Anal. Chem. 1986, 58,591-594
(27) Pomianowski, A.; Leja, J. Can. J. Chem. 1963,41, 2219-2230. (28) Kirbitova, N. V.; Anurkina, N. V.; Eiiseev, N. I. I z v . Vyssh. Ucbebn. Zaved., Gorn. Zh. 1978, 7 , 134-137. Chem. Abstr. 1979, 9 1 , 143956f. (CSIRO Translation No. 13098). (29) Williams, W. J. “Handbook of Anion Determination”; Butterworths: London, 1979;p 587. (30)Thumm, B. A,; Tryon, S. J. Org. Chem. 1964,2 9 , 2999-3002.
59 1
(31)Rolia, E. Trans.-Inst. Min. Metall., Sect. C 1970, 79, C207-C214. (32) Jones, M. H.; Woodcock, J. T. Anal. Chem. 1975,47, 11-16. (33) Jones, M. H.; Woodcock, J. T. Talanta 1979,26, 815-820.
RECEIVED for review July 15,1985. Accepted October 4,1985.
Microdetermination of Phosphate in Water by Gel-Phase Colorimetry with Molybdenum Blue Kazuhisa Yoshimura* Chemistry Laboratory, College of General Education, Kyushu University, Ropponmatsu, Chuo-ku, Fukuoka 810, J a p a n Masatoshi Ishii a n d Toshikazu T a r u t a n i Department of Chemistry, Faculty of Science, Kyushu University, Hakozaki, Higashiku, Fukuoka 812, J a p a n
The blue specles of molybdophosphate are strongly adsorbed on Sephadex gels. Almost all the blue species in a 50-cm3 sample solution are concentrated in 0.20 g d Sephadex 0 2 5 (fine) wlthln 10 mln. Direct absorptiometry of the heteropoly acid concentrated in the gel phase was developed for the determlnatlon of the phosphate at parts per billion levels In natural waters. The colored gel beads, on which the blue specles reduced by ascorbic acid In the presence of antimonyl Ions were adsorbed, are packed Into a 10mm cell; the attenuances at 836 and 416 nm are measured; and the attenuance dlfference Is used for the determination of traces of phosphate. The use of cells of other length (5, 2, and 1 mm) gave a wide concentration range for calibration from parts per billlon to parts per million levels. This method is simple In operatlon and has a high reproduclbillty of measurements.
Phosphate reacts with molybdate in a strong acidic solution to produce the yellow 12-molybdophosphate (12-MF’A) species (1). In the determination of the amount of phosphate in various samples, the yellow species derived by chemical reaction is absorptiometrically employed in many laboratories. It is, however, difficult to determine phosphate a t microgram per cubic decimeter levels in water, even though a long light-path cell is used after reduction of the yellow species. A preconcentration step such as solvent extraction (2) or coprecipitation with metal hydroxides (3) is usually necessary to prolong operation time and lower the precision of analysis. It has been shown that direct absorptiometry of an element concentrated in the solid phase leads to a simple method of high sensitivity (4-7). For phosphate determination, anionexchanger phase absorbance of the molybdenum blue has already been used, but it takes a rather long time for color development (8),and the color in the resin phase is not stable (9,10). In the previous paper, a sensitive method for silicic acid was reported that used solid-phase absorptiometry after concentration of the blue species on Sephadex G-25 (11).With this method, the amount of silicic acid at the parts per billion level or lower could precisely and simply be determined for high-purity industrial waters. This paper describes the ap-
