Anal. Chem. 1984, 56,603-605
603
Liquid Chromatographic Determination of Ammonium in Water Munehiko Mizobuchi,* Kikuo Tamase, Yoshimi Kitada, Michiko Sasaki, and Kaoru Tanigawa Nara Prefectural Institute of Public Health, 57-6 Ohmori-cho, Nara 630, J a p a n In waters and wastewaters the forms of nitrogen of greatest interest are nitrate, nitrite, ammonia, and organic nitrogen. All of these forms of nitrogen are biochemically interconvertible and are components of the nitrogen cycle. Ammonia is produced largely by the deamination of nitrogen-containing organic compounds and is naturally present in surface and groundwater and in wastewater. Ammonia is commonly determined as an index of water pollution by organic compounds. Ammonium is currently determined by the ammonia electrode method (1-4), by the molecular absorption method (5,6),and by the ion chromatographic method (7-10). It is also determined by phenate or titration method after distillation to remove interfering substances in water (11). This method requires a substantial amount of water for the distillation. Several factors make fluorescamine suitable for assaying primary amines, including amino acids, peptides, and proteins (12-16). Fluorescamine reacts almost instantaneously with primary amines a t specific pHs to form stable fluorophors. In addition, the reagent, as well as its hydrolysis products, does not exhibit fluorescence behavior, so extensive cleanup or chromatographic separation is not required. This paper describes the determination of ammonium in environmental water samples by high-performance liquid chromatography for the separation of the ammonium fluorophor from others.
EXPERIMENTAL SECTION Chemicals. All chemicals were reagent grade and were obtained from Wako Pure Chemicals Co., Ltd., Osaka, Japan, except fluorescarnine (Hoffmann-La Roche Diagnostics, Basel, Switzerland). Fluorescamine solution (3 mg/mL) was prepared by dissolving 75 mg of fluorescamine in 25 mL of acetone and was stored at room temperature. A 0.05 M phosphate buffer solution (pH 2.0) was prepared by dissolving 6.80 g of potassium dihydrogen phosphate in 1000 mL of water and adjusting to pH 2.0 with phosphoric acid and a pH meter. A 0.2 M borate buffer solution (pH 9.5) was prepared by adding 1M sodium hydroxide to 0.2 M boric acid solution and adjusting pH with a pH meter. Standard Solution. A 2.9654-g portion of ammonium chloride, dried at 105 O C , was dissolved in deionized, distilled water and diluted to 1000 mL; 1.0 mL = 1.0 mg NH4+. Apparatus. Chromatography was carried out with a Shimadzu LC-3A liquid chromatograph (Kyoto, Japan) with a 250 mm X 4 mm i.d. stainless steel column prepacked with LiChrosorb RP-18 ( 5 fim, E. Merck, Darmstadt, Germany). A 50 mm X 4 mm i.d. precolumn packed with Permaphase ODS (Du Pont, Wilmington, DE 19898) in our laboratory was used to assure stability of the analytical column. Fluorometric detection was accomplished with a Shimadzu Model RF-510LC spectrophotofluorometer. Excitation and emission wavelengths were set at 390 nm and 470 nm, respectively, and both slit bandwidths were 10 nm. Chromatographic Conditions. The mobile phase consisted of 0.05 M phosphate buffer solution (pH 2.0) and acetonitrile (65:35, v/v). The flow rate was 1.0 mL/min and the column temperature was set at 40 "C during the analysis. Other spectrophotofluorometer conditions were as follows: range, 2 or 1; response, fast; gain, 10; sensitivity control, 8. Collection of Water Samples. A btalof 106 samples of river water were gathered from three main rivers in Nara Prefecture in plastic bottles. Twenty effluents were collected from wastewater treatment facilities in Nara. Sixteen rainwater samples were collected at our laboratory and stored at below 4 "C in a refrigerator in plastic bottles before analysis. Procedure. All samples were filtered through acetylcellulose 0003-2700/84/0356-0603$01.50/0
membranes (Millipore Co., Type HA, 0.45 fim pore size, and 13 mm diameter). Up to 1.0 mL of the filtered sample was placed in a 10-mL test tube and made up to 2.0 mL with 0.2 M borate buffer solution. Two hundred microliters of fluorescamine solution was added and the solution was mixed vigorously for 20-30 s until the color of the solution changed from white to light yellow. This solution was used for the analysis of ammonium fluorophor by HPLC. A sample ranging from 5 to 20 pL was injected into the HPLC system. Calculation. The concentration of ammonium was determined from the peak height by comparison to standards which were run throughout the procedure. The standard curve was linear in the concentration range of 0.2-5.0 fig NH4+/2.2mL. Retention time was obtained by using an integrator of a Shimadzu Chromatopack E1A.
