
Substituted benzophenones, under appropriate reaction conditions, react to form strong fluorescent species with a number of Ions such as boron, vanadium, chromium, and nitrate. The proposed method utilizes 2,2'-dlhydroxy-4,4'-dimethoxybenzophenone as a new and sensitive fluorometric reagent for the determination of nitrate. The procedure to eliminate possible Interferences from high concentrations of chloride, sulfide, and humic acid substances Is also incorporated In the automated method. The proposed method has been applied to a wide variety of natural waters and sediments. The analysis may be performed at a rate of 20 samples per hour. The method can be used to detect nitrate as low as 5 wg/llter nitrate-nitrogen. The proposed method has also been compared with the most wldely used colorimetric method for the determination of nitrate.
It is generally accepted that the majority of substituted benzophenones tend to produce phosphorescence instead of fluorescence (1-3). The main reason for this is that the majority of these compounds possess the lowest excited singlet state of (n,r'+) character; therefore, intersystem crossing to triplet manifold is usually very efficient. It is also well established that certain environmental factors such as substitution, solute-solvent interaction, nature of the catalyst, etc., can alter these compounds and the nature of transition, energies, and intensity of luminescence ( 4 ) .
Acetophenone and related compounds are known to react in concentrated sulfuric acid, and produce polymeric species (5). Therefore, it is possible to alter the transition probabilities of these molecules by changing the reaction conditions, suitable substitution, solvent, etc., and produce species which may result in fluorescence instead of phosphorescence. In fact, in our laboratories, it was found that benzophenone, generally considered to produce phosphorescence, was made to produce strong fluorescence when dissolved in concentrated sulfuric acid. In addition to that, it was also found that the fluorescence of various substituted benzophenones was markedly affected by the nature and position of substituents in the benzophenone molecule. It was further observed that the fluorescence of some substituted benzophenones in the presence of trace quantities of other ions such as boron, nitrate, chromate, and vanadate was enhanced considerably. Therefore, it was decided to systematically investigate these compounds as possible fluorometric reagents for the analysis of various constituents in water, waste waters, and sediments. The fact that these reactions can be carried out in a media with relatively high acid concentration offers an additional advantage, particularly when analyzing sediments or other solid samples. If these reagents can be optimized to selectively determine various contaminants, they will prove advantageous in the routine analysis of sediments, since the error due to contamination will be minimized considerably. During the analysis of sediments, the majori-
ANALYTICAL CHEMISTRY, VOL. 47, NO. 14, DECEMBER 1975
2347
CAM
- 10 SAMPLES PER HOUR. 1:2 SAMPLE TO WASH
RATIO
COLOUR MLS CODE MIN
AIR
JACKETED
*PURPLE
*
1 71 I
GREEN1
144
TECHNICON PROPORTIONING PUMP
I
OIL BATH
1-1 HEWLETT -PACKARD 680 STRIP CHART RECORDER RANGE - 50 rn v CHART SPEED - 8 in PER HOUR
FAR RAND RATIO FLUOROMETER
II
-
PRIMARY FILTER 355 n rn NARROW PASS SECONDARY FILTER- 460n m NARROW PASS SAMPLE APERTURE 2 or 3 REFERENCE APERTURE 2 RANGE - 1 ( 0 - 4 p p m N 0 3 )
-
-
PULSE SUPPRESSORS
-
1 0 025 in I D
ACIDFLEX MANIFOLD PUMP TUBE 2- ORANGE-YELLOW 0 020 in ID 3- ORANGE- BLACK 0 005 in ID
x. ACIDFLEX MANIFOLD PUMP TUBES
NOTE ACIDFLEX TRANSMISSION TUBING AND SLEEVING USED FOR SULFURIC ACID TRANSMISSION NOTE TECHNICON FLUOROMETER FILTERS USED
Flgure 1. Manifold for proposed fluorometric method
ty of samples are initially digested in concentrated acids and a particular component is analyzed after neutralizing the excess acid. This practice normally introduces the chance of considerable error, particularly when analyzing trace constituents in sediments or other solid materials. The possible use of the above reagents may eliminate this problem and provide relatively accurate results. This series of papers will investigate the application of various substituted benzophenones and related compounds for the analysis of trace contaminants in water, waste water, and sediments. This paper deals with a systematic survey of hydroxy and methoxy substituted benzophenones and the use of 2,2f-dihydroxy-4,4f-dimethoxybenzophenone as a sensitive reagent for the direct determination of nitrate in natural waters and sediments. The method is optimized to analyze natural waters and sediments for nitrate in the range of 5.0 pg/liter-1.0 mg/liter nitrate-nitrogen. The sensitivity of the method has been compared with the most widely used colorimetric method for the determination of nitrate which depends upon the reduction of nitrate to nitrite and the determination of the resultant nitrite by colorimetry (6). In the proposed method, other forms of nitrogen such as nitrite or organic nitrogen compounds do not react; hence, this method can be used to determine nitrate directly without any subtraction of background due to other forms of nitrogen. 2348
EXPERIMENTAL Apparatus. All AutoAnalyzer equipment used consisted of standard Technicon modules. A Neslab RTE-3 circu!ator, manufactured by Neslab Instruments Ltd., was used to maintain a temperature of 20 O C during fluorescence measurement in the flow cell. Fluorescence was measured using a Ratio Fluorometer-2, manufactured by Farrand Instruments Ltd., in conjunction with a Hewlett-Packard 680 strip chart recorder. Preparation of Column to Remove Organic Matter. The XAD-2 column consists of 15 cm X 0.32 cm i.d. glass tubing packed with amberlite XAD-2 polymeric adsorbent. Initially, the glass tubing was plugged a t one end with glass wool and an N-6 nipple was butted against the plugged end using a lh-in. length of %-in. i.d. Tygon tubing for sleeving. A short length of Technicon lh-in. i.d. transmission tubing was attached to the nipple and fitted with a hosecock. The other end of the column was connected to a 65-mm filtering funnel using 3/x-in. i.d. Tygon tubing. The column assembly was secured in a vertical position and filled with distilled deionized water making sure no air bubbles were trapped between the funnel and hosecock. A washed aqueous slurry of the resin was poured into the funnel and packed into the column by draining water through the bottom of the column. The funnel and Tygon tubing were removed from the top of the column. The N-6 nipple assembly complete with the transmission tubing and hosecock was filled with distilled deionized water and forced over the open end of the column making sure no air bubbles were trapped inside. The column was then inserted while running the manifold as shown in Figure 1. Reagents. All the reagents used were of reagent grade. The vari-
ANALYTICAL CHEMISTRY, VOL. 47, NO. 14, DECEMBER 1975
TABLE I
REACTIVITY
HYDROXY-AND METHOXY - BENZOPHENONES,
AND FLUORESCENCE OF VARIOUS
REACTIVE IONS
REAGENT
Benzophenone (BPI
(Highly Fluorescent)
RELATIVE FLUORESCENCE INTENSITY (DIVISIONS)
None
0
Cr6+
5 IO 15
OH
@!+J @- !
2- Hydroxybenzophenone
(HBP)
2 - Hydroxy - 4- rnethoxybenzophenone (HMBP)
b
O
C
;:: H
~ 5 : ~ r 6 + B3'
3
IO
50
OH
-
H3C0
2'-Hydroxy 4'- rnethoxyacetophenone (HMAP)
- -
- -
4'-Chloro 2 hydroxy 4 methoxybenzophenone (CHMBP)
C@ -l
2- Hydroxy - 4- methoxybenzophenone-5-sulfonicocid
d! - CH,
! @!