plication of this technique to determine trace levels of phosphate. EXPERIMENTAL SECTION Reagents. All reagents were of analytical grade and deionized-distilled water. A standard phosphate solution (50 mg of P/dm3) was prepared by dissolving 0.220 g of potassium dihydrogen phosphate and diluting it to 1 dm3. A combined reagent was prepared by mixing 50 cm3 of 2.5 mol/dm3 sulfuric acid solution (70 cm3of concentrated sulfuric acid diluted to 500 om3),5 cm3of potassium antimonyl tartrate solution (0.274 g of potassium antimonyl tartrate hemihydrate in 100 cm3of water), 15 cm3of 4% (w/v) ammonium molybdate solution, and 30 cm3of 0.1 mol/dm3 ascorbic acid solution (0.76 g of ascorbic acid in 100 cm3 of water). Sephadex G-25 (fine) was purchased from Pharmacia Fine Chemicals. Apparatus. Absorbance measurements were made with a Nippon Bunko spectrophotometer, Model UVIDEC-320. An inside-mirrortube (12 mm i.d., 40 mm length) was placed between the cell and the detector window for recovering the scattered light from the gel layer (7,12).A perforated metal plate ( A = 1.0) was used in the reference beam to balance the light intensities. The sample cell was similar to that described in the previous paper (11). The thickness of the gel layer was varied by inserting quartz spacers of varying thicknesses into an ordinary 10-rnm quartz cell. An acrylic resin spacer was also used to make certain the entire light beam struck only the packed area. Procedure for Gel-Phase Colorimetry of Phosphate. Natural waters were filtered through a 0.45-pm membrane filter paper (Millipore). First, 8 cm3of the combined reagent and then 0.20 g of the gel beads were added to a 50-cm3water sample containing 0.05-5 pg of phosphate-phosphorus in a poly(ethy1ene) container. After the mixture was stirred for 10 min at 15-20 OC, the colored gel beads were allowed to settle. Cell lengths (1,2, 5, or 10 mm) were selected depending on the degree of the color intensity of gel beads. The gel was transferred to the sample cell with a pipet. The cell was set in an ordinary holder in the spectrophotometer. The attenuances at 836 nm and 416 nm were measured with air as a reference. The differences between the attenuances (AA) were used for determining the level of phosphate. A calibration graph of each cell length was obtained by taking standards throughout the procedure described above. Distribution Measurement. To a 250-cm3 water sample or 1.67 X lo4 mol/dm3 phosphate and 34.5 containing 2.78 X cm3of the combined reagent solution, an appropriate amount of the gel (0.01-0.05 g) was added. The mixture was stirred for 4 h at 20 “C. Then the concentration of the blue species in the
0003-2700/86/0358-059 1$01.50/0 @ 1986 American Chemical Society
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ANALYTICAL CHEMISTRY, VOL. 58, NO. 3, MARCH 1986
1
i
Stirring time i
IO
800 Wavelength / nm
/
min
Time dependence of color development: (A) Sephadex G 2 5 (fine),0.20 g; (B) Sephadex G-25 (medium), 0.20 g. The sample parameters were 10 pg of P/dm3, 50 cm3. Flgure 2.
,
1000
Absorption spectra of the molybdenum blue species of phosphate. Solid lines represent solution spectra (cell, 10 mm): (A) 1 mg of P/dm3, (B) blank. Dotted lines represent gel spectra (cell, 10 mm) (gel, Sephadex G-25,0.20g; solution, 50 cm3): (C) 80 pg of P/dm3, (D) 40 pg of P/dm3, (E) blank. Flgure 1.
Table I. Distribution Ratios of the Blue Species of MPA W(initia1 concn), mol/dm3
wt of gel, g
10-30, dm3/kg
2.78
0.0101 0.0181 0.0324 0.0403 0.0506 0.0105 0.0208 0.0314 0.0404
6.33 7.21 8.53 9.44 11.0 10.5 11.7 13.9 17.2
0
1.67
24
Flgure 3. Effects of standing time on color development: sample, 13 pg of P/dm3, 50 cm3; gel, Sephadex G-25 (fine), 0.20g.