RESULTS AND DISCUSSION Analytical Conditions by HPLC. Fluorescamine reacts with primary amines (12,13,16) such as amino acids, peptides, and proteins but not with secondary amines such as proline, hydroxyproline, and sarcocine. Primary amine fluorophors sometimes interfere in the analysis of ammonium in water. Two reversed-phase columns, pBondapak CI8 and LiChrosorb RP-18, and various mobile phases (pH 2.0-7.0) were tested with regard to their ability to effectively separate the ammonium fluorophor. The retention times of 18 primary amines (17 amino acids and histamine) were measured as potential interferences. The amino acids were Gly, Ala, Val, Leu, Ileu, Ser, Thr, Cys, Met, Asp, Glu, His, Arg, Lys, Phe, Try, and Tyr. LiChrosorb RP-18 was a useful column with the mobile phase given in the Experimental Section. Under our analytical conditions, the peak for the ammonium fluorophor appeared 7.8 min after injection of the sample and was separated from those of Gly, Ala, and Tyr. The excitation and emission wavelengths were set at 390 nm and 470 nm, respectively, to get maximum fluorescent intensity. They were slightly different from those used for measuring amino acids (12), chlorodiazepoxide (14), and sulfadiazine (15). The effect of pH on fluorescent intensity of the fluorophor is shown in Figure 1. The maximum value was obtained at pH 9.5. Identification of Ammonium. One milliliter of river water and a few drops of 0.2 M borate buffer solution were taken in a test tube of 10-mL capacity and mixed. The test tube was placed in a water bath at about 80 OC and connected to an aspirator and the content was evaporated to dryness. Ammonium completely disappeared in the resulting chromatogram although the other components remained. This showed that the ammonium fluorophor was satisfactorily separated from other components under our analytical conditions. Standard of Ammonium. In ref 11, anhydrous ammonium chloride was used to make stock ammonium solution. The interference of chloride ion on the reaction of ammonium and fluorescamine was tested before defining the reagent as a standard reagent of ammonium in our proposed method. It was found that chloride did not interfere below a concentration of 500 fig of Cl/mL. This result showed that the reagent could be used as a standard reagent. Stability of Ammonium Fluorophor. The stability of ammonium fluorophor was measured by using a standard solution containing 4.0 pg/2.2 mL of ammonium. The result 0 1984 American Chemical Society
604
ANALYTICAL CHEMISTRY, VOL. 56, NO. 3, MARCH 1984
Table 11. Summary of Ammonium Recovery Experiments
5 r
recovery, %
amt of NH,t added,lg
n
4.0
5
I
0.4
0
4.0
5
Effluent 99.8
1.9
0.4
5
Rainwater 98.8
5.4
0 a, a
5
6
0
IO
PH o f borate b b t f e r
Figure 1. Effect of pH on fluorescent Intensity of fluorophor formed with fluorescamine.
Table I. Relationship between Volume of Fluorescamine and Fluorescent Intensity with 3.0pg of S H , - / 2 . 2 m L
50
200 250
intens, cm/&
n
2.2 4.65 6.6
0.73
1 2
10.6
1.55
3 4 5 6
2.20 2.83 3.53
showed that the fluorescent intensity slightly decreased with time at a rate of 6% in 5 h. It was found, however, that even if the intensity of fluorophor could not be immediately measured by HPLC after adding fluorescamine, the intensity of ammonium fluorophor was almost stable within a few hours and gave good analytical results. Volume of Fluorescarnine. The relationship between the fluorescent intensity and the volume of fluorescamine was measured with the standard solution (Table I). The fluorometric intensity increased proportionally with the volume of fluorescamine added. We found that 200 pL of fluorescamine was enough to detect ammonium at concentrations of 0.1 pg of NH4+/2.2 mL. The addition of more than 200 pL of fluorescamine increased the fluorescent intensity but was accompanied by formation of an unknown product on the surface of the solution which might be a hydrolyzed product of fluorescamine. Buffer Capacity. There are undoubtedly many compounds or chemicals with an ability to change the pH value of river water. To find out whether the buffer capacity of 0.2 M borate buffer solution was adequate, pH values of 51 samples were measured before and after the addition of the buffer solution. Before the addition, the pH values of the river water were distributed from 7.5 to 8.3. By addition of buffer solution, the distribution of the pH values was minimized to a range of from 9.4 to 9.5. This showed that mixing water sample and buffer solution in a ratio of 1:l assured