OCH,
OH
-
@-!&OH
-
, 4,4'-
Tetrahydroxybenzophenone
-
H
O
G OH
- 4 - methoxybenzophenone
2,2'-Dihydroxy-4,4'-dirnethoxybenzophenone (DHDMBP)
v5: c r 6 + B3'
50
B3+
20
Cr6+ v5
40 00
+
(TTHBP)
2,2', 4 Trihydroxy (THMBP)
50
HO
4 & OH
OCH,
'd
io
SO,H
2,2' Dihydroxybenzophenone ( 0 , O ' - DHBP)
2,2'
5
v5:cr 6+ B 3+
bOC%
(HMBPS)
2,4 Dihydroxybenzophenone (DHBP)
4
B3'
G
O
H
No3
io(-ve)
No 3
15
No3
125
*
OH b
O
c
H
3
OH
h o c , ,
*
ous substituted benzophenones used in this study were manufactured by the Aldrich Chemical Company, Inc. Mercuric Sulfate Solution. A 0.4% solution was prepared by dissolving 2 g of mercuric sulfate in distilled deionized water containing 5 ml of concentrated sulfuric acid and then diluted to 500 ml with distilled deionized water. Stock Solutions of Substituted Benzophenones The 10-3M solutions of various hydroxy and methoxy substituted benzophenones were prepared by dissolving the appropriate amounts in concentrated sulfuric acid. Nitrate Stock Solution The stock solution was prepared by dissolving 0.6850 g of sodium nitrate in distilled deionized water and diluting to 1 liter. Working solutions were prepared daily by diluting the appropriate volumes of the above solution. Amberlite X A D - 2 Polymer Adsorbent. Amberlite XAD-2 polymeric adsorbent, 20-50 mesh, was obtained from B.D.H. Chemicals. The resin was purified by solvent extraction and stored under methanol ( 7 ) .The resin was washed several times, prior to packing the column, with distilled deionized water to remove the methanol. Fuluic acid (FA). The organic matter used was an FA originating from Bh horizon of the Armsdale soil in Prince Edward Island, Canada. The ash and water content of the purified FA was 6.5% and 8.0%, respectively. FA was chosen to represent the water soluble organic matter which is frequently present in surface and ground waters and imparts yellow coloration to most natural waters.
INDICATES RELATIVE FLUORESCENCE QUENCHING.
Other reagents used in the colorimetric method for nitrate were prepared as described in Riley's method (6). Procedure. The manifold and AutoAnalyzer equipment were connected as shown in Figure 1. This involved two steps: (i) the dilution of samples and the addition of mercuric sulfate to remove any interference due to chloride and sulfide, and (ii) the development of fluorescence for the determination of nitrate. The sample was first diluted and mixed with mercuric sulfate to remove any possible interference due to a high concentration of chloride and sulfide. Because of the high sensitivity of the reaction, it was necessary to dilute the sample to cover the most common range, viz., 0.025-4.0 mg/liter nitrate, prior to introducing the sample for fluorescence development (Figure 1). Samples were analyzed a t the rate of 10 samples per hour using a 1:2 sample-to-wash ratio. It was possible to analyze samples containing as low as 0.5 wg/liter of nitrate-nitrogen by eliminating the dilution step prior to the determination. However, the above-mentioned range was chosen since the nitrate content of the majority of natural water samples lies in that range.
RESULTS AND DISCUSSION Fluorescence Characteristics and Reactivity of Various Benzophenones. The substituted benzophenones listed in Table I were investigated under various reaction and solvent conditions. All of the benzophenones showed fluo-
ANALYTICAL CHEMISTRY, VOL. 47, NO. 14, DECEMBER 1975
2349
90
90 i
1
80 -
70 -
70
-i
60
A
60
-
50
-
50
-
A0
-
A0
-
30
-
i
200
-
WAVELENGTH MILLIMICRONS
i l
250
300
350
A00
A50
I \
500
WAVELENGTH -MILLIMICRONS
WAVELENGTH - MILLIMICRONS
Figure 2. Uncorrected fluorescence spectra of solutions run through the manifold in Figure 1
+
( A ) Emission spectrum of DHDMBP. (6)Emission spectrum of 1 X 10-6M nitrate 4- DHDMBP. ( C ) Excitation spectrum of 1 X 10-6M nitrate DHDMBP. (0) Emission spectrum of 1 X 10-6M quinine sulfate in 0 . l N sulfuric acid. For all spectra, coarse sensitivity = 1; fine sensitivity = 80; excitation and emission slits = 2.0. For ( A ) and (4, exitation A = 385; for (C), emission h = 440; for ( D ) ,excitation X = 350.
