can be considered as a real “alcohol electrode”, whose behavior is, for example, governed by a n order-disorder structural transition. This transition is the one responsible for the response, and among the various soapwater systems so far studied, the Potassium stearate-water systemseems to be the most powerful indeed for analytical applications.
LITERATURE CITED (1) G. Eisenman. Anal. Chem., 40, 310 (1968). (2) J. M. Vincent and A. Skoulios, Acta Crysta//ogr., 20, 432, 441, 447 ( 1966).
(3) V. Luzzatti, “X-Ray Diffraction Studies of Lipid-Water System” in “Biological Membranes”, D. Chapman, Ed., Academic Press, New York. NY, 1968. n 71. (4) C. Both, M. Mascini, and A. Memoli, Anal. Chem., 44, 1371 (1972). (5) c . Botre, s. Borghi, and M. Marchetti, Biochim. Biophys. Acta, 135, 208 (1967). 16) A. M. Liauori and C. Botre. J. Phvs. Chem.. 71.3765 (1967). . . (7) P. Mueller and D. 0. Rudin, Natbe 213,603 (1967). (8) T. Narahashi, D. T. hazier, and M. Yamada. J. Pharmacol. fxp. Ther., 171, 32 (1970). (9) T. Narahashi, D. T. hazier, T. Deguchi, C. A. Cleaves, and M. C. Ernan, J. fharmacol. Exp. Ther., 177, 25 (1971).
for review October 21, 1974. Accepted February 6, 1975.
Micro Semi-Automated Analysis of Surface and Wastewaters for Chemical Oxygen Demand Andrea M. Jirka’ and Mark J. Carter Central Regional Laboratory, Environmental Protection Agency, Chicago, lL
A micro sample digestion technique for the determination of chemical oxygen demand (COD) is described. An automated spectrophotometric measurement of the appearance of chromium( 111) after sample digestion completes the method. Adequate sensitivity at 600 nm is achieved by using a 50 mm flowcell to measure COD values in the range of 3-900 mg/l. The semi-automated method is compared to the standard method with respect to precision, accuracy, ease of analysis, and comparability of data.
The oxidation of organic and inorganic wastes in a receiving water depletes the dissolved oxygen supply, which can have a profound effect on aquatic life (1).A meaningful determination of the natural oxygen demand of wastewaters has been a problem since the previous century. The biochemical oxygen demand (BOD) test was developed to measure the natural oxygen demand of wastes under laboratory conditions similar to those found in receiving waters (2-4). The advantage of the BOD test is that it is a good indicator of the biodegradability of a waste. The major disadvantages of the BOD test are the long time required for analysis, the poor precision, and the indeterminable accuracy of the method ( 4 , 5 ) . T o substantially reduce the time required to estimate the ultimate oxygen demand of a wastewater, the chemical oxygen demand (COD) test was developed (6). The addition of silver sulfate (7, 8) and mercuric sulfate ( 9 ) to the acidic dichromate digestion solution increased the reliability of the COD test (10, 11).However, since chemical oxidation does not differentiate between biologically stable and unstable wastes, a correlation between COD and BOD values must be developed for each sample type (12). Stenger and Van Hall reported a very rapid method for measuring the total organic carbon (TOC) content of water samples, which can be related to oxygen demand (13). However, the advantage in being able to rapidly determine TOC values is offset by the high initial equipment cost. In Author to whom correspondence should be addressed.
60609
addition, the informational content of the TOC analysis is less useful than that gained from the BOD or COD methods (14). The TOC test does not differentiate between compounds with the same number of carbon atoms, but which are in different stages of oxidation and, hence, have different oxygen demanding potentials. Since the COD and BOD tests measure the amount of oxygen required to stabilize waste samples their values inherently reflect the original oxidation state of the chemical pollutants. The standard COD test ( 4 ) is widely used because it provides a good balance between the value of the information gained and the speed of analysis when compared to the BOD and TOC tests (12). However, the standard method for determining COD has limitations which are not inherent in the concept of the test. The back-titration of dichromate after sample digestion is an insensitive method of detection. This lack of method sensitivity has been partially alleviated by using two different concentrations of dichromate and a relatively large sample volume. In addition, the consumption of large quantities of expensive reagents, the extensive and limiting bench space required, and the difficulty of disposing of large quantities of highly acidic mercury, silver, and chromium wastes, are serious problems for most industrial laboratories ( 1 5 ) . Recently, there has been considerable interest in simplifying the rather tedious standard COD method (16-19). Unfortunately, procedures using a much shortened digestion period or lower digestion temperatures produce results equivalent to those obtained by the standard method only for very readily oxidized wastewaters (20-22). Bloor (23) and Johnson (24) determined the organic content of biological materials by using dichromate as an oxidant and then measuring the excess dichromate spectrophotometrically. This procedure eliminated the tedious detection procedure of the standard method. The spectrophotometric procedure has been applied to the analysis of water samples in which the COD was determined by measuring the appearance of Cr(II1) after manual digestion (25-2 7 ) .
