Anal. Chem. 1983, 55, 1620-1622
1620
Continuous Flow Fluorometric Determination of Ammonia in Water Toyoakl Aokl," Satoshl Uemura, and Makoto Munemori Laboratory of Environmental Chemistry, College of Engineering, University of Osaka Prefecture, Mozu-umemachi, Sakal 59 1, Japan
In general, colorimetric methods (1, 2 ) are used for the determination of ammonia in an aquatic environment. However, color or turbidity interferes with these methods and, therefore, a time-consuming preliminary distillation step (3) is required before analysis. Thus, a fast and highly sensitive method is desired. At present, an ammonia selective electrode method ( 4 , 5 ) has been widely used for determining ammonia in environmental samples. However, this method is susceptible to interferences by amines (6). More recently, a gas-phase molecular absorption method (7,8) has been reported, but this method is not quite as sensitive. Roth (9) developed a highly sensitive fluorometric method for the determination of amino acids, in which the amino acids are reacted with o-phthaldialdehyde (OPA) in the presence of 2-mercaptoethanol (MCE) in alkaline medium to produce fluorescent adducts. Similarly, OPA reacts with ammonia to produce fluorescent isoindole fluorophore, whose structure is unknown. Danielson and Conroy (10) recently used this reaction for the determination of ammonia (10-4-10-2 M) in water. We previously reported on the continuous flow method (11) for determining free chlorine, utilizing separation of chlorine by means of tubular microporous poly(tetrafluoroethy1ene) (PTFE) membrane. In this paper, the application of this technique to the fluorometric determination of ammonia with OPA reagent is presented.
EXPERIMENTAL SECTION Apparatus. The configuration of a continuous flow system is shown in Figure 1. Sample solution and OPA reagent were pumped into a separation unit by a peristaltic pump, P. The pH of the sample solution could be changed by adding various concentrations of NaOH. The values of pH, in the following discussion, are those measured on effluent from T3. The construction of the separation unit is the same as shown in a preceding paper (11). Unless otherwise indicated, the length of the separation unit is 400 mm. The OPA-ammonia reaction product is detected by the fluorometer (Japan Spectroscopic Co.) as shown in Figure 1 which is equipped with a flow cell of quartz tubing (1.0 mm i.d., 2.4 mm 0.d.). Reagents. The o-phthaldialdehyde and 2-mercaptoethanol were of biochemical grade (Wako Pure Chemical Co.). All reagents were of analytical reagent grade except the amines which were of reagent grade. An ammonium chloride solution was used as a stock solution. Working standards were prepared by serial dilution. OPA reagent was prepared by the procedure reported by Lindroth and Mopper (12). Doubly distilled water was used in the preparation of all solutions, the second distillation being carried out from alkaline permanganate in an all-Pyrex still. Procedure. Flow rates of the sample solution, 1N NaOH, and OPA reagent were 2.1,0.38, and 0.23 mL/min, respectively. The pH values of effluents from T2 and T3 were 9.5 and 13, respectively. Molecular ammonia, NH3,liberated by mixing the sample solution with 1N NaOH permeates through a microporous PTFE membrane into a buffered OPA reagent stream in the inner tube. The reaction product is fed to the flow cell of a fluorometer and the fluorescence intensity was measured at 486 nm with the excitation at 370 nm. A sample solution was allowed to flow into L1, and the fluorescence intensity was recorded. Before each measurement of sample solution, redistilled water was flushed through T3 for 5 min. The fluorescence intensity of this run is taken as a blank. The fluorescence intensity of a sample, in the subsequent discussions, is given as the difference between the ~
intensity of the sample and that of the blank.
