T h e organic carbon is a useful measure of the energy potential of raw shale. If required, the oil yield (galhon),as determined by Fischer assay, can be calculated from the organic carbon. Finally, the organic carbon content on retorted shale is a useful measure of an energy, or heat source, that is wasted. Although most of the material used in this study was mine-run raw shale (that is, samples were a n aggregate composite of shale from many strata), several of the samples are from cores obtained from the Piceance Creek basin. Some changes in the inorganic matrix may have occurred in these samples, yet the organic carbon data are valid. Since gross changes in the inorganic (mineral) matrix may affect the validity of the direct determination of organic carbon, a thorough study of other shale deposits should be made before applying this method as a routine procedure for organic carbon determinations.
LITERATURE CITED (1) T. F. Yen, “Facts Leading to the Biochemical Method of Oil Shale Recovery’’, Anal. Chem, Pertaining to Oil Shale and Shale Oil, Washington, D.C., June 24-5, 1974. (2) J. W. Smlth, L. G. Trudeii, and W. A. Robb, U S . , Bur. Mines, Rep. lnvest. 7693, 1972. ( 3 ) N. B. Young, J. W. Smith, and W. A. Robb, US., Bur. Mines, Rep. invest. 8008, 1975. (4) N. E. Vanderborgh, “Characterization of Oil Shales by Laser Induced Pyrolysis”, FACSS, Atlantic City, N.J., Nov. 19, 1974. (5) K. E. Stanfield, i. C. Frost, W. S. McAulev, and H. N. Smith, U.S., Bur. Mines. Rep. lnvest4825, 1951. E. W. Cook, Fuel, 53, 16-20 (1974). R. F. Cuimo, Mikrochim. Acta, 175-180 (1969). A. B. Hubbard, U.S., Bur. Mines Rept. Invest. 6676, 1965. K. E. Stanfieid, i. C. Frost. W. S. McAuiey, and H. N. Smith, U.S., Bur. Mines Rep. invest. 4825 (195 1). J. W. Smith, Bull. Am. Assoc. Petrol. Geol., 50, 167-70 (1966). A. W. Decora, F. R. McDonald, G. L. Cook, US., Bur. Mines, Rep. lnvest. 7523, 1971. B. L. Beck, D. Liederman. and R. Bernheimer. Am. Chem. SOC.,Div. Fuel Chem. Prepr., 15, 31-37 (1971).
ACKNOWLEDGMENT These studies were conducted a t the ERDA Anvil Points facilities located on the Naval Oil Shale Reserve near Rifle, CO. The authors wish to thank ERDA for permission t o publish.
RECEIVEDfor review February 2, 1976. Accepted April 8, 1976.
Simplified Kjeldahl Nitrogen Determination for Seawater by a Semiautomated Persulfate Digestion Method J. M. Adamski Suffolk County Department of Environmental Control, 1324 Motor Parkway, Hauppauge, N. Y. 11787
Persulfate digestion in combination with indophenol colorlmetry via a Technlcon AutoAnalyzer II was used as an alternative to the standard Kjeldahl nltrogen method as specified in “Standard Methods”. Application of this method to the analysis of seawater samples demonstrated that a large quantity of samples can be rapidly processed wlth relative ease in a small envlronmental laboratory. A detection limit of 0.06 mg/l. as nitrogen was calculated for the operating range 0-5.6 mg/l. Seawater samples spiked wlth 3.00 mg/l. of ammonia nltrogen were analyzed wlth a precision of f0.25 mg/l. and a spike recovery of 105 f 6.8%. Seawater samples splked with 3.35 mg/l. of Kjeldahl nitrogen contained in raw domestic waste were analyzed with a precision of f0.30 mg/l. and a spike recovery of 100 f 8.1 %. The results of this study indicated that the persulfate method greatly simplifies the determination of Kjeldahl nitrogen in seawater while successfully malntalnlng low level requirements for sensltivity, precision, and accuracy.