Several COD methods which use a spectrophotometric ANALYTICALCHEMISTRY, VOL. 47, NO. 8, JULY 1975
1397
GLASS
TRA\SMISSIOV T U R I X I ? 11)Sampler h a s h
To S a m p l e r i i Wash R e c e p t a c l e
;l.h''
116-0246-01
Sample
i l . b l R ~ u g e n th a t e r
Figure 1. Automated system for chemical oxygen demand Numbers in parentheses correspond to the flow rate of the pumptubes in mllmin. Numbers adjacent to glass coils and fittings are Technicon Corp. part numbers
t
750
I TIME
-
Figure 2. Recorder trace for chemical oxygen demand of potassium hydrogen phthalate standards analyzed in duplicate
means of detection and an automated sample digestion procedure have also been reported. Sample digestion was accomplished using a continuous digestor (28,29) or a high temperature bath (30-32). However, Tifft and Cain reported data which show that these automated procedures do not produce results equivalent to the standard method for all sample types (33).The incomparability of data was attributed to incomplete sample oxidation caused by the short digestion times in the automated methods. In addition, the higher concentration of sulfuric acid used in most automated systems requires that a smaller amount of mercuric sulfate be used to avoid its precipitation in the sample lines and the flowcell. The lesser amount of mercuric sulfate caused chloride to be more of an interference in these automated methods than in the standard method. The method reported here combines the advantages of the reliability of the standard digestion procedure (41, with the superior sensitivity and precision of an automated procedure based on the spectrophotometric measurement of Cr(II1) (27). Use of the resultant micro semi-automated COD method has increased the productivity of this laboratory threefold and reduced the consumption of very expensive reagents and the production of wastes twentyfold.
EXPERIMENTAL Apparatus. Samples were digested in Corning No. 9949 16 X 100-mm screwcap (cap No. 9998) culture tubes. Spectrophotometric measurements were made with the apparatus shown schematically in Figure 1. The automated system was fabricated using Technicon Corporation AutoAnalyzer I1 equipment consisting of a Sampler IV, Pump 111, Colorimeter 11, Recorder 11, and a single channel Digital Printer. The colorimeter was used in the direct mode and equipped with 600-nm interference filters and 50-mm 1398
ANALYTICAL CHEMISTRY, VOL. 47, NO.
8, JULY 1975
flowcells. The Standard Calibration control was set a t 228 to attain a reading of 1000 mgh. of COD at full scale on the recorder. A glass capillary was used as a sample probe. The sampler was operated a t 40 sampleshr with a 3:1, sample to wash ratio. Wastewater samples with particulate matter were blended with a Teckmar Model SDT homogenizer before taking an aliquot for analysis. An adjustable 0-5 ml Oxford pipet with disposable polypropylene tips was used for aliquoting samples and for addition of reagents. Reagents. Unless otherwise noted, all chemicals were ACS reagent grade. All reagent water was de-ionized and distilled. Digestion solution was prepared by adding 10.216 g of K2Cr207, 167 ml of concd H2S04 and 33.3 g of HgS04 to 500 ml of water and diluting the cooled solution to 11. Catalyst solution was prepared by dissolving 22 g of Ag~S04in a 9-lb bottle of concd H2S04. Sampler wash solution was 50% sulfuric acid by volume. A stock potassium acid phthalate solution, equivalent to 10 g/l. COD, was prepared by dissolving 8.500 g of a dried portion of NBS standard reference material 84 h in water and diluting to 1 1. Working standards of 25, 50, 75, 100, 250, 500, and 750 mg/l. COD were prepared by diluting 2.5, 5, 7.5, 10, 25, 50, and 75 ml of stock solution to 1 l., respectively. Procedures. It was necessary to wash all culture tubes and screw caps with 20% HzS04 before their first use to prevent random contamination. Digestion was carried out by placing 2.5 ml of sample and 1.5 ml of digestion solution in a culture tube. Three and one-half ml of catalyst solution were added carefully down the side of the culture tube so that the acid formed a layer on the bottom. The tube was capped tightly and then shaken to mix the layers. Two blanks and a set of standards were prepared in the same manner and were analyzed with each sample set. All samples, blanks, and standards were heated in an oven a t 150 "C, which is the observed reflux temperature of 50% sulfuric acid. After two hours, the tubes were removed from the oven, cooled, and placed in the Sampler IV tray.
Table I. Comparison of the Precision of the Semi-Automated and Standard Chemical Oxygen Demand Methods
IIOL
[COD1 ,m:i I. Simple \ a . Method
Manual
SemiAutomated
[cooj
Figure 3.
"I
,
Calibration curve for automated chemical oxygen demand
Each point is the average of duplicate determinations
The analytical manifold and reagents were set up as indicated in Figure 1. Two digested blanks were analyzed a t the beginning of each sample set to zero the base line. A mid-scale standard was used to calibrate the recorder and printer ( 3 4 ) . Standards were rerun periodically during the course of an analysis run to ensure that the system remained in calibration. The COD values of unknown samples were obtained by direct printout. A typical recorder trace for standards is shown in Figure 2.
RESULTS A N D DISCUSSION Sample Digestion. It has been shown that COD procedures using a shortened digestion period and/or a reduced digestion temperature do not attain the same degree of sample oxidation as the standard method (20-22, 33). Any alternate test procedure used to analyze wastewaters for COD must produce results equivalent to or better than the current standard method (35). Therefore, to ensure data comparability, the temperature, the period of sample digestion, and the concentration of reagents used in the standard method were adopted for use in the semi-automated procedure described here ( 4 ) . Since the micro colorimetric detection technique required only 2 ml of digested sample, the quantities of Sample and reagents used were reduced twentyfold in comparison to the standard method (4, 11). Use of the standard COD digestion apparatus was eliminated and instead all samples, blanks, and standards were digested in small screw-cap culture tubes. The potential for sample contamination from large glass surfaces was consequently reduced. Screw caps with phenolic resin liners were found to be unacceptable since they were attacked by the digestion solution giving erroneously high COD values. Teflon-lined caps greatly reