Ultramicro Semiautomated Method for Simultaneous Determination of Total Phosphorus and Total Kjeldahl Nitrogen in Wastewaters Andrea M. Jirka', Mark J. Carter, Dorothy May, and Frederic D. Fuller U S . Environmental Protection Agency, Central Regional Laboratory, 18 19 West Pershing Road, Chicago, 111. 60609
An ultramicro technique for the digestion of organic nitrogen and phosphorus compounds in wastewater samples is described. The digests are analyzed simultaneously for phosphate and ammonia in the range of 0.02-2.00 mg PA. and of 0.06-10.00 mg N/l. using automated spectrophotometry a t the rate of 30 sampledh. The accuracy, precision, and tolerance to common interferences of the ultramicro method are discussed. Comparability testing of the ultramicro and standard methods shows a small but statistically significant difference for the phosphorus but not for the nitrogen results. The decomposition products of many organic phosphorus and nitrogen compounds are nutrients which serve to encourage the growth of algae when discharged into water bodies ( I ) . High nutrient loadings in a restricted water body can have an adverse effect by greatly accelerating the eutrophication process (2). For this reason, the industrial and municipal waste discharge permits, issued as a result of the Federal Water Pollution Control Act Amendments of 1972, have set limitations on the concentration of nitrogen and phosphorus containing wastes that can be discharged (3).The need to monitor a large and diverse number of discharges has greatly increased the requests for analysis of wastewater samples for total albuminoid or Kjeldahl nitrogen (TKN) and total phosphorus (TP) ( 4 ) .This study was initiated to develop a rapid, smallscale method for the simultaneous analysis of T K N and TP that is tolerant to the interferences commonly found in wastewaters. An additional requirement was that the new method must produce data that are comparable to data produced by the standard methods. In recent years, a number of reviews have appeared concerning methods for the analysis of nitrogen and phosphorus in organic matrices (5, 6) and environmental water samples (7-9). The vast majority of methods reviewed involve the destruction of organic matter by chemical oxidation with the conversion of phosphorus and nitrogen to phosphate and ammonia. The standard technique for analysis of ammonia in digested samples involves a distillation to avoid interferences followed by titration or nesslerization (10). However, this method is slow, requires large sample volumes, and has been criticized because of poor precision and lack of sensitivity ( I 1-14). In the indophenol blue reaction, discovered by Berthelot, ammonia reacts with phenol and hypochlorite to form a blue color (15).Since the use of this method eliminates most of the difficulties of nesslerization and titration, the indophenol blue procedure has become increasingly popular. Harwood and Huyser have reviewed the extensive literature on the application of this method to the manual (16) and automated (17) analysis of wastewaters. Most of the common spectrophotometric methods for phosphate determination are based on the formation of 12molybdophosphoric acid from phosphate and molybdate in acid solution and subsequent reduction to a blue heteropoly compound (6, 20). Ascorbic acid, stannous chloride, and 1amino-2-naphthol-4-sulfonic acid have been used as reducing agents (6). Murphy and Riley showed that only the method using ascorbic acid was not subject to error when applied to water samples with high ionic strengths (21). 1038
Environmental Science & Technology
No technique which is applicable to wastewaters has been reported for which TP and T K N can be analyzed simultaneously out of a common sample digestion. Sample digestion using potassium persulfate (10, 24-29), perchkoric acid (32-35), or ultraviolet irradiation (43,44) has been successfully applied to phosphorus analysis. It has been suggested that these methods be applied to TKN analysis (28,30,3640, 45). However, these nitrogen digestion methods have not gained wide usage because they have not been proved reliable (5, 31, 41, 42, 45). The most widely used method to determine organic nitrogen is the Kjeldahl method (46). Wilfarth (47) added a mercuric oxide catalyst, and Gunning (48) added potassium sulfate to the Kjeldahl method to increase the digestion rate. Laws has suggested applying the Kjeldahl digestion to the determination of total phosphorus (6).Recent work has shown that the Kjeldahl method can serve as a common digestion for determining organic nitrogen and phosphorus in water samples (49-53). The method described here involves a smallscale modification of the Kjeldahl method for sample digestion, followed by the simultaneous automated spectrophotometric determination of orthophosphate and ammonia.