Although the analysis of Kjeldahl nitrogen has received widespread acceptance as an important parameter for assessing the effects of domestic wastes on receiving waters, its use is often limited by the expensive nature of the analysis, especially in cases where a large data base is necessary to satisfy the statistical requirements of routine monitoring surveys and computer models. Employing the classical Kjeldah1 technique as described in “Standard Methods” ( I ) on a large scale is a tedious and time-consuming proposition which can seriously limit the analytical capabilities of small environmental laboratories, both in terms of manpower as well as bench space requirements. This classical procedure often lacks the necessary precision and accuracy for detecting the 1194
ANALYTICAL CHEMISTRY, VOL. 48, NO. 8, JULY 1976
low nitrogen levels commonly encountered in marine samples even when large sample aliquots are utilized to increase the sensitivity. The following study proposes the use of an alternative procedure for determining Kjeldahl nitrogen in seawater samples which provides better sensitivity, better precision and accuracy, greater simplicity in sample preparation, and a greater capacity for handling a large quantity of samples with minimal manpower and space requirements. This procedure employs a modified persulfate digestion procedure as described in the routine methods manual of the Ontario Ministry of the Environment (2) in combination with indophenol colorimetry for ammonia nitrogen as described in the works of Rossum and Villarruz ( 3 ) and Weatherburn ( 4 ) . Applying these principles to the analysis of marine samples using an adaptation of the automated Grasshoff and Johannsen ( 5 )procedure as specified by O’Connor and Miloski (6) completes the analytical scheme.
EXPERIMENTAL Apparatus. A modified version of the indophenol procedure as described by O’Connor and Miloski ( 6 )was employed using a Technicon AutoAnalyzer I1 system with the appropriate accessories as shown in Figure 1. Sample digestion was accomplished on a hot plate in standard 125-ml Erlenmeyer flasks. Reagents. Phenate reagent was prepared by dissolving 35 g of phenol and 0.4 g of sodium nitroprusside in approximately 250 ml of distilled water and diluting to 11. The phenate reagent was stored in an amber glass bottle to inhibit decomposition and refrigerated when not in use. Preparation of the phenate reagent with fresh reagent grade phenol was necessary for proper color development. Once a bottle of phenol has been opened, reagent quality can be maintained by storing the remainder of the phenol under nitrogen gas. Hypochlorite reagent was prepared by dissolving 20 g of sodium hydroxide and 2 g of sodium dichloro-S-triazine-2,4,6-( lH,3H,5H)-trione (MCB Manufacturing Chemists catalogue number SX503) in approximately 250 ml of dis-
I70 -010 3
DI
A10
WASTE
170-0103
32
157
60'C
A
I
5 TURNS
157- 8273
-
DI
--
4 116 0 4 8 8 01
32 2 00
SAMPLE
63 DISTILLED WATER
2.00
A
SAMPLE ASSIST
I20
--
w
23
A
-
- BOBS
AIR
DISTILLED W A T E R
AIR
3o/~R. 1.1
I
.32
RESAMPLE
I 20
7
5 TURNS
A
WASTE
WASTE
4
Table I. Effects Due to Extended Digestion Time on Ammonium Chloride Standards a Prepared in Distilled Water 2o
Sample
20-minute digestion
50-minute digestion
42
w
10
HYPOCHLORITE
PHENATE
.IO
PULL THROUGH
.BO
ABSORBANCE
N concentration, mg/l. 10-minute digestion
BUFFER
VS.
WAVELENGTH
1
1s
z
1 2 3 4 5 6 Std dev, mg/l Re1 std dev, %
1.51 1.48 1.50 1.51 1.48
1.44 1.58 1.47 1.47 1.55
...
...
10.016 fl.1
10.06 14.0
1.06 1.75 1.46 0.91 0.90 1.61 10.37 *29.
m a (r
0 .IO
4
05
Known concentration of standards, 1.40 mg/l. 400
tilled water and diluting to 1 1. This reagent is generally stable for about three days if refrigerated when not in use. Buffer reagent is prepared by dissolving 9 g of boric acid, 2 g of sodium hydroxide, and 120 g of sodium citrate in 500 ml of distilled water and diluting to 1 1. A wash solution approximating the sodium sulfate content and final color of a digested blank was prepared by adding 20 ml of concentrated sulfuric acid and six drops of 0.1% methyl red in ethanol to about 500 ml of distilled water and neutralizing to the methyl red end point with 4M sodium hydroxide (1:190 ml). Back titration with 4 M sulfuric acid was then required to duplicate the pink color of digested samples. The final volume was adjusted to 1 1. with distilled water. This solution was maintained in the wash reservoir of the automatic sampler throughout routine analytical procedures. Procedure. A 25-ml aliquot of seawater sample and 1.0 ml of concentrated sulfuric acid were pipetted into a 125-ml Erlenmeyer flask and boiled down on a hot plate until the white fumes of sulfur trioxide first appeared. Boiling must be accomplished in the presence of glass beads or boiling chips at low heat to avoid violent bumping. While the sample was sufficiently warm to maintain the salt residue in solution, a measured amount of doubly recrystallized potassium persulfate (approximately 1g) was added to the digestion mixture. The sample digest was then swirled gently to ensure complete contact between the persulfate and the sample residue. If salt precipitation occurred prior to persulfate addition, gentle heat was again applied
600
700
WAVELENGTH
so0
900
1000
(nm)
Figure 2. Spectral scans for two digested 0.50 mg/l. ammonium chloride standards (as N) using the indophenol method in the presence and absence of methyl red. The solid line indicates a standard with no residual color while the dashed line represents the same standard containing methyl red
until all of the precipitate redissolved. After persulfate addition was completed, the digestion mixture was strongly heated at fuming for 10 additional min. No special safety precautions were required when digesting with persulfate other than those generally employed when handling strong acids and bases. Too much heating beyond the 10-min ' limit resulted in a loss of precision. The data in Table I for a series of replicate distilled water standards digested over various time intervals indicate the undesirable effects of prolonged digestion. After allowing sufficient time for cooling, 15 to 20 ml of distilled water was added to the residue with gentle application of heat until solution was complete. The digest was then neutralized with 4 M sodium hydroxide t o the yellow methyl red end point (1 drop 0.1% methyl red in ethanol) and back-titrated dropwise to the first pink color (final pH approximately 5.5). The sample was then quantitatively transferred to a 50-ml volumetric flask and brought to volume with distilled water. This final step accomplisheda twofold dilution ANALYTICAL CHEMISTRY, VOL. 48, NO. 8, JULY 1976
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Table 11. Nitrogen Determinations on Digested Ammonium Chloride Standards Prepared in Distilled Water a n d Seawatera
Sample
N concentration, mg/l.
Sample
a
No.
In distilled water standards
In seawater standards
2.95 2.93 3.09 3.04 2.98 3.01 2.90 2.87 2.83 2.80 2.96 2.91 2.85 2.88 2.77 2.74
3.09 3.12 3.09 2.98 2.98 2.95 3.09 3.12 2.82 2.85 2.82 2.72 2.71 2.69 2.74 2.86
Known concentration of standards, 2.80 mg/l.
Table 111. Precision and Accuracy Summary f o r Digested Ammonium Chloride Standards Prepared in Distilled Water a n d Seawater Distilled water Seawater standards standards Colorimetry precision (mg/l.) Total precision (mg/l.) Relative precision for colorimetry (%) Total relative precision (%) Accuracy as percent recovery
f0.035 AO.099 A1.2 h3.4 103
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20
Ammonium spike recovered mg/l. % 3.15 3.12 3.06 3.03 3.02 2.96 3.40 3.43 3.15 3.18 3.01 3.25 3.20 3.29 3.02 3.19 2.89 2.95 3.77 3.29
Sewage spike recovered % mg/l.
105 104 102 101 101 99 113 114 105 106 100 108 107 110 101 106 96 98 125 98
RESULTS A N D DISCUSSION A series of replicate digestions were conducted on known ammonium chloride standards prepared in both distilled water a n d seawater. T h e known concentration of each standard was 2.80 mg/l. as nitrogen. Each digested aliquot was analyzed in duplicate. T h e results of these replicate analyses ANALYTICAL CHEMISTRY, VOL. 48, NO. 8 , JULY 1976
3.52 3.78 2.88 3.23 3.08 3.21 3.60 3.76 3.55 3.57 3.58 3.38 3.22 3.63 3.38 2.94 3.43 2.76 3.29 3.43
105 112 86 96 92 96 107 112 106 107 107 101 96 108 101 88 102 82 98 102
Mean 3.15 105 3.35 100 Std dev h0.21 k6.8 10.27 h8.1 a Known concentration of spikes; ammonium spike, 3.00 mg/l. sewage spike, 3.35 mg/l.
Table V. Precision Summary f o r Spiked a n d Unspiked Marine Samples
h0.076 h0.156 f2.6 f5.4 104
on the initial sample aliquot to ensure that all of the salt residue remained in solution. Blanks must be analyzed with each set of digested samples in order to correct for any nitrogen contamination in the sulfuric acid and potassium persulfate reagents. The nitrogen concentration in the blank was independently determined and this result subtracted from all sample analyses before any final results were reported. The seawater digest now containing nitrogen as ammonium ions was finally analyzed colorimetrically using the Technicon AutoAnalyzer I1 system schematically illustrated in Figure 1. Calibration of the automated system was accomplishedby employing a dilution loop to maintain a routine operating range from 0-5.6 mg/l. as N. The nitrogen concentration in digested distilled water blanks varied between 0.21 and 0.31 mg/l. as N. In the operating range indicated, a digested blank is approximately equal to a 5% chart recorder deflection. To confirm that the residual color of digested standards due to the presence of methyl red indicator does not interfere with the colorimetric determination of ammonium ions at 630 nm, absorbance vs. wavelength scans were derived in the spectral region between 400 and 1000 nm for two identical digested 0.50 mg/l. ammonium chloride standards (as nitrogen). Neutralization was accomplished for one standard in the previously described manner with 4 M sodium hydroxide using the methyl red end point while the other was neutralized with the aid of a pH meter (final pH of 5.5).The scans were conducted on a Turner model 350 spectrophotometer using a 1.0-em cell and distilled water as a reference (see Figure 2). No interference effect was encountered.
1196
Table IV. Spike Recovery Summary on Marine Samplesa
ColorTotal imetry precisi- precion sion Standard deviation of samples spiked with ammonium chloride (mg/l as N)
f0.25
&0.090
Relative standard deviation of samples spiked with ammonium chloride (%)
h7.4
f2.6
Standard deviation of samples spiked with sewage (mg/l. as N)
f0.30
f0.098
Relative standard deviation of samples spiked with sewage (96)
f8.2
h2.7
Standard deviation of unspiked samples (mg/l. as N)
f0.12
f0.04
Relative standard deviation of unspiked samples (%)
A52.
h17.
are given in Table 11. The total precision (digestion plus colorimetry) was determined by finding the standard deviation from t h e mean for a series of replicate digestions. T h e colorimetry precision was evaluated independently of the total precision by finding the difference between duplicate colorimetric analyses on each 'digested standard and then determining t h e standard deviation of these differences. T h e application of this statistical technique is described by Youden ( 7 ) .Accuracy is expressed as a percent recovery of the known standard concentration. Precision and accuracy calculations for the analyses given in Table I1 are summarized in Table 111. A standard deviation of f0.02 mg/l. as nitrogen was calculated for the mean of 17 digested distilled water blanks. The detection limit of the persulfate procedure was then defined as three times t h e standard deviation of t h e blank reading or 0.06 mg/l. as nitrogen for t h e routine operational range between 0 and 5.6 mg/l.
The precision and accuracy of the persulfate digestion method for actual marine samples were calculated by running a series of duplicate ammonium chloride and sewage Kjeldahl nitrogen spikes on a set of 20 marine water samples taken from the coastal waters of eastern Long Island. T h e concentration of nitrogen in the sewage spike was predetermined using the standard Kjeldahl distillation procedure described in “Standard Methods” ( 1 ) for five replicate aliquots. T h e reagents as described in “Standard Methods” ( 1 )were appropriately adjusted in strength for use in a Labconco microdistillation apparatus. Final analysis was accomplished titrimetrically using boric acid and standard sulfuric acid ( I ) . Each marine sample was spiked separately in duplicate with 3.00 mg/l. of ammonium chloride as nitrogen and 3.35 mgh. of sewage Kjeldahl nitrogen (based on the mean of five replicate analyses as specified in “Standard Methods”). T h e amount of spike recovered from each of the samples is given in Table IV along with the calculated standard deviation from the mean recovered spike. This later value represents the variation in accuracy for the 20 samples analyzed. Precision results based on duplicate digestions of the same 20 marine samples are summarized in Table V. Each digest was again colorimetrically determined in duplicate such that a total precision and an independent colorimetry precision were both calculated using the procedure described by Youden (7).
