Determination of Oxygen-Consumed Values of Organic Wastes A Comparative Study of Methods W. ALLAN MOORE, F. J. LUDZACK, AND C. C. RUCHHOFT Environmental Health Center, Public Health Service, Cincinnati, Ohio
During the past few years several methods have been proposed for the determination of oxygen consumed in organic wastes. In order to evaluate the proposed methods a comparative study was made. When silver sulfate is used as a catalyst in conjunction with the Moore procedure, higher oxidation values are obtained than with the other methods studied. This
D
is especially true on straight-chain acids, which are not attacked by the ordinary methods. Statistical evaluation of the data indicated that the Moore acid dichromate method is applicable to a larger variety of wastes with advantages in precision and reproducibility. It also has advantages in ease of manipulation and time required.
URIKG the past few years several methods have been pro-
run a t the same time when the iodic acid method was used. In each posed for the determination of oxygen consumed in organic case an equal number of blanks were run, except when the check wastes. The shortcomings of the hmerican Public Health Assoresults on blanks assured that a smaller number would be sufficiation's permanganate method are well known and there is a cient. Tests were run by each method on various sample volumes definite need for a better method for determining oxygen conwithin the range permitted by the amount of available oxidizing sumed in stream sanitation and industrial waste studies. __ In order to evaluate the variTable I. Statistical Comparison of Methods for Determining Oxygen Consumed ous proposed methods, a comCoefficient parative study was made by Sample Compound or Waste No. of Standard Confidence of % of so. a n d Method Used Detn8.a Mean Range Deviation Limits, ib Variation Theoretical this laboratory. I n addition 1 to the permanganate or standOrganic dye waste .. 110 0.041 IodateU 39.4 99 960 223 ard method ( I ) , the methods 0.064 .. 73.6 166 96 1,153 591 Ingols 466 0 . 3 0 6 .. Madison 1,184 211 99 690 studied included the dichro75 0,072 .. 169 33.3 98 463 Permanganate 100 1,183 113 15.9 36 0.013 .. Moore mate method proposed by Ingols and Murray ( 4 ) , the die o-Cresol 91.4 39 0.024 99 575 79 14.0 IodateJ chromate method of Madison 86.3 0.024 12.9 29.1 98 543 80 Ingols 72.9 Madison 6 8 . 9 0 . 0 6 6 99 459 174 3 0 . 5 ( 6 ) , the iodic acid method of 0.051 22.8 51.5 70.5 99 443 100 Permanganate Dzyadzio ( 2 ) as modified by 84.2 100 530 31 5.1 11.6 0.01 Moore Johnson, Halvorson, and Tsu3 Oil refinery caustic waste 6,322 0.045 .. Iodaten 49 50,900 8,182 2,274 chiya ( 5 ) , and the dichromate 1,976 0.019 .. Ingols 50 46,000 3,680 874 0 . 0 8 8 .. Madison 50 2 , 8 3 8 6 , 4 1 3 3 2 , 2 0 0 1 9 , 3 6 0 method proposed by Moore, 1,232 50 2 784 0.046 2 9 , 1 0 0 10,040 ,. Permanganatec Kroner, and Ruchhoft ( 7 ) of 347 0,007 .. 50 48,400 1,843 783 Moore 0.013 1,676 .. Moore-Ag,SOd 29 742 57,500 3,230 this center. Shortly after the 4 Textile dye waste study was started, the use of 20 27,477 6 , 6 7 4 1,634 4,543 0.060 .. Iodatea Ingols 20 35,209 2,313 569 1,285 0.016 .. silver sulfate as a catalyst as .. 20 36,599 786 204 461 Moore 0.006 proposed by Muers (8) was Moore-AgzSOd .. .., . .,,. ..... ..... ... .. added to the modifications 5 Glycocoll 10 . .4 i 7 '"76 ..... ..... Iodate", e used. 6512 20 18.3 41.4 0:0k4 Ingols ~
EXPERIMENTAL
In the present study, four organic compounds which are typical of those that might be found in sewage and industrial wastes, and four industrial wastes, were used. Industrial wastes were selected that would be stable over the period necessary to carry out the comparison. Ten replicates a t a time were run by each of the methods studied, except that only five replicates could be conveniently
6
7
Moore Moore-AgzSOd Distillery waste Ingols hloore hloore-4gL304 2-Amino-8-naphthol-6sulfonic acidf Ingols Moore Moore-AgzSO4
20 20
592 607
19 20
4.5 4.5
10.2 10.2
0.008 0.007
90.8 94.8
20 20 20
356 344 902
38 19 24
14.8
33.4 13.6 11.9
0.041 0.018 0.006
.. .. ..