rescence only in a very strong acidic medium. In other media, such as aqueous solutions between pH 1-12, and in organic solvents, such as ethanol and dioxane, no measurable fluorescence intensity was obtained. It was also found that the fluorescence of these substituted benzophenones was quenched with excessive water content. No fluorescence was obtained when the water content exceeded 30%. In addition, it was also found that the reactivity of various substituted benzophenones with metals and other anions differed with the nature and position of the substituent. The sensitivity of the reaction was also strongly dependent upon the temperature of fluorescence development as well as on the percentage of water in the final solution prior to the fluorescence development. In our preliminary investigations, using the manual method, the reaction was strongly exothermic resulting in erratic high temperatures when different ratios of water and sulfuric acid were mixed. Therefore, it was decided to investigate these compounds using the automated method. Preliminary experiments were carried out using a manifold similar to that described in our earlier work (8). Qualitative screening of various substituted benzophenones with metals and anions was carried out by passing solutions containing 1-5 X IO-jM solutions of individual ions through the manifold. These included aluminum, antimony(III), arsenic(III), beryllium, bismuth(III), boron, cadmium, calcium, cerium(IV), chloride, chromium(II1 and VI), cobalt, copper(II), iron(I1 and 111),lead, magnesium, manganese(I1 and VII), mercury, molybdenum(VI), nickel, nitrate, selenium(1V and VI), silver, tin(I1 and IV), titanium(II1 and IV), vanadium(1V and V) and zinc. Fluorescent characteristics and reactivity of various substituted benzophenones are tabulated in Table I. Benzophenone (BP) produced strong fluorescence when dissolved in sulfuric acid. I t did not, however, react with any of the ions listed above. 4‘-Chloro-2-hydroxy-4-methoxybenzophenone (CHMBP), 2,2’-dihydroxy-4,4’-dimethoxybenzophenone (DHDMBP) and 2,2’-dihydroxybenzophenone (0,O’-DHBP) gave the highest response for boron, nitrate, and vanadium(\;), respectively. 2,2’,4,4’-Tetrahydroxybenzophenone (TTHBP) reacted with boron, chromium(VI), and vanadium(V) and produced increased fluorescence. However, the presence of a nitrate ion showed quenching of the fluorescence intensity of T T H B P and the magnitude of quenching was proportional to the concentration of nitrate in solution. 2350
The fluorescence excitation and emission spectra for all substituted benzophenones and their product, after reaction with various cations, were shown to possess similar excitation and emission characteristics with excitation a t 380 nm and emission a t 445 nm. Figure 2 shows uncorrected excitation and emission spectra of 2,2’-dihydroxy-4,4’-dimethoxybenzophenone and its reaction product with nitrate under similar experimental conditions described in Figure 1. The sensitivity of the reaction was also compared with similar concentrations of quinine sulfate. The use of CHMBP for the determination of boron has already been reported (8).This paper will deal with the analytical potentialities of DHDMBP as a reagent for the determination of nitrate in natural waters and sediments. Optimization of P a r a m e t e r s for Quantitative Analysis of Nitrate. In our preliminary studies, it became apparent that the reaction of substituted benzophenones with metals and various anions was strongly dependent upon the water-to-sulfuric acid ratio, the temperature, and the time of fluorescence development. Therefore, the effect of these parameters was studied in detail. The studies were carried out using a manifold similar to that described in our earlier work (8). The effect of sulfuric acid/water (v/v) ratio was investigated by interchanging sample pump tubes to give desirable ratios. Solutions containing 0-500 wg/liter nitrate were sampled through the manifold for 10 minutes and the steady state values were compared using various ratios of sulfuric acid to water. Fluorescence development did not take place in a medium containing less than 70% sulfuric acid. The sensitivity and the concentration range where the response was linear, varied with the concentration of sulfuric acid. A maximal fluorescence was obtained between 83-86% sulfuric acid. Nonlinear calibration curves were obtained when the ratio of sulfuric acid to water exceeded 92%. Typical results are shown in Figure 3. Therefore, an 80% sulfuric acid medium was used for further studies. The effect of temperature on the fluorescence intensity of the blank and nitrate with DHDMBP is shown in Figure 4.The fluorescence intensity of the blank and the reaction product in the presence of nitrate increased with temperature. In the presence of nitrate, the optimum temperature was found to be 50 f 2 “C. In addition, the fluorescence of the blank increased rapidly above 60 “C. Therefore, further investigations were carried out at 50 f 2 “C. Special precautions were taken to ensure that there was no change in