CONCLUSION Precision and accuracy data have confirmed that the semiautomated persulfate digestion method is an acceptable
routine laboratory procedure for determining Kjeldahl nitrogen in seawater samples. Excellent recovery of Kjeldahl nitrogen in the form of a sewage spike demonstrated that the technique is suitable for detecting the presence of Kjeldahl nitrogen a t low levels in seawater samples contaminated with domestic waste. Greater simplicity in analytical procedure allows for greater speed in sample processing and a lower cost per analysis when compared to the classical Kjeldahl method. The persulfate procedure becomes especially advantageous when it is necessary to analyze a large number of samples. At maximum efficiency, one person could perform approximately 100 Kjeldahl nitrogen analyses per day using the persulfate digestion method while only 18analyses could be performed per day using the classical macrodistillation method.
LITERATURE CITED (1) American Public Health Association, “Standard Methods for the Examination of Water and Wastewater”, 13th ed., APHA, Washington, D.C., 1971 p 244. (2) Ontario Ministry of the Environment, “Handbook of Analytical Methods for Environmental Samples”, Ontario Ministry of the Environment, Laboratory Services Branch, Toronto, Ont., 1975. (3) J. R. Rossum and P. A. Villarruz, J. Am. Water Works Assoc., 55,657 (1963). (4) M. W. Weatherburn, Anal. Chem., 39, 971 (1967). (5) K. Grasshoff and H. Johannsen, J. Cons., Cons. Int. Explor. Mer, 34, 516 (1972). (6) B. O’Connor and W. Miloski. unpublished work, Suffolk County Department of Environmental Control, Oct. 1974. (7) W. J. Youden, “Statistical Methods for Chemists”, Chapman and Hall, London, 1951, p 16.
RECEIVEDfor review February 9, 1976. Accepted April 15, 1976.
Characterization and Appl cat on of FerroZ ne Iron Reagent as a Ferrous Iron Indicator Charles R. Gibbs Hach Chemical Company, P.O. Box 907, Ames, Iowa 50010
FerroZlne Iron reagent, 3 4 2-pyridyl)-5,6-bis( 4-phenylsulfonlc acid)-1,2,4-trlazine, monosodium salt, monohydrate has been further characterized. Details of its synthesis, purlfication, and analysis are given. The conditional formation constant of its trls complex with ferrous Iron, a study of complex formation vs. pH for a number of buffer systems, and the effects of temperature on the complex are reported.
Since its introduction by Stookey ( 1 )in 1970, FerroZine iron reagent, 3-(2-pyridyl)-5,6-bis(4-phenylsulfonic acid)-1,2,4triazine, monosodium salt, monohydrate, (hereafter referred to as FZ) has been applied to the determination of iron in a wide range of situations. Its advantages include its water solubility, low cost per gram, and high sensitivity. This paper will report the data gained in efforts aimed a t producing a well-characterized and pure product. The widest use of FerroZine reagent has been in the determination of serum iron and total iron-binding capacity (2-19). T h e reagent has been applied in a number of automated analyzers (7-10,15,16) and in a test kit (11)for these purposes. A variety of systems have been used for reducing the iron to the ferrous state and for successfully overcoming minor interference by abnormally high serum copper levels.
Other reported uses of FZ for determination of iron have been with potable water ( I ) , seawater (20), plant nutrient solutions ( 2 I ) ,plant materials (22),and high purity reagent chemicals (23). Procedures have also been described for the sequential (24)or simultaneous (25)determination of iron and copper using FZ in conjunction with bathocuproine sulfonate or by itself. In addition, procedures have been reported for the determination of cobalt(I1) (25)and ruthenium(II1) and osmium(VIII) (26) in which FZ was used as the colorimetric reagent. Several analytical uses take advantage of the fact that FerroZine reagent forms a colored complex with ferrous but not ferric iron. Thus, species which reduce ferric iron can be indirectly determined. Ascorbic acid has been determined in fruit juices (27) and in serum and urine with an automated system (28).Methods for determination of sulfur dioxide in liquid samples and upon absorption from gases have been reported (29,30). NADH (reduced nicotinamide adenine dinucleotide) levels have been similarly monitered to follow enzymatic activities in NADH/NAD redox systems (31).
EXPERIMENTAL S y n t h e s i s of F e r r o Z i n e I r o n R e a g e n t . Production of 3-(2-pyridyl)-5,6-diphenyl-l,2,4-triazine (PDTZ) is carried out using the method of Case (32) as modified in the following procedure (33). ANALYTICAL CHEMISTRY, VOL. 48, NO. 8, JULY 1976
1197