20 20 20
1,320 1,336 1.356
52 25 35
31.1 13.9 23.4
0.010 0.005
79.4 80.4 81.8
6.0
5.3 13.8 6.1
10.3
0.008
Sodium lauryl sulfate0 20 1,436 62 16.9 38.1 0.012 71.8 Ingols 76.4 34.9 0.010 20 1,527 68 15.3 Moore 90.7 20 1,812 106 23.0 52.0 0.013 Moore-AgzSOa a T e n replicates were run on all methods except iodate, where five replicates were run. b 95% confidence limits of single determination. C American Public Health Association standard method. d Silver sulfate could not be used as a catalyst on this waste, resulted in 40 t o 60% lower value t h a n regular procedure. e Replicate determinations varied from 45 t o 133 p.p.m. a n d less t h a n 10% of theory was obtained. f Technical grade. 0 Technical grade, probably a mixture of lauryl sulfates. 8
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1298
ANALYTICAL CHEMISTRY
Tahle 11. Ninety-Five Per Cent Confidence Limits of Mean on Number of Replicates Indicated in Table I Sample Iodate Organicdye waste 5.5 o-Cresol 3.3 Oil refinery c a m tic waste 894 Textile dye waste 1016 Glycocoll ... Distillery waste .. 2-Amino-8naphthol-6. . sulfonic acid Sodium lauryl sulfate ...
195$Zo Confidence Limits of Alean -~ PermanMadison ganate Bloore 10 4 4.1 2.8 4.0 3.4 1.6
Ingols 6.2 2.9 282 287 9.2 7.5
9 7
...
7 0
. .
8.5
...
394 ,
.
111 103 2.3
, .
3.1
...
...
MooreAgZSOr
.. . .., 311 i:3 2.6
3.1
5,2
7.8
11.6
agent. Over one half the samples vere so tested in order to check the effect of sample size. The effect of different reflux times on the oxygen-consumed values was evaluated by the Moore method to determine whether the specified 2-hour period could be shortened. The iodic acid method mas checked by adding various amounts of distilled water to the reaction mixture before refluxing, to determine the effect of the volume of the reflux mixture (with a constant sample volume) on oxygen-consumed results. RESULTS
Table I presents the statistical data obtained on all samples. On the first two samples 100 replicates were run by each method. Because of accidental factors which gave results not representative of the method or sample, a few determinations were discarded and statistical data were calculated on the basis of the remaining replicates. Sample 1 indicated an oxygen-consumed value of nearly the same magnitude for both the !doore and Ingols procedures. However, the range of results of the Ingols and particularly the Madison method was excessive. Although the standard deviation of the permanganate method places it second on this sample, the order of magnitude of its coefficient of variation indicates that it is inferior to all the methods except that of Madison. On sample 2 the iodic acid method gave the highest percentage recovery, although in reproducibility it was inferior to both the Moore and Ingols methods. iLs with the first sample, the order of magnitude of the coefficients of variation places the permanganate method fourth, only slightly better than the Madison procedure. On sample 3 the iodate method again gave the highest oxygenconsumed value of all except the -Moore procedure using silver sulfate as a catalyst. The reproducibility of the iodate procedure was verv poor, 3orer reproducibility being shown only by the Madisor; metho$. The coefficients of variation on this sample were in the same order LLS with the previous two samples. The data on confidence limits included in Tables I and I1 are limits, which means that either the single determination or the mcan as indicated in the table will be within the limits stated 19 out of 20 times. On the basis of the data assembled on the first three samples, the Madison and permanganate procedures were dropped and another procedure, the basic Moore method modified by the addition of silver sulfate in the reflux mixture, was added to the list of methods given further study As a result of this work, it was judged feasible to decrease the number of replicates for each method to 20 for the remainder of the study with each group of five or ten replicates obtained on different days. This made it possible to use industrial wastes which were not stable enough to be used over the long period of time required for the greater number of determinations used in the first three samples