ANALYTICAL CHEMISTRY, VOL. 47, NO. 14, DECEMBER 1975
82 6
0
1 00
200
300
90%
400
500
NITRATE ( Hg/ LITRE
Figure 3. Effect of sulfuric acid to water ratio, in percent, on the fluorescence of nitrate-DHDMBP
temperature when the reagent, sulfuric acid, and sample were mixed in the manifold, since the exothermic reaction between sulfuric acid and water would raise the temperature. The samples were mixed with sulfuric acid and the resultant mixture was immediately cooled, using a jacketed mixing coil with cold water before passing through a heating bath. The reagent and the acidified sample were then preheated to optimum temperature in the oil bath before mixing. These precautions were necessary to eliminate any leaks in the manifold and to obtain reproducible results. The time required for the maximum fluorescence development was investigated by changing mixing coils and adding suitable delay coils in the oil bath which was maintained at 50 f 2 " C . There was no significant increase in fluorescence intensity with time. Therefore, only a double mixing coil was used during the fluorescence development. Variation of DHDMBP concentration, between 10-3M did not significantly alter the fluorescence intensity in the range 0-100 pg/liter of nitrate. However, above 10-3M reagent concentration, a high reagent blank was obtained. Therefore, lC)-3MDHDMBP was chosen during the nitrate analysis. The order of addition of reagents which was found to give maximum fluorescence and reproducible results is shown in Figure 1. With optimum conditions and dilution of a sample, as illustrated in Figure l, the response was linear between 0.025-4 mg/liter of nitrate. The range could be altered by varying the ratio of water to sulfuric acid, by varying the temperature, or with the use of appropriate dilution of the sample. It is possible to detect as low as 0.5 Fglliter of nitrate in the original sample. Precision and recovery of the proposed method was checked by running a series of replicate determinations of known amounts of nitrate in the range 0.05-4 mg/liter in both synthetic lake water and actual lake water spiked with an appropriate concentration of nitrate. The relative standard deviation varied between 2-10% in the above concentration range of nitrate. Interferences. The proposed method was tested in the presence of a number of major and minor ions as well as other substances normally found in natural waters and sediments. A 10- to 100-fold molar excess of major ions and minor ions was added to a solution containing 0.05 mg/liter of nitrate. The samples were run using a similar manifold except for the dilution of the sample. The metal ions investigated included all the metals previously listed in this paper. None of the major ions and minor ions such as alkaline earths, bicarbonate, or trace
I
100,
CK $5
35
45
55
011Bath Temp
65
75
- OC
Figure 4. Effect of temperature on the fluorescence of (0)blank and
(0)nitrate 4- DHDMBP
metals interfered except chloride, fulvic acid, and sulfide. These substances decreased the fluorescence intensity of the DHDMBP-nitrate product when present above 10, 1, and 0.1 mg/liter levels, respectively. Interferences from other organic compounds were also investigated. These included ammonia, glycine, cysteine, methionine, D-glucose, xylose, nitrilotriacetic acid, EDTA, aniline, a,a-dipyridyl, pyridine, urea, and detergents. None of the above compounds reacted to produce any significant interference. Removal of Interferences. In the majority of automated methods, the most convenient way of eliminating interferences is to dilute the sample in the manifold prior to the determination. Because of the high sensitivity of the proposed method, it was possible to incorporate a dilution step into the manifold as shown in Figure 1,to tolerate concentration as high as 100, 10, and 1 mg/liter of chloride, fulvic acid, and sulfide, respectively. The dilution step was sufficient for the removal of these interferences for the majority of natural waters. However, in some instances, particularly in the case of sediments and ground waters, concentrations of chloride, sulfide, and fulvic acid substances may exceed the above levels. Therefore, it was necessary to devise an alternate method for the removal of these interferences prior to the determination of nitrate. The number of properties of chloride, sulfide, and fulvic acid are known, viz., precipitation of chloride and sulfide
ANALYTICAL CHEMISTRY, VOL. 47, NO. 14, DECEMBER 1975
2351
Table 11. Effect of Interfering Substances and Their Removal Results after the addition of maskResults prior t o the removal of interferences
Interfering substances, mg/l.
Distilled water Chloride (250) Chloride (500) Chloride (1000) Sulfide (10) Sulfide (25) Sulfide (50) Fulvic acid (50) Fulvic acid (100) Fulvic acid (250)
Nitrate added,
h i t r a t e recovery,
mdl.
mg/l.
R e c o v e v , L6
mdl.