equilibrated solution was determined by the conventional solution method. The distribution ratio of molybdophosphate, D, was calculated by means of the equation [(mol of molybdophosphate adsorbed)/ (kg of dry gel)]
D=
12
Standing time / h
[(mol of molybdophosphate) /(dm3 of solution)] (1)
RESULTS AND DISCUSSION Absorption Spectra of the Molybdenum Blue Species. Absorption spectra of the blue species of 12-molybdophosphate (12-MPA) are shown in Figure 1. Maximum absorbances of the molybdenum blue in the solution are at 890 and 710 nm, whereas the blue species in the gel phase have absorption maxima a t 836 and 698 nm. The difference (AA) of the attenuances a t the absorption maximum wavelength (836 nm) and the absorption minimum wavelength (416 nm) for the blue species was used for the determination of phosphate at the sacrifice of sensitivity, because AA is unaffected by a small packing difference in each sample layer. If necessary, the net absorbance of the blue species in the gel phase can be obtained by subtracting AA for the blank from AA. The use of a mirror tube lowered background attenuance by about l ( 7 , 12). Adsorption Isotherm of 12-Molybdophosphate. At the low ranges of adsorption, almost all the blue species were adsorbed on the gel. Distribution measurements were performed in a linear calibration range of the conventional solution method (Table I). By assuming uniform complex formation of the blue species with the gel, constant adsorption capacity, and no interaction among the adsorbed blue species, the obtained data are fitted by a linearized Langmuir-type isotherm, in analogy with 12-molybdosilicic acid (IO). The adsorption capacity is 0.167 mol/kg of dry gel, and the formation constant is 1.80 X lo5 dm3/mol. Although experi-
mental conditions were not strictly the same for 12-MPA and 12-molybdosilicate, the numbers of adsorption sites in the gel are not so different from each other. Higher adsorbability of 12-MPA compared to that of 12-molybdosilicic acid is mainly due to the stronger interaction between 12-MPA and the gel. Optimization of Conditions. Amount of Combined Reagent. Due to the importance of acidity the final acid concentration should be maintained constant. The concentration of molybdate and acid suggested in the literature (13) was adopted. Addition of 8 cm3 of the combined solution, which contains molybdate, acid, and reducing agent, was found to be appropriate to develop the color of the molybdenum blue. The solution is stable for 4 days if stored in a refrigerator, but it is preferable to prepare the solution just before its use. Stirring Time. The molybdenum yellow species are readily reduced by ascorbic acid in the presence of antimonyl ions (24). The color of the molybdenum blue was developed within 4 min of the addition of the combined reagent. For simplified operation, the coloring agent and the gel were therefore added simultaneously. As shown in Figure 2, the use of finer gel particles leads to a faster sorption rate. The particle diffusion of 12-MPA may partially occur in its adsorption on Sephadex gel, although 12-MPA is a comparatively large molecule. The stirring time can be reduced to only 10 min when a finer gel is used. Standing Time. The absorbance by the adsorbed blue species was constant for at least 1 h. Allowing it to stand for a long time after the adsorption, however, results in an increase of AA, because of progressive color development. It is recommended that AA be measured within 1 h or at a constant time interval after color development (Figure 3). Temperature. Calibration graphs obtained at different temperatures (15-20 "C) gave the same straight line. At temperatures lower than 15 "C and higher than 20 "C, the graphs had the same slope but different AA values (for the blank), which were -0.109 at 10 "C, -0.100 a t 15-20 "C, and -0.078 a t 25 "C for a 10-mm cell. The temperature should
ANALYTICAL CHEMISTRY, VOL. 58, NO. 3, MARCH 1986
593
___ Table 11. Effects of Foreign Ions on the Determination of Phosphate'
species added
molar ratio, foreign ion/P
amt of P found, ,ug/dm3
100
9.8
-2
1000
8.1 10.1
-19
9.7
-3 0
N as nitrate S as sulfate
100 1000 100 1000
10.0 10.8
Ge(1V) MV)
10
9.8
Al(II1) Ti(1V) Fe(II1)
10
Si as silicic acid P concentration / pg/drns
Flgure 4. Calibration curves of phosphate by gel-phase colorimetry with cells of different cell lengths: sample, 50 cm3;gel, Sephadex G 2 5 (fine), 0.20g; cell length (A) 10 mm, (B) 5 mm, (C) 2 mm, (D) 1 mm.