Experimental Apparatus. Samples were digested in 1 X 8-in. heavywalled pyrex tubes. The tubes were held in a Technicon # 114-0009-02 rack with 2 in. cut off the bottom. The tubes were heated in a Technicon BD-40 block digestor. Acidwashed Chemware TFE boiling stones were used to prevent bumping during sample evaporation. Spectrophotometric measurements were made using the apparatus shown schematically in Figures 1-3. The system includes the following Technicon AutoAnalyzer I1 components: Sampler IV with a 30/h 2:l cam; proportioning pump I11 with a dilution manifold; phosphate manifold; ammonia manifold; colorimeter equipped with 15-mm flowcells (F/C), S10 phototubes and 630-nm interference filters for TKN; colorimeter equipped with 50-mm flowcells, S1 phototubes, and 880-nm interference filters for TP; dual-pen recorder; and double-channel digital printer. The air intake lines on the dilution tray and ammonia manifold were attached to a dilute sulfuric acid air scrubber. A Techmar Model SDT homogenizer was used to homogenize samples. An Oxford 5-10-ml adjustable pipet with disposable polypropylene tips was used to withdraw sample aliquots. A Kimble DM-73 dispenser fitted with 2- and 10-ml barrels was used for addition of digesting solution and digestion tube dilution water. A Vortex genie mixer was used to help in dissolving digested samples. A Precision Vari-HiSpeed centricone centrifuge was used to clarify turbid digested samples. For spectrophotometric analysis, samples were contained in 15 X 85 mm disposable glass culture tubes. Reagents for Sample Digestion. All chemicals were ACS reagent grade, and all reagent water was deionized and distilled. Digesting solution was prepared by dissolving 2.0 g of HgO in 25 ml of 6 N H2S04.Then 200 ml of conc H2S04was carefully added to 500 ml of water. While the strong acid solution was still hot, 134 g of K2S04 was dissolved in it. The HgO solution was added. The solution was cooled, diluted to 1 l., and stored above 20 "C. It is extremely important that
precipitation of the K2SO4 be avoided, since this will result in low recoveries for TKN. The digestion tube dilution water was nitrogen and phosphorus free. Reagents for Automated Dilution Manifold in Figure 1. The sampler wash solution was prepared by adding 35 ml of conc HzSO4 to 500 ml of water and diluting to 1 1. The dilution manifold solution was prepared by diluting 12.5 ml of 10 N NaOH to 1 1. with water. Reagents for Automated Ammonia Manifold in Figure 2. Complexing reagent was prepared by dissolving 33 g of potassium sodium tartrate and 24 g of sodium citrate in 900 ml of water, diluting to 1 l., and adding 0.25 ml of Brij-35 wetting agent (Technicon Co. No. T21-0110). Alkaline phenol solution was prepared by dissolving 83 g of phenol and 36 g of sodium hydroxide in 900 ml of water, cooling, and diluting to 1 1. The solution was stored a t 4 "C. Sodium hypochlorite solution was prepared by diluting 200 ml of Chlorox (5.25% available Clz) to 11. with water. Sodium nitroprusside reagent was prepared by dissolving 0.5 g of sodium nitroprusside in 900 ml of water and diluting to 1 1. The solution was stored at 4 "C. Reagents for Automated Phosphate Manifold in Figure 3. Sodium chloride solution was prepared by dissolving 5 g of sodium chloride in 900 ml of water, diluting to 1l., and adding 0.25 ml of Levor IV wetting agent (Technicon Co. No. T210332). A 4.9 N sulfuric acid solution was prepared by adding 136 ml of conc H2SO4 to 500 ml of water, cooling, and diluting to 1 1. Ammonium molybdate solution was prepared by dis~ ~900 - ~mlHof~ water O and solving 40 g of ( N H ~ ) ~ M o ~ O in diluting to 1 1. Ascorbic acid solution was prepared by dissolving 18 g of ascorbic acid in 900 ml of water and diluting to 11. The solution was stored at 4 "C and prepared fresh weekly. Antimony potassium tartrate solution was prepared by dissolving 3.0 g of K(Sb0) C4H40~1/2H20in 900 ml of water and diluting to 1 1. A combined color reagent for total phosphorus was prepared by combining the following solutions in order: 50 ml of 4.9 N sulfuric acid solution, 15 ml of ammonium molybdate solution, 30 ml of ascorbic acid solution, and 5 ml of antimony potassium tartrate solution. This reagent was prepared fresh daily. Standards. A stock nitrogen standard containing 0.100 mg N/ml was prepared by dissolving 1.050 g of glutamic acid, which