RESULTS AND DISCUSSION Fluorescence of Reaction Product. The reaction product of ammonia with OPA reagent has the optimum excitation wavelength (Aex) at 370 nm and the optimum emission wavelength (A,) a t 486 nm. The effect of pH on the reaction between ammonia and OPA reagent was studied in a batch system. The reaction products of amino acids with OPA reagent are known to be stable (9). However, the reaction product of ammonia is unstable as shown in Figure 2. The fluorescence intensity increases to the maximum and then almost immediately decreases with time. The maximum intensity was obtained at 4 min after the mixing of sample and reagent solutions at pH 9.5. From these results, the pH of the OPA reagent was adjusted to 9.5 with 0.1 M borate buffer and the flow rate of this reagent solution was adjusted so that the reaction product reaches the flow cell at 4 min after the reaction by adjusting the length of tube and speed of the peristaltic pump. Under this condition, it took 6 min after sample solution began to flow into the system for a constant fluorescence intensity to be reached. Figure 3 shows the effect of OPA concentration on the fluorescence intensity. The fluorescence intensity increased with the increase in OPA concentration. However, the fluorescence intensity of blank also increased. Moreover, with the increase in OPA concentration, the time for the fluorescence to reach the maximum intensity decreased so that the control of reaction became difficult. Therefore, loe3M OPA was selected to be satisfactory. The fluorescence intensity also changes with the concentration of MCE. The effect of MCE was studied over a range of 5.0 X to 4.0 X M. Fluorescence intensities of the sample and the blank increased with the increase in MCE concentration. As a compromise, 1.5 X low3M was chosen as the MCE concentration. Optimum pH of Sample Solution. Ammonia exists in aqueous systems as both NH3 and NH4+. The pH of natural water in aquatic environments is in the range of 6-8. In this range, ammonia exists primarily in the cation form, NH4+. Therefore, the conversion of NH4+ to NH3 was studied by measuring the fluorescence intensity at various pH values of the sample solutions fed into the separation unit. The fluorescence intensity increased with the increase in the pH of the sample solutions and became constant above pH 12 as shown in Figure 4. The relative fluorescence intensity expected from the distribution of ammonia is also shown in Figure 4 as a function of pH. The equilibrium constant used for the calculation of distribution was taken from Sillen and Martell (13). The theoretical values agree very closely with those obtained experimentally. In the following experiments, sample solutions were adjusted a t pH 13 by addition of 1 N NaOH. Optimum Flow Rate, With constant flow rate ratios of sample solution to OPA reagent at about 10,4.5, and 2.5, the fluorescence intensity changed with the flow rate of the OPA reagent. The fluorescence intensity increased as the flow rate of the OPA reagent decreased and the ratio increased. This can be explained by an increase in the amount of ammonia which can react with the OPA reagent in a unit of time. On the other hand, with the decrease in flow rate of OPA, reagent
0 1983 American Chemical Soclety 0003-2700/83/0355-1620$01.50/0
ANALYTICAL CHEMISTRY, VOL. 55, NO. 9, AUGUST 1983
i,r-F 1 ... ..
T1
1 N NaCH
,LI~
/"
g a s t e
~j
T 2 T3
Sepuration unit
Fluorometer
Sample OPA
Reagent
Flgure 1. Schematic diagiram of continuous flow system: P, peristaltic pump; L1, L2, Tygon tubing: T1, T3, PTFE tubing: T2, microporous PTFE
tubing.
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Table I. Interference of Various Compounds compounds RFI a compounds RFI a glycine 99 tartaric acid 104 aspartic acid 99 humic acid ( 2 0 ppm) 96 109 lysine 100 sodium chloride 105 phenylalanine 102 sodium nitrate 104 albumin (100 ppm) 103 sodium sulfate 101 calcium chloride 100 glucose 98 acetic acid 103 magnesium chloride 112 citric acid 114 hydrazine sulfate a Relative fluorescence intensity, fluorescence intensity of 1.0 X M ammonia solution containing 1.0 x M compound except for albumin (100 ppm) and humic acid (20 ppm)/fluorescence intensity of 1.0 x M ammonia in %. ~
Table 11. Interference of Various Amines compounds ammonia methylamine
iqI LO
ethylamine
e 20
15
20
Reaction t i m e
,min
5
0
10
Figure 2. Effect of pH arid reaction time on tlhe fluorescence interisity of ammonia (batch experiments). Concentrations of ammonia, OPA, and MCE are 4.0 X M, 1.0 X M, and 1.5 X M, re-
spectively.
1o
-~
1o - ~ OPA, M
1 0-2
Figure 3. Influence of OPA concentration on the fluorescence intensity of ammonia (0)and time (A)to reach the maximum intensity (batch experiments),OPA reagent blank (0).Concentrations of ammonia and MCE are 4.0 X lo-' M and 1.5 X M, respectively.
loot----....
.ri
Flgure 4. Relative fluorescence Intensity (0)and calculated fraction
of ammonia species in aqueous solution as a function of pH. For concentrations see Figure 2.
(---)
RFI' pK,, concn, (25 C) M pH 1 3 pH 9 9.24 10-5 100 100 10.68 355 117 124 102 10.63 552 140 149 101 4.96 104 NDb 10.77 100 ND 10.93 102 ND 9.80 100 ND
aniline dimethylamine diethylamine trimethylamine a Relative fluorescence intensity, fluorescence intensity M ammonia solution containing amine as of 1.0 x shown in table/fluorescence intensity of 1.0 X M ammonia in 7%. Not determined. response time increased. As a compromise, the ratio of the flow rates and the flow rate of the OPA reagent were selected to be about 10 and 0.2 mL/min, respectively. Effect of Temperature. The fluorescence intensity increased with an increase in temperature (10-25 "C). This is probably due to the decrease in the solubility and increase in the permeation rate of ammonia with the increase of temperature. Therefore, it is necessary for the temperature of solutions to be held constant. Calibration Curves and Precision. The fluorescence intensity was proportional to the ammonia concentration from 2X up to 2 X lo4 M. The detection limit ( S I N = 3) at pH 13 was 1.8 X lo-* M. The relative standard deviations (n M, 2.1% at 1.0 X = 5) a t pH 13 were 4.4% at 1.0 X M, and 1.8% a t 1.0 X M, respectively. The sensitivity of this method depends to a great extent on the dimensions of the separation unit. When the length of the unit was reduced to 300 mm and 200 mm, the relative fluorescence intensities were reduced, respectively, to 80% and 67% of that with the length of 400 mm. If the length of the separation unit was short, the response time was shorter, but the sensitivity was lower. Interference Studies. Table I lists levels of interference with 1.0 X lo4 M ammonia from organic and inorganic species. The concentration of species except for albumin protein (100 ppm) and humic acid (20 ppm) was M, being 100 times higher than that of ammonia. Excess inorganic salts slightly positively interfered. This may be due to salting out. Hydrazine also interfered, since it permeates partly through microporous P T F E membrane. Among the organic species, amino acids and albumin protein which react with the OPA reagent did not interfere with this method. Organic acids except for citric acid did not interfere. The concentration of hydrazine and citric acid in aquatic environments is probably less than lo4 M and, therefore, they would not cause any serious problem in the
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Anal. Chem. 1983, 55, 1622-1624
Table 111. Results of a Comparison Study for Present, Phenate, and Selective Electrode Methods for Determination of Ammonia in River Samples concn of NH,? MM selective sample present phenate electrode Kizu River 2.28 4.0 3.6 (Dec 13, 19$82) (2.2;)c Uzi River 14.5 16.5 16 (Dec 13, 1982) (1!.3)' Katzura River 227 232 250 (Dec 20, 1982) (227J' Yodo River 84.2 88.5 96 (Dec 20, 1982) (84.0)' a Average of duplicate determinations. Determinations were performed at pH 13. Determinations were performed at pH 9. determination of ammonia by this method. The interference of amines is shown in Table 11. Primary amine such as methylamine and ethylamine among the amines interfered positively a t p H 13, since they permeate through microporous P T F E membrane and react with the OPA reagent similarly to ammonia. As they are more basic than ammonia, their permeability is expected to decrease with decreasing p H more rapidly than that of ammonia. The results a t p H 9 in Table I1 indicate that this expectation is the case. At p H 9, primary amines did not interfere at the concentration level 10 times smaller than that of ammonia. However, the fluorescence intensity of ammonia derivative was reduced to three-fifths of that a t pH 13. From the above results, it is said that no primary amine exists in a sample solution when the value obtained at pH 9 coincides with that obtained at pH 13. On the other hand, if the value obtained at pH 9 is lower than that at pH 13, the contribution of primary amines must be considered for the determination of ammonia in the sample solutions. Secondary and tertiary amines which interfere with the determination by an ammonia selective electrode (6) did not interfere at the concentration level 10 times higher than that of ammonia, since they do not react with OPA reagent, even if they permeate through microporous P T F E membrane. Application. To demonstrate real sample application, recovery studies of ammonia on a spiked river sample were carried out. The percentage recovery ranged from 95 to 105% over the concentration range of lo4 to M.
Several river samples were analyzed by the present method. The results are compared with those obtained by phenate and ammonia selective electrode methods in Table 111. Below M ammonia, the calibration curve for the selective electrode method displayed some nonlinearity. Because of the considerable time required for the electrode potential to stabilize at these low levels, the accuracy of data obtained from this method is poor. The phenate method requires preliminary distillation to separate ammonia from interfering substances. I t took 45 min to collect above 95% of the ammonia by the distillation step. The total analytical time was approximately 70 min. Compared with these methods, the present method is very fast and the time required for analysis was 6 min. The concentration of ammonia in the Katzura River was exceptionally high. This may be because the Katzura River receives the discharge of effluent from the domestic waste treatment plants. For all samples, the values obtained at pH 13 were in agreement with those at pH 9 within experimental error. Therefore, primary amines do not exist a t a level of lo4 M or more in these rivers. According to the present method, the concentrations of M ammonia in rainfall a t Sakai were found to be 3.0 X and 2.3 X M. These values are in the same order of magnitude as those reported by Vijan and Wood (7) (2.9 x M), and Hendry and Brezonik (14) (0.9 X M) a t Gainsville, FL. Registry No. Ammonia, 7664-41-7; poly(tetrafluoroethylene), 9002-84-0; o-phthaldialdehyde, 643-79-8; water, 7732-18-5.
LITERATURE CITED (1) Patton C. J.; Crouch S. R. Anal. Chem. 1977, 4 9 , 464-469. (2) Ngo T. T.; Phan P. H. A,; Yam F. C. Anal. Chem. 1982, 5 4 , 49-51. (3) "Standard Methods for the Examination of Water and Wastewater", 15th ed.; American Public Health Association: Washington, DC, 1980; pp 351-366. (4) Midgiey D.; Torrance K. Analyst (London) 1972, 9 7 , 628-633. (5) Beckett M. J.; Wllson A. C. Water Res. 1874, 8 , 333-340. (6) Lopez M. E. Rechnitz G. A. Anal. Chem. 1982, 5 4 , 2085-2089. (7) Vijan P. N.; Wood R. G. Anal. Chem. 1981, 5 3 , 1447-1450. (8) Vijan P. N.; Wood R. G. Anal. Lett. 1982, 75 (B8), 699-707. (9) Roth M. Anal. Chem. 1971, 4 3 , 880-883. (10) Danleison N. D.; Conroy C. M. Manta 1982, 29, 401-404. (11) Aoki T.; Munemori M. Anal. Chem. 1983, 55, 209-212. (12) Lindroth P.; Mopper K. Anal. Chem,; 1979, 5 1 , 1867-1674. (13) Sill6n, L. G., Martell, A. E., Eds. Stability Constants of Metal-Ion Complexes"; Burlington House: London, 1984. (14) Hendry, D. C.; Brezonik, L. P. Envlron. Scl. Techno/. 1980, 14, 843-849.
RECEIVED for review Janurary 27, 1983. Accepted April 19, 1983.
Linearity Testlng of Ultravlolet Detectors in Liquid Chromatography C. D. Pfelffer," J. R. Larson, and J. F. Ryder Analyilcal Laboratories, Dow Chemlcal U S A . , Mldland, Mlchlgan 48640
The ultraviolet spectrophotometer has become the single most useful detector for high-performance liquid chromatography (LC) because of its sensitivity, versatility, and broad dynamic linear range (1). However, in a detector of this type, there must be a compromise involving the slit width between sensitivity and broad linear dynamic range (2). Increasing the slit width permits a larger amount of energy to pass through the flow cell leading to a reduction in noise. Therefore, the slit width may be increased to improve de-
tectability by improving the signal-to-noise ratio up to the point when stray light increases significantly, and a linear response is no longer obtained. Conversely, the maximum linear dynamic range necessary for highly accurate assay-type analyses is achieved by using a narrow slit width. A theoretical discussion of the relationship between sensitivity and linearity, published by Stewart (3), reached a similar conclusion. Due to the theoretical complexity in the relationship between sensitivity and linearity in ultraviolet detectors, it would
0003-2700/83/0355-1622$01.50/00 1983 Amerlcan Chemical Society