!%yoconfidence
With sample 4 (Table I), the iodate method resulted not only in very poor reproducibility but also in a lower oxygen-consumed
value. The use of the Moore procedure, modified by addition of the silver sulfate, on this waste, resulted in a reduction of oxygen consumed of 40 to 60%. Definite signs of incompatibility between catalyst and sample were noted as soon as refluxing began. The iodate method oxidized sample 5 from 5 to 20%, whereae the dichromate methods resulted in uniform high oxidations. M'ith two different standard solutions of iodate, the results on sample 3 ranged from 45 to 133 p.p.m. of oxygen consumed. A similar situation was evident in a filtrate from a waste containin naphtholsulfonic acid. On the latter material, the iodate metho! would not give a consistent oxygen-consumed value, whereas the dichromate methods yielded consistent results on the order of 60,000 p.p.m. As this sample changed from day to day, it could not be included in this study. After the work on these samples, the iodate procedure was dropped from the study. A distillery waste, sample 6 , was high in organic acids such a~ acetic, propionic, and lactic. Hence, the silver sulfate method responded very well from the standpoint of bot,h recovery and reproducibility. The results obtained with samples 7 and 8 indicate no definite superiority for any of the dichromate reflux methods used. The minimum safe number of replicates (S), required to produce a mean (95% confidence limits) {Tithin 2% of the mean of a large number of determinations on the same sample, is given in Table 111. The data prwrnted in Table I11 were calculated according to the follo\~-ingformula:
0.02%
d;=< s
t" _-
where s = standard deviation on a large number of individual determinations, n = indicated minimum number of replicates, t , = Fisher's t value for n replicates a t 95% confidence limit, and 3. r mean of a large number of individual determinations.
Table 111. AIinimum Safe Number of Replicates for Each Method with Each Sample Sample Ingols Organicdyewaste 42 o-Cresol 8 Oil refinery caustic waste 6 Textile dye filtrate 6 Glycocoll 21 1Y Distillery waste 2-Arnino-8naphthol-6sulfonic acid 4 Sodium lauryl sulfate 4
Iodate
Xadison
Permanganate
18
10045
52
9 22 37
75 .. ..
22
. .b
..
, .
28
..
Moore 5
hloore.ig?804
4
3 3 3 6
..
3
3
2
5
5
3 3
No results obtained with silver catalyst on firat two samples. b Iodate method too variable and gave less than 10% theoretical recovery.
a
-
The allowance of 2% of the mean is an arbitrary figure used on all samples; the remainder of the values are taken from tables or calculated from the data obtained on the samples. This value is exacting in its evaluation of a method and rapidly inclyases in severity when the degrees of freedom for n approach 1, as an inspection of a table of t values \Till phow. For this reason a minimum safe number of replicates of less than three would be unusual. The results as given in Table I11 indicate by the use of this parameter that, with the exception of the last two samples, both the unmodified Moore method and the catalyst modified method are statistically superior to any of the other methods tested. I t was partially on the basis of these results that the iodate method was eliminated from further consideration. FACTORS AFFECTING OXYGEN CONSUMED RESULTS
The time required for determination of the number of replicates and blanks indicated is given in Table IV, which includes both total elapsed time and manipulation time. The reflux time required no personal attention except in the iodate method, where bath temperatures must be held a t 190a =!= 5' C. In the Madison method closer attention is required than in any of the others, as
1299
V O L U M E 2 3 , NO. 9, S E P T E M B E R 1 9 5 1 the determination of the end point in the digestion cycle is vague, and the heating rate must be carefully controlled. Other factors such as recognition of titration end points, size of sample used, rate of heating, etc., are important in the selcction of the most efficacious method. The following discussion is designed to bring out points that would be noticeable only to those \Tho wed the methods repeatedly.