be maintained constant within f l "C,and the calibration graph obtained at the same temperature should be used. Alternatively, the calibration graph can be constructed by using the value of AA (for the blank) at the operating temperature and the constant slope value. Calibration. The effectiveness of long light-path cells has already been discussed for higher sensitivity solid-phase absorptiometry (7,12,15). Increasing the cell length gives only a moderate increase in the background attenuance, whereas it gives a proportional increase in the absorbance of the sample species. The calibration curves obtained by using cells of various cell lengths are shown in Figure 4. AA values (for the blank) were -0.037, -0.058, -0.100, and -0.100 for 1-,2-, 5-, and 10-mm cells, respectively. It is convenient to use the 5-mm cell from the viewpoint of sensitivity and linearity of the calibration curve. In the case where the 10-mm cell is used, the linear range of the calibration curve is up to 20 pg of P/dm3. The phototube of the instrument may be out of proportional detection to the light intensity, due to the large light loss by the sample layer. The use of cells of different cell lengths gives a wide concentration range for calibration. The sensitivity of the gel-phase colorimetry with the lO-mrn cell is 32-fold higher than the conventional solution method. The larger the sample volume, the higher the sensitivity. The volume effect on sensitivity can be evaluated by the equation previously reported (16). For the present system the value of D is 3.01 X lo4at low coverage. The sensitivity may be 1.98 times higher for the 100-cm3sample and 3.91 times higher for the 200-cm3 sample than that of the 50-cm3 sample. Effect of Foreign Ions. Table I1 shows the effects of foreign ions. Most ions except for arsenate did not cause more than 5% error when present in up to 10 times the concentration of phosphate. Arsenate did not ipterfere when present in a 0.1-fold ratio to phosphate. Nitrate, sulfate, and silicic acid caused little interference when present in less than 2 mg of N/dm3, 10 mg of S/dm3, and 10 mg of Si/dm3, respectively. In the case of silicate determination (IO),copper(1I) caused negative errors. The present method does not suffer from the interference of copper(I1). Iron(II1) interfered when present in more than a 10-fold ratio to phosphate, if the pH of the sample solutions were above 3. Hydrolyzed iron(TI1) species may adsorb phosphate and make the recovery of phnsphate incomplete. The concentration of iron in natural waters filtered through a 0.45-c~mfilter paper is generally much lower than that of phosphate. Therefore, by using the filtered sample solutions, the concentration of dissolved orthophosphate cap be determined by the preseqt method. If sample solutions were stored at pH 2, the presence of iron in a 1000-fold ratio to phosphate did not interfere. Humic acid interfered when more than 0.1 mg/dm3 was present because the adsorbed humic acid contributes to absorbance at 416 nm. Only the attenuance at the absorption
relative error, %
+1
+8 -2 +5
lp.5
0.1 1
19.8
+98 -4
9.6
10
-2 +4
9.8
lb
10.4
lob
loooc
1.7 9.7 10.0
-23
1boc 100
10.3
+3
9.8
-2
Mn(I1) Cu(I1)
1%
-3 0
50-cm3sample solution containing 0.500 gg of P. solution is above 3. The solution is stored at pH 2.
* pH of the
Table 111. Determination of Phosphate in Natural Waters concn, ,ug of P/dm3 at the following cell lengths samplea
10 mm
5 mm
Underground Water ( n = 5 ) A B
9.0 f 0.3 14.0 i 0.7 Seawater
C surface water bottom water D surface water bottom water E surface water bottom water
1.4 4.4 15 10
49b 48
7.2 4.2 16
14 48 49
a A, a runoff from Akiyoshi-do Cave, Yamaguchi; B, a small spring in Akiyoshi-dai Plateau, Yamaguchi; C, seawater at the mouth of Hakata Bay, Fukuoka; D, seawater at the middle part of the bay; E, seawater at the inner part of the bay. Determined after 10 cm3 of the sample was diluted to 50 cm3 with 3% sodium chloride solution. _ i -
--
maximum wavelength (836 nm) can be used in such cases, although reproducibility i s lowered. The amounts of interfering foreign ions that occur in natural waters are generally tolerable, and therefore the present method may be directly applied to natural waters. If the sample solution has a saline concentration found in seawater, the absorbances are affected by the change of the effective light-path length due to shrinkage of the gel beads. It is preferable to use a calibration curve prepared with solutions at the same saline concentration as the sample solution. Determination of Phosphate in N a t u r a l Waters. The present method was applied to the determination of orthophosphate in seawater qnd underground water. Low concentration of phosphate in natural waters could be easily de@rmined with high precision (Table 111). Analytical results obtained by using cells of different lengths were in close agreement with each other. As a result, phosphate at parts per billion levels can be determined in a conveniently short time. The procedure is simple, and a t least 20 samples can be analyzed within 1 h.