Recovery, r6
0.25 0.50 0.25 0.50 0.25 0.50 0.25 0.50 0.25 0.50 0.25 0.50 0.25 0.50 0.25 0.50 0.25 0.50 0.25 0.50
0.250 0.500 0.170 0.380
100
0.250 0.500 0.240 0.500 0.250 0.490 0.240 0.485 0.255 0.510 0.245 0.525 0.260 0.490 0.240 0.515 0.23 5 0.525 0.250 0.500
100 100 96 100 100 98 96 97 102 102 98 105 104 98 96 103 94 105 100 100
Nitrate recovery,
100 68 76 56 55 26 28 0 0 0 0 0 0 50 59 22 32 6 8
0.140 0.275 0.065 0.140
0 .oo 0 .oo
0.00 0 .oo 0 .oo
0 .oo 0.125 0.295 0.055 0.160 0.015 0.040
by silver, complexation of chloride with mercury and antimony (9, 10) and the ability of fulvic acid to precipitate in acid media (11, 12) as well as its ability to be adsorbed on synthetic polymeric adsorbents (13).Attempts were made to utilize the above approaches for the removal of these interfering substances. Precipitation of chloride and sulfide with silver was not successful because the excess silver gave positive interference during the analysis. Use of antimony to remove chloride resulted in the quenching of the fluorescence of the DHDMBP-nitrate product. The most satisfactory approach to eliminate interferences was the addition of mercuric sulfate, via the dilution line, prior to the development of fluorescence (9). The removal of fulvic acid interference could be accomplished by manual precipitation with cupric or aluminum ions and is outlined in detail elsewhere ( 1 4 ) . However, this involves time consuming manipulations such as titration and centrifugation. Therefore attempts were made to effectively remove fulvic acid using activated charcoal and/or XAD-2 polymeric adsorbent manufactured by Rohm & Haas Company. Activated charcoal adsorbed nitrate in both neutral and acidic solutions. XAD-2 resin, on the other hand, adsorbed nitrate in neutral solutions, but not in acid media. Fulvic acid substances are quantitatively adsorbed in acidic media (13).As in the above procedure, the sample is diluted with 0.4% (w/v) mercuric sulfate in 1% (v/v) sulfuric acid; it was found convenient to employ the XAD-2 column after the sample dilution step to eliminate interference from fulvic acid substances. When mercuric sulfate and XAD-2 resin were used, as illustrated in Figure l, it was possible to remove up to l%of the chloride, 100 mg/liter of sulfide, and 250 mg/liter of fulvic acid as interferences. Table I1 shows the effect of the above approaches to removing these interfering substances. Comparison of Colorimetric Methods and the Proposed Fluorometric Method for Nitrate Analysis. The majority of the methods used in water quality laboratories to determine nitrate in water and sediments utilize Riley’s procedure (6). This method has recently been modified to analyze sediments and some natural waters containing high 2352
ing agent and XAD-2 column
concentrations of calcium and organic matter ( 1 4 ) . In this study, the above methods as well as the proposed fluorometric procedure were critically evaluated. The criteria selected for the comparison study included reproducibility, freedom of interference, selectivity, limit of detection, and convenience. The performance of the methods, using synthetic lake water spiked with 0.05-4 mg/liter nitrate, indicated that all methods gave reproducible results with a relative standard deviation of 1-10% in the above concentration range. During the interference study, it was found that none of the other forms of nitrogen, such as ammonia, glycine, urea, amino acid, etc., gave any response with the proposed fluorometric procedure. Interference of nitrite in Riley’s method is well known ( 6 ) ,since the method first reduces nitrate to nitrite with a cadmium coil and then utilizes the resultant nitrite for color development. Therefore, in cases where the sample contains nitrite, the absorbance resulting from nitrite must be first determined by repeating the analysis without the cadmium coil. However, in the proposed fluorometric method, it is not necessary to rerun the sample since nitrite does not react with DHDMBP. Fulvic acid interfered in both methods; however, the interference was more serious in the colorimetric procedure (6) than in the proposed fluorometric method. For example, a t 50 mg/liter of fulvic acid, nitrate standards gave only 10-15% response as compared to similar standards without fulvic acid. On the other hand, the above solutions gave quantitative recovery with the proposed fluorometric procedure. The typical results of the comparison of various methods are illustrated in Table 111. Analysis of Natural Waters and Sediments. In view of the great sensitivity and selectivity obtained by the use of DHDMBP, it was decided to apply this reagent for the determination of nitrate. The results of the analysis of some natural waters and sediments are shown in Table 111. Natural waters were analyzed directly without any pretreatment. Sediments were extracted with distilled deionized water for 20 minutes on a Burrell wrist-action shaker. The extract was centrifuged a t 2500 rpm for 20 minutes, and the clear supernate was analyzed directly.
ANALYTICAL CHEMISTRY, VOL. 47, NO. 14, DECEMBER 1975
Table 111. Comparison of Colorimetric and Proposed Fluorometric Methods R i l e y ' s method
Nitrate added, Sample
Lake water
mgll.
Kitrate,a mg/l.
1.o 2 .o
River water Well water
Modified colorimetric method
Recovery, '4
1.3@ 14.25
10.0
99 0.26
Sediment- 1B (sand)
7.05 2.60
Sediment-2B (clay) 10.0
ACKNOWLEDGMENT The authors thank M. Schnitzer of the Canada Department of Agriculture, Ottawa, Ontario, Canada, for providing us with a purified sample of fulvic acid.
LITERATURE CITED (1) A. A. Lamola and G. H. Hammond, J. Chem. Phys., 43, 2129 (1965). (2) D. R. Kearns and W. A. Case, J. Am. Chem. SOC.,88,5087 (1966).
106 105 4.45
103 106
101 99
4.50 97 103
denotes 10 g of sediment e x t r a c t e d w i t h 200
Distilled deionized extraction gave quantitative recovery with minimum interferences as compared to 0.1N sulfuric acid extraction ( 1 4 ) . The use of sulfuric acid as an extractant resulted in the leaching of metals such as iron and manganese since these metals are present in very high concentrations in most sediments ( 1 5 ) .The high concentration of these metals interfered in the method. The only way to remove this interference was to neutralize the extract prior to analysis. The detection limit for sediments was in the neighborhood of 100 wg/kg of nitrate. The figures for sediments are based on dissolving 20 g of sediment, dry weight, in 100-ml volume.
99 105 6.55
4.40 22 24
A
7 .OO
4.50
1.25
101 104
104 101
47 54
5 .O a T h e results are t h e average of 5 determinations. sediment e x t r a c t e d w i t h 200 ml of d i s t i l l e d water.
0.69
6.50
5 .O 10.0
99 99
100 106
44 62
5 .O 10 .o
Sediment-2A (clay)
15.35
6.55
10.0
96 107
99 102
92 a7
Recovery, %
1.29
0.75
6.30
Proposed fluorometric method Pzitrate,a mg/l.
97 100
42 47
5.0
O/O
104 99 15.50
100
Sediment-lAb (sand)
Recovery,
1.35 101 99
5 .O 1.o 2 .o
Kitrate,a m g / l .
105 99 ml of distilled water. B denotes 40 g of
(3) R. Schimada and L. Goodman, J. Chem. Phys., 43, 2027 (1965). (4) S. K. Lower and M. A. El-Sayed, Chem. Rev., 66, 199 (1966). (5) D. J. Cram and G. S. H. Hammond, "Organic Chemistry", 2nd ed., McGraw-Hill,New York, 1959, p 297. (6) P. G. Brewer and J. P. Riley, Deep Sea Res., 12, 765 (1965). (7) G. A. Junk, J. J. Richard, M. D. Grieser, et al., J. Chromatogr., 99, 745 (1974). (8) B. K. Afghan, P. D. Goulden. and J. F. Ryan, Water Res.. 6, 1475 (1972). (9) D. W. W. Andrews, Anaiyst(London), 89, 730 (1964). (10) P. W. West and T. P. Ramachandran, Anal. Chim. Acta, 35, 317 (1966). (11) M. A. Rashid, SoilSci., 111, 298 (1971). (12) P. Dubach, N. C. Mehta. and H. Devel, Z.Pflanzenernaehr.. Dueng., Bodenkd., 102, 1 (1963). (13) Technical Bulletin, Amberlite XAD-2, Rohm & Haas Company, International Division, Philadelphia, Pa. (14) B. K. Afghan and J. F. Ryan, Environ. Lett., 9 (2), in press. (15) A. Murdrock. "The Feasibility of Using Dredged Bottom Sediments as an Agricultural Soil". Master's Thesis, McMaster Univers;ty, Hamilton, Ontario, Canada, 1974.
RECEIVEDfor review June 20, 1975. Accepted August 21, 1975. This paper was presented by B. K. Afghan a t the 58th Chemical Institute of Canada, Annual Conference, May 26-28,1975, Toronto, Ontario, Canada.
ANALYTICAL CHEMISTRY, VOL. 47, NO. 14. DECEMBER 1975
2353