Table IT. Time Study on LMethodsfor Determining Oxygen Consumed Ingols
Iodate
Madison
Permanganate Moore
6 0 . of replicates deter10 10 10 10 5 mined" 1.75 7 3 4.50b Total hours elapsed 3.75 I 7 2.50 2.50 Manipulation time, hours 2.75 I n each case a n equal number of blanks were run in time indicated. b Use of silver catalyst involves only solution of silver sulfate in concentrated sulfuric acid stock; otherwise time and manipulation are s a n e as regular procedure. Used 1 gram of Ag9604 for each 7 5 nil. of acid.
Although the permanganate method requires less time than any of the other methods, it is very difficult to obtain uniformity in results. Heating in a water bath is subject to variation, especially when a large number of replicates are run. I n some cases a given sample may boil more rapidly than others and concentrate the mixture, with resulting difference in the oxygenconsumed value. The effect of agitation is also very noticeable, on the oxygen consumed, as the reaction mixture tends to precipitste manganese dioxide to a cert,ain extent and if not agitated frequently variable results are obtained. I n some cases, this precipit,ate is difficult to redissolve before the sample is titrated. The titration end point is definite, but because the permanganate color fades slowly the titration has to be conducted slowly and carefully to obtain permanent end points. Different analysts following the same methods of manipulation produce results which vary appreciably. The effect of sample size is significant; hence the use of as close to 507, of the oxidizing agent as possible is desirable in order to avoid variable results. I n the Madison method the amount of oxidizing agent available is small, which limits the size of the sample that can be used. This t,cnds toward large errors when strong industrial wastes are tepted. The major objections to this method are in the close attention required over a prolonged period and the difficulty in detecting the digestion end point. The digestion period is terminated when the concentrated sulfuric and phosphoric acid misture has fumed for exactly 4 minutes. It is imperative that the first traces of fuming be detected; if funied too long, the oxidizing reagent is decomposd. Blank determinations give poor agreement as a result of the difficulty in stopping the digestion at the right time. The titration using sodium diphenylamine sulfonate as an indicator requires a lapse of time between additions of the ferrous ammonium sulfate before there is any noticeable change in the color of the indicator. There is no chance to judge whrn the end point is being approached, so that back-titration is frequently necessary. Use of different sample volumes gives good reproducibility, if not more than 50% of the oxidizing agerit is used. The volatile acid iodate method requires more equipment, time, and manipulation than any of the other methods tried. The use of thc phosphoric acid bath to control reflux temperature is a definite hazard. The substitut,ion of a bath wax reduces this hazard to a certain extents,although no improvement is effected in manipulation or time involved. The large volume of solution required for the steam distillation makes lat,er cooling a problem, but if a smaller volume is used in this stage, recovery is low because of incomplete separat,ion of the iodine formed during the reflux period. A 500-ml. aliquot of sample is cumbersome to titrate. Poor checks were obtained on blanks throughout the
study. The agreement in oxygen-consumed values for different sample volumes was good, providing total sample volume did not exceed 40 ml. Above this volume, there was a definite drop in the oxygen consumed, even though only distilled water was added to the reaction mixture. This is due to the fact that such mixtures boiled vigorously below the 190" C. temperature specified in the method. The method is not applicable to as large a variety of samples as is the case in the dichromate oxidations; however, if the iodic acid is effective on a given sample its percentage recovery may be higher than those by other methods. The Ingols method gives fair results with a wide variety of samples and requires little time and manipulation. However, reproducibility is