almost identical pH value of about 9.5. Precision and Accuracy. The analytical recovery was examined by spiking river water, effluent, and rainwater with known amounts of ammonium. Table I1 summarizes the results of these recovery studies. Average recoveries were from 95.8% to 100.7% on five replicate samples and their standard deviations were from 1.9 to 5.4. Reproducibility was measured with standard solution, river water, effluent, and rainwater (Table 111). The results showed that their relative standard deviations were from 1.22% to 2.28%. Detection Limit. The detection limit was obtained from the peak of chromatogram and noise of the base line. A 5-pL sample containing 2.0 of pg NH4+/2.2 mL and 20 pL con-
3.4
fluorescent intens. cm
fluorescent intens, cm
8.5
2.0
Table 111. Reproducibility of Standard and Real Samplesa
re 1
vol of
fluorescamine,
100 150
s
River Water 100.7 5 95.8
1
VL
av
7 8
9 10
std (5.0 l g / 2.2 mL)
river water
13.2 13.2 13.45 13.1 13.05 12.8 13.25 13.15 13.0 13.3
effluent
rainwater
9.6 9.95 9.95 9.9 9.75 9.7 9.85 9.8 9.75 9.7
10.05 10.4 10.05
11.7 11.7
10.1 10.15 10.1 10.35 10.25
10.05 9.9
11.45 11.4 11.45 11.05 11.2 11.0 11.3
10.14 0.15 1.45
11.40 0.26 2.28
13.15
9.80
S
0.18
0.12
RSD (%)
1.37
1.22
3
11.7
Measuring condition: standard, river water, and effluent range x2, sample 5 pL, rain water range x l , sample 1 0 WL.
u04812 04812 r e t e n t i o n time ( r n i n )
Figure 2. Liquid chromatograms of river water (left) and rainwater (rlght). Measuring condltions were as follows: (river water) sample, 1 mL; range, 2; inJectbnvolume, 5 pL; (rainwater) sample, 1 mL; range, 1; injection volume, 20 pL.
taining 0.2 pg of NH4+/2.2 mL were used to determine the detection limits under the measuring conditions of the spectrofluorophotometer at range 2 and range 1,respectively. When the detection limit was assumed to be twice the signal to noise ratio of the base line (SIN = 2), these limits were 0.1 pg of NH4+/2.2mL and 0.03 pug of NH4+/2.2mL, respectively. Comparison with Colorimetric Method. We used river water, effluent from wastewater treatment facilities, and rainwater to compare the analytical results of our proposed method with those of the phenate method. Typical chro-
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Anal, Chem. 1084, 56,605-608
matograms of river water and rainwater are shown in Figure 2. River water and effluent were distilled and measured colorimetrically ( I I ) , and rainwater was directly measured by the colorimetric method (18) after filtration with a Millipore filter. It was found that there was a close relationship between our HPLC method (X)and the colorimetric one (Y), showing the following relation: log Y = 0.982 log X - 0.129, r = 0.986 on river water; log Y = 0.964 log X , r = 0.995 on effluents, and log Y = 1.075 log X - 0.073, r = 0.993 on rainwater. The concentration measured by our HPLC method was higher than that of the distillation-colorimetric method. But 3 of 106 river water samples showed much higher concentrations by the distillation-colorimetric method than those obtained by our HPLC method in spite of the fact that the levels were within the detectable limit. Those results showed that it was difficult to distill at a same distillation rate of 6-10 mL/min with distillation sets as defined (11). So, each distillation set having different rates gave a different recovery of ammonia gas into sulfuric acid or boric acid solution. The river water samples were sampled after heavy rain and consequently contained a very high amount of solid matter suspended in water, over 300 Mg/mL. The ammonium, combined to the surface of the matter, might be released during the course of the distillation step resulting in an unreasonably high concentration in the colorimetric method. The conventional distillation-colorimetric method (11,17) requires up to 500 mL of water and also requires much apparatus, space, and reagents. Furthermore it is difficult to distill a t the same distillation rate and maintain it. Our proposed HPLC method requires only 1 mL of sample and
a filtration procedure and is accurate and rapid. Registry No. NH4+,14798-03-9;fluorescarnine, 38183-12-9; water, 7732-18-5.
LITERATURE CITED (1) Hikuma, Motohlko; Kubo, Tatsuru; Yasuda, Takeo; Karube, Isao; Suzuki, Shuichi Anal. Chem. 1980, 52, 1020-1024. (2) Meyerhoff, Mark E. Anal. Chem. 1980, 5 2 , 1532-1534. (3) Karube, Isao; Okada, Tadashi; Suzuki, Shuichi Anal. Chem. 1981, 53, 1852-1854. (4) Fraticeill, Y. M.; Meyerhoff, M. E. Anal. Chem. 1983, 55, 359-364.