had been dried at 105 "C for 1 h, in 900 ml of water, adding 2 ml of conc H2S04, and diluting to 11. A stock phosphorus standard containing 0.100 mg P/ml was prepared by dissolving 0.4394 g of KH2P04 which had been dried at 105 "C for 1h, in 900 ml of water, adding 2 ml of conc H2S04 and diluting to 11. Combined working standards containing 0.40 mg P/1. and 2.00 mg N/L, 1.00 mg P/1. and 5.00 mg N/l., and 2.00 mg P/1. and 10.00 mg N/1. were prepared by diluting 4.00 ml phosphorus stock and 20.0 ml nitrogen stock, 10.0 ml phosphorus stock and 50.0 ml nitrogen stock, and 20.0 ml phosphorus stock and 100.0 ml nitrogen stock to 1 l., respectively. Each working standard was preserved with 2 ml of conc HzS04 and stored in the refrigerator. A blank was also prepared by adding 2 ml of conc HzSO4 to 11. of water. Two intercalibration standards were prepared at 40 and 60% of full-scale concentrations of phosphorus and nitrogen by an independent analyst using independent reagents to verify the accuracy of the original standards. Procedures. All glassware was rinsed with 1:l HC1 to prevent phosphorus contamination. No commercial detergents were used. Wastewaters were collected in high-density polyethylene containers. They were preserved with 2 ml of H2S04/l. Raw sewage samples were stored at 4 "C in addition to being preserved with HzS04.Any nonuniform samples were blended for about 30 s. Representative 10-ml aliquots of samples were
placed into digestion tubes in a rack along with 2-4 Teflon boiling stones and 2 ml of digesting solution. A pair of blanks and a series of standards were prepared in the same manner. It was helpful to precede the two blanks with a "set standard" to locate the maximum of the peak while setting the printer. The rack of tubes was placed in the block digestor. The samples were allowed to evaporate at 200 "C for about % h and then were digested for h after the block reached a temperature of 370 "C which usually required about Y2 h. The tubes were removed from the digestor and allowed to cool for at least 5 min. Then 10 ml of distilled water was added. The samples were mixed well with a vortex mixer, then transferred to clean
Glass Transmission Tubing
I (To Wash Sampler Receptacle) IV
lOTC
.
116-0489-01
116 0246-01
( 2 501 Sampler Wash Sol n
(0 321 Air (0 231 Digested Sample
I (1 601 Dilulion Loop Sol n
(TO PO, Manifold)
(TO NH, Manifold)
.
(0 421
1
116-0200 P O 2
10 421
Figure 1. Automated total phosphorusand total Kjeldahl nitrogen dilution manifold Numbers in parentheses correspond to flow rate of pumptubes in mlhin. Numbers adjacent to glass coils and fittings are Technicon Corp. part numbers
Recorder
Printer
Figure 2. Automated ammonia manifold, 0.06-10.00 mg N/I. For explanation of manifold numbers, see Figure 1
157-0273-03 37 5 - c 7 7 ml
5TC 170.0103
A,o
5TC 170-0103
10 321 AIR ( 0 321 NaCl
I 116-0489.01 10 4 Waste
(To Waste) 4
421 (From Dil Manifold)
(0 601 From F/C
FHT] Recorder
Printer
Figure 3. Automated phosphate manifold, 0.02-2.00 mg PA. For explanation of manifold numbers, see Figure 1 Volume 10, Number 10, October 1976
1039
15 X 85 mm test tubes. Samples containing clay-like particles were centrifuged until clear. Samples containing dark material indicating incomplete digestion were redigested at a greater dilution. The tubes were placed in the sample tray. The analytical manifolds and reagents were set up as shown in Figures 1-3. The colorimeters, recorder, and printer were allowed to warm up for M h with the pump running. The spans of the instruments were synchronized. A midscale "set standard", placed a t the beginning of each sample set, was used to locate the maximum of the peak. It was followed by two blanks which were used to zero the baseline. A midscale standard was used to calibrate the recorder and printer. Full-scale and 20% standards were included to check linearity. For quality assurance, two intercalibration standards and one sample duplicate were analyzed with each sample set. The TP and TKN concentrations were obtained directly from the Digital Printer. For samples which were out of range, the original sample was diluted and carried through the entire digestion procedure again. Blank values in reference to the baseline were below 0.25 mg Nfl. and below 0.05 mg PA. If the values were higher, contamination in the digesting solution was usually the cause and it was reprepared.