poor, especially on strong wastes, where only a small sample can be used. The reagent mixture is unstable and must be freshly prepared about every 2 weeks. The titration of iodine with thiosulfate to the starch-iodide end point in the presence of chromic salts is particularly poor, as the end point is masked by the confusing colors. Different analysts can obtain appreciable differences due to this fact,or. To treat a blank differently from the sample itself is generally considered poor technique. However, in the case of the Ingols method if the blank is refluxed, more or less of the dichromate is decomposed, the amount of such decomposition depending upon several factors. For this i'eason, the cold blank was used as recommended in the original procedure and the results do not appear to be affected to any appreciable extent. The use of different sample volumes has little effect on the oxygen-consumed values. The Moore method is intermediate in time required and apparatus involved. The end point is very sharp, and after a little experience an analyst knows approximately how close to the end point he is as the color changes from yellow through various shades of green to blue, then sharply to red-blue a t the end point. J'ery little effect on the results obtained is noted regardless of sample sizc; in fact, close checks have been obtained when the back-titrat,ion was only 2 to 3 drops. The ferrous ammonium sulfate requires standardization daily, as the acid concentration is kept purposely low (20 ml. of concentrated sulfuric acid per liter) and its normality gradually decreases. However, the standard dichromate solution is stable over long periods of time. The end point is sharp when the concentration of sulfuric acid in the titrating mixture does not exceed 30%; hence the reaction mixture must be diluted before titration and the acid content of t,he standard ferrous ammonium sulfate must be controlled in order to obtain a satisfactory end point. Little difference was observed in results obtained by different analysts. Sample volume is not critical, although results on purified effluents may not be too reliable because of the small consumption of the oxidant with the consequent high back-titration. Refluxed blanks check very well and normally do not show a depletion of more than 0.10 ml . As the 2-hour digestion period was subject to some question, digestion periods of 0.5, 1, and 2 hours were tried on several samples. On most samples, 0.5 or 1 hour would be sufficient; however, the 0.5-hour reflux time on sample 3 in Table I resulted in an 18% lower, and the 1-hour digestion in an 11%lower oxygen consumed than results obt'ained on the 2-hour reflux period. Glycocoll (aminoacetic acid) exhibited similar behavior. Therefore, it is well to check different reflux times on a given sample before attempting to shorten the 2-hour reflux period. If no significant difference in the results is obtained, the shorter time may be adopted. EFFECT O F SILVER SULFATE A S A CATALYST
In Table 5' the effect of 1 gram of silver sulfate in conjunction with the regular Moore method was checked on 17 different compounds in order to evaluate its applicability. Acetic acid is virtually unaffected by acid dichromate alone, whereas with the silver sulfate oxidation is better than 95y0 theoretical. Other
ANALYTICAL CHEMISTRY
1300 Table V.
Effect of Silver Sulfate on Moore Method for Determining Oxygen Consumed
Regular . With AgzSOc % of % of Compounda Mean Range theory Mean Range theory 26 Acetic acid 2.4 1014 62 95.1 38 362 17 A 1anin e 33.5 870 80.6 25 1790 97.7 1804 26 a-Amino-n-caproic acid 98.5 40 240 260 28 7.8 8.1 Benzene 18 1306 1748 18 34 71.8 96.1 n-Butyric acid 252 410 95 26.4 Chlorobenzene 41.4 176 2095 2413 43 83.2 o-Cresol 95.8 54 619 1672 33 29.7 80.1 67 E t h y l alcoholb 1074 1264 14 83.6 97.6 57 Furoic acid 626 17 78 63.9 100 Glutamic acid 980 882 13 45.4 484 82.7 Lactic acid0 28 2248 83 62.4 1805 77.7 150 Oleic acid fi 17 34 20 0.8 1.3 Pyridine 2018 7 4 . 2 2494 36 91.7 124 Sodium stearate 671 22.5 704 21.4 89 40 Toluene 1528 1206 46.8 36.9 839 706 Turpentine 1295 1573 94 95.9 78.9 77 Valene 5 Ten replicates of each compound run with and without silver catalyst. b 87% CxHnOH. 0 Impure grade of lactic acid.