(5) Grleve, Steven; Syty, Augusta Anal. Chem. 1981, 53, 1711-1712. (6) Anlgbogu, Vlncent C.; Dletz, Mark L.; Syty, Augusta Anal. Chem. 1983, 5 5 , 535-539. (7) Small, Hamish; Stevens, Timothy S.; Bauman, Wllliam C. Anal. Chem. 1975, 47, 1801-1809. (8) Bouyoucos, Spiros A. Anal. Chem. 1977, 49, 401-403. (9) Fritz, James S.; Gjerde, Douglas T.; Becker, Rose M. Anal. Chem. 1080, 55, 1519-1522. 10) Mlzobuchi, M.; Ohmae, H.; Umoto, F.; Tanaka, T.; Ichimura, K.; Ueda, E.; Itano, T. Clln. Chem. (Winston-Salem, N . C . )1983, 29, 408-409. 11) "Standard Methods for the Examlnation of Water and Wastewater", 14th ed.;American Public Health Association: Washington, DC, 1975; pp 406-418. 12) Stein, S.; Boehlen, P.; Stone, J.; Dairman, W.; Udenfriend, S. Arch. Blochem. Slophys. 1073, 155, 203-212. 13) Felix, Arthur M.; Terkelsen, Gene Arch. Blochem. Siophys. 1973, 157, 177-182. (14) Stewart, James T.; Wliliamson, Jonathan L. Anal. Chem. 1978, 48, 1 182-1 185. (15) Sigel, Carl W.; Woolley, Joseph L., Jr.; Nichol, Charles A. J. fharm. Sci. 1975, 64, 973-976. (16) Udenfriend, Sidney; Steln, Stanley; Boehlen, Peter; Dairman, Wallace; Lelmgruber, Wllly; Weigeie, Manfred Sclence 1972, 178, 87 1-872. (17) "Testing Methods for Industrlai Wastewater, JIS K0102, 1981"; Japanese Standards Assoclatlon: Tokyo, 1981; pp 126-127. (18) Kanno, Saburo; Fukui, Syozo; Naito, Syojl; Kaneko, Mlkihiro Eisei Kagaku 1988, 14, 280-284.
RECEIVED for review June 22, 1983. Accepted December 1, 1983.
Determination of Strontium-90 in Urine by Extraction without Ashing Vladimir SEasniir'
Institute of Experimental Pharmacology, Slovak Academy of Sciences, 842 16 Bratislava, Czechoslovakia The elements Na, Ca, Co, Zn, Sr, Cs, Y, Zr, Ru, Ce, and Pm, if ingested or inhaled, will be retained in the body for an appreciable length of time concentrating in the bone and the liver. From the radiotoxicity point of view the radionuclide strontium-90 is the most dangerous component. A survey of the literature shows that most laboratories carry out gross /3 urine analysis. This method detects the presence of radioactive fission products that do not emit y-rays. Better estimates of the retention and the subsequent doses in an actual contamination case can be made if an analysis method to measure strontium-90 specifically in urine is available. However the determination of strontium-90 is usually laborius and time-consuming. Therefore a simple and selective extraction method has been developed for this purpose. The most tedious operation in radiostrontium determination is concentrating it from urine and separating the radionuclide from the mixture of other radionuclides. The methods of precipitation (I-4), ion exchange (5-79, and extraction (8-11) are frequently used for this purpose. Extraction procedures are especially advantageous due to their speed and high selectivity. V i s i t i n g scientist. Present address: U n i v e r s i t y of Montreal, Nuclear Physics Laboratory, Montreal, Quebec, Canada H3C 357.
It was shown (12-14) that dicarbolides of transitions metals possess high selectivity toward cesium, whereas the extraction of strontium is not significant. Dicarbolides of cobalt were used for separation of cesium from the mineralizates of biological tissues (15-1 7), contaminated excreta (18),and milk (19). This paper demonstrates that PEG strongly increases the extraction of microamounts of strontium into nitrobenzene, when the univalent hydrophobic anion of dicarbolide-H+ was used. The mechanism of the synergistic effect of PEG in the extraction of strontium cation is explained by Rais (20). The magnitude of the synergistic effect >lo3 creates good conditions for the extraction of strontium after previous extraction of cesium. Since both radionuclides are most significant components of fission mixture and the extraction of other fission and activated radionuclides using dicarbolide-H+appears to be low, the conditions for a selective and quantitative separation of radiostrontium from contaminated urine were investigated in this work. Dicarbolide-H+ with high stability constant of its complex anion, fully dissociated and stable also in a high acidic media, together with PEG of mean relative molecular mass 300 was used for this purpose. Urine is a complex matrix. Any analytical separation that attempts to isolate an almost insignificant mass of material
0003-2700/84/0356-0605$01.50/00 1984 American Chemlcal Socletv