Results and Discussion Sample Digestion. The wastewater sample digestion technique of Morgan et al. was adapted to an ultramicro scale (54). Since less than 10 ml of digested sample was required for spectrophotometric analysis of ammonia and phosphate by the method described here, the volume of mercury waste that must be treated before disposal was 25 times less than in the standard manual methods (IO, 28). The advantage of small scale, combined with the fact that mercury has been shown to be the best catalyst (47,55-60),contravenes recent arguments against the use of mercuric oxide (61, 62). Careful control of the digestion temperature between 360-370 "C is required to assure complete sample digestion without incurring any loss of nitrogen (55,56,63).In the ultramicro method, the boiling point of the samples during digestion was fixed at 370 "C by adding a digestion reagent that contained 0.66 g K2S04/ml conc H2S04. The potential problem of sample overheating was eliminated by using a Technicon Co. BD-40 block heater for digesting samples (49). The block has a built-in thermocouple and temperature controller which was set to maintain 370 "C. The accuracy of the temperature controller was f 5 "C in reference to a Thermolyne Model PM-1K50 pyrometer with a Chromel-Alumel thermocouple. Bremner (56)showed that a 10-min digestion period after the samples became clear gave complete recovery of nitrogen from the common amino acids. However, to ensure sufficient time for clearing and digestion of particulate matter in the ultramicro method, the samples were held at 370 "C for h. Acid-washed Teflon boiling stones provided very effective antibumping action so that the samples could be evaporated at 200 "C in % h. By use of this procedure, forty 10-ml samples containing no more than 100 wg nitrogen and 20 Fg phosphorus could be completely digested in 1%h. Phelps (64), Shirley and Becker (65), and Bremner (56) have shown that nicotinic acid is difficult to digest, and for this reason, it has become one of the primary standards used for evaluating new TKN methods ( 5 ) .The efficiency of digestion using the ultramicro technique was evaluated by testing the recovery of nitrogen from nicotinic acid added to wastewaters. The mean recovery from 13 wastewater samples was 104% (Table I). In comparison, the recovery of nitrogen from ammonium sulfate and nicotinic acid in distilled water was 99% for both. These results show that the ultramicro digestion technique efficiently recovered nitrogen from a wide variety of wastewater matrices. 1040
Environmental Science & Technology
Based on work by others, the recovery of phosphorus from adenosine 5'-monophosphoric acid (AMP) was used to assess the utility of the ultramicro digestion technique (26,40,44). The mean recovery of phosphorus, added as AMP, from 12 waste samples was 98% (Table 11).In comparison, the recovery of phosphorus from AMP added to distilled water was 102%. These results show that the ultramicro technique effectively digested organic compounds without causing the loss of phosphorus. Automated Spectrophotometric Analysis of Ammonia and Phosphate. Since organic nitrogen and phosphorus compounds are converted to ammonia and phosphate during digestion, the problem of analysis is reduced to determining the latter two compounds in a strongly acidic solution. The automated ammonia analysis system described by Harwood and Huyser ( I 7 ) and based on the indophenol blue reaction (15)was adapted for use with highly acidic samples. The indophenol blue chemistry is pH sensitive with a substantially reduced sensitivity for samples below pH 2 ( I 7). Table I . Recovery of Nicotinic Acid Added to Wastewater Samples with Ultramicro Semiautomated Total Kjeldahl Nitrogen Method Total Kjeldahl nitrogen, mg N/I.
Sample type
Sample
Nicotinic acid added
Sample + nicotinic acid
Distilled water 3.00 yso,the material is said to be oil wet. Because its attractive interaction with oil is stronger than with water,