compounds resulted in varying degrees of improvement on oxygen-consumed values, but substantial improvement was obtained in 14 of the 17 compounds used. Particularly good results are obtained when using the catalyst on short-chain carbon acids. Certain compounds tend to precipitate the silver and either nullify its effects or actually result in a lower oxygen-consumed value than the method without catalyst. This is generally true when the sample has a high concentration of chlorides. The precipitated silver also causes some difficulty in determining the end point, olving to turbidity effects. Sample 3 in Table I was one of those which decreased the oxygen-consumed value upon addition of silver sulfate. Silver sulfate as a catalyst is recommended as a complement to the regular procedure rather than a substitute, as apparently many wastes show incompatibility. If work is to be done on a given type of waste, it would be beneficial to use both the regular and the catalyzed methods and select the one giving the better results. Neither the Ingols nor the iodate method is applicable
with the silver sulfate because of the reaction with the added iodide before titration. CONCLUSIONS
Application of statistical procedures to the data leads to the conclusion that the Moore method is preferable to the other methods tested for the determination of oxygen consumed in organic wastes. This method also provides greater ease of manipulation and applicability to a large variety of samples and requires less time. The use of silver sulfate as a catalyst for dichromate reactions in determination of oxygen consumed appreciably extends the useful range of the procedure. On most compounds or mixtures and especially with straight-chain acids an increase in the theoretical oxidation is noted. With a few compounds, or in the presence of high chloride concentration, the use of the catalyst is precluded, making i t necessary to check both modifications. Certain materials such as benzene, toluene, and pyridine are not oxidized by either procedure. LITERATURE CITED
(1) Am. Public Health Assoc., New York, “Standard Methods for the Examination of Water and Sewage,” 9th ed., p. 122, 1946. (2) Dzyadzio, A. &Vodosnabzhenie I., i Sanit. Tekh., No. 8-9, 117-25 (1938). (3) Ettinger, M. B., U. S. Public Health Service, Environmental Health Center, Cincinnati, Ohio, unpublished memoranda, 1950. (4) Ingols, R. S., and Murray, P. E., Water & Sewage Works, 95, 113-17 (1948). (5) Johnson, P. W., Halvorson, H. O., and Tsuchiya, H. M., AbSOC., p. 2S, Atlantic City, stracts of 109th Meeting, AM.CHEM. 1946. (6) Madison, K. M., Division of Water, Sewage, and Sanitation Chemistrv. 113th Meeting AM. CHEM.SOC.. Chicago. 1948. (7) Moore, W. k:,Kroner, R. C, and Ruchhoft, C: C., A N ~ LCHEM., . 21, 953-7 (1949). (8) Muers, M. M., J. SOC.Chem. Id., 55, 71T (1936). RECEIVEDSeptember 13, 1950. Presented before the Division of Water, Sewage, a n d Sanitation Chemistry at the 118th Meeting of the AMERICAN CHEMICAL SOCIETY,Chicago, 111.
Determination of Glutamine and Asparagine in Plant Tissue Extracts G . W. BUTLER‘ Plant Chemistry Laboratory, Department of Scient@ Industrial Research, Palmerston North, New Zealand
V
ARIOUS methods for the estimation of glutamine are based
on its anomalous decomposition to form pyrrolidone carboxylic acid and ammonia. The most widely used is the modification of Vickery, Chibnall, and coworkers (16) of the original method of Chibnall and W-estall (3). This consists of measuring the ammonia formed when a plant extract is heated a t 100” C. and pH 6.5 for 2 hours. This method is not specific, as other constituents of plant extracts such as urea, allantoin, and asparagine may also liberate ammonia under these conditions. To overcome this lack of specificity, Pucher and Vickery ( I S ) developed a method for the quantitative extraction of pyrrolidone carboxylic acid, with its subsequent estimation through an amino nitrogen determination of the glutamic acid formed by acid hydrolysis, Measurement of the carbon dioxide liberated by ninhydrin before and after hydrolysis a t 1 Present address, Botanical Laboratory, University of Lund, Lund, Sweden.
100” C. and pH 6.5 has been made the basis of a method ( 8 , 1 8 ) . Archibald (1) and Krebs (10) used hydrolysis with glutaminase preparations from dog kidney and Clostridium welchii, respectively, Krebs claiming a high degree of specificity for his method. The position with regard to asparagine is much less satisfactory, in that the only method available is through hydrolysis with 1N sulfuric acid at 100’ C. for 2 hours; the asparagine concentration is calculated by subtracting the values for glutamine amide nitrogen and ammonia nitrogen from the total amide nitrogen value. Vickery et al. (16), in their original description of the method, emphasized its lack of specificity and pointed out that errors in the determination of glutamine must be reflected in asparagine values. That this caution is justified is amply demonstrated by results with rhubarb (19) and tobacco (18), which were inexplicable on current hypotheses of amide metabolism. Vickery and coworkers stress that results using indirect