594
Anal. Chem. 1986, 58,594-597
Registry No. Sephadex G-25,9041-35-4;phosphate, 1426544-2; water, 7732-18-5.
(IO) Matsuhlsa, K.; Ohzeki, K.; Kambara, T. Bull. Chem. SOC.Jpn. 1982,
LITERATURE CITED
(11) Yoshlrnura, K,; Motomura, M.; Tarutani, T.; Shlrnono T. Anal. Chem. 1984, 56, 2345-2349. (12) Yoshimura, K.; Waki, H. Talanta, In press.
(1) Kircher, C. C.; Crouch, S. R. Anal. Chem. 1982, 5 4 , 879-864. (2) Heslop, R. E.; Person, E. F. Anal. Chlm. Acta 1987, 39, 209-221. (3) Harry, L.; Rowe, J. J.; Grirnaldi, F. S. Anal. Chem. 1955, 27, 258-262. (4) Yoshirnura. K.; Waki, H.; Ohashi, S. Talanta 1978, 2 3 , 449-454. (5) Yoshirnura, K.; Nigo, S.; Tarutani, T. Talanta 1982, 2 9 , 173-176. (6) Waki, H.; Korkisch, J. Talanta 1983, 30,95-100. (7) Yoshimura, K.; Waki, H. Talanta 1985, 32,345-352. (6) Tanaka, T.; Hilro, K.; Kawahara, A. BunseklKagaku 1979, 28,43-47. (9) Matsuhlsa, K.; Ohzekl, K.; Karnbara, T. Bull. Chem. SOC.Jpn. 1981, 2675-2677.
3335-3336.
(13) Horwltz, W., Ed. “Official Methods of Analysis of the Association of Official Analytical Chemists”, 12th ed.; AOAC: Washington, DC, 1975; p 622. (14) Going, J. E.; Eisenreich, S. J. Anal. Chim. Acta 1973, 70,95-106. (15) Waki, H. “Ion Exchange Technology”; Naden, D.,Streat, M., Eds.; Ellis Horwood: Chichester, 1984; pp 595-602. (16) Yoshimura, K.; Ohashi, S. Talanta 1978, 25, 103-107.
RECEIVED for review April 3, 1985. Resubmitted October 4, 1985. Accepted October 4, 1985.
Automated Fluorometric Method for Hydrogen Peroxide in Air Allan L. Lamus,* Gregory
L. Kok, J o h n A. Lind, Sonia N. Gitlin, B r i a n G. Heikes, a n d Richard E. S h e t t e r
National Center for Atmospheric Research, P.O. Box 3000, Boulder, Colorado 80307
A fluorometrlc method for measurlrlg H,O, vapor in air utlllzes peroxidase enzyme to catalyze the reactlon In which hydroperoxides cause dlmerlratlon of (p-hydroxypheny1)acetic acld. I n a second channel, H,O, is seiectlvely decomposed by catalase so that the fluorescence slgnal Is due only to organic hydroperoxldes. The difference between the two slgnals Is a measure of H20, vapor. The H 2 0 2 vapor Is collected by means of a glass coil through which air and water flow concurrently. The coefflcent of variatlon Is 0.5 % at 2.5 parts per blllion by volume. The standard deviation of the base line is 10 parts per trllilon by volume (pptv) under iaboratory condltlons. Thls standard deviation has varied between 3 and 33 pptv during ground-based field missions, and was 70 pptv on alrcrafi flights. Thirty seconds is requlred for the slgnal to change from 10 to 90% of Its maxlmum value.
a peak emission wavelength of 400 nm (11). The peroxide concentration is directly proportional to the fluorescence intensity. Peroxidase also catalyzes the reaction of organic hydroperoxides to form the fluorescent dimer. In order to distinguish HzOzfrom organic hydroperoxides, the technique utilizes a dual-channel flow system with a dual-cell fluorometer. The reaction described above yields a measure of both the hydrogen peroxide and organic hydroperoxides in the first channel. In the second channel, the enzyme catalase is added to selectively destroy hydrogen peroxide before the peroxidase-catalyzed reaction occurs. The second channel therefore provides an analytical blank for the determination of HzOP The two signals are produced simultaneously by the system, which is automated by means of a Technicon AutoAnalyzer peristaltic pump.
Hydrogen peroxide is believed to dominate oxidation processes that convert SOz to HzS04in clouds a t pH values less than 4.5. The predominant source of HzOzdissolved in cloud water is H 2 0 zvapor formed photochemically in the atmosphere (1-5). Concentrations of H20zvapor observed by the described technique have ranged from 30 parts per trillion by volume (pptv) to 4 parts per billion by volume (ppbv) in the eastern U.S.A. Attempts to collect HzOz from air by scrubbing with water-filled impingers have been hindered by artifact formation of HzOz in the solution (6-9). Earlier methods utilized detection of the dissolved HzOzby a luminol technique (6). Interferences observed in the measurement of HzOz in cloud water by the lurninol method may pose a problem in the measurement of the vapor (10). This paper describes an alternative way to measure HzOZ vapor in real time. A stripping coil is used in combination with a fluorometric analysis of HZOp The method as applied to aqueous samples has been described in detail (10). The HzOZvapor technique incorporates changes that accommodate a lower and narrower concentration range of HzOz in the solution used for stripping HzOz from air. The technique is based on the selective catalysis of HzOz decomposition with (p-hydroxypheny1)acetic acid (POPHA). The reaction products are water and the dimeric product, 6,6’-dihydroxy-3,3’-biphenyldiacetic acid. The dimeric product fluoresces with a peak excitation wavelength of 320 nm and
EXPERIMENTAL SECTION The H202 is stripped from the atmosphere by means of concurrently pulling the air sample and scrubbing solution through a Technicon AutoAnalyzer coil (6, 12). The scrubbing solution M potassium acid phthalate in water adjusted to pH is 5 X 6.0 with NaOH. The tube length of the 10-turn, 2-mm4.d. coil is approximately 50 cm. Air sampled at 2 L/min has a residence time of 0.05 s. Scrubbing solution is pumped into the inlet of the coil at a rate of 0.42 mL/min, giving an air-to-water ratio of 4800 (Figure 1). The scrubbing solution, which is impelled by the air as it flows through the horizontal coil, forms a thin film on the glass, providing a large surface for gas exchange. The system is provided with a stripping coil for each channel. For maximum accuracy, the pump tubes introducing water into the coils should be calibrated for delivery rate. Air and scrubbing water are pumped from the collection coil into a vertical separator tube. The water plus about 0.3 mL/min of the scrubbed air is pumped by the AutoAnalyzer from the bottom of the separator. The sum of the flow rates of the pump tubes delivering the scrubbing water and the reagents into the system is approximately 0.3 mL/min less than the sum of flow rates of the pump tubes leading from the fluorometer cell and the debubbler exiting the system (Figure 2). This difference causes about 0.3 mL/min of the scrubbing air t o be pumped through the system. The air segments the reaction stream with bubbles helping to maintain sharp concentration gradients along the stream. The sample conditioning reagent is added (0.16 mL/min) to each stream of scrubbing solution. The conditioning reagent M formaldehyde and 0.02 M phthalate (Table I) contains 5 X buffer adjusted to pH 6.0. The former, by forming hydroxy-
0003-2700/86/0358-0594$0 1.5010
@ 1986 American Chemical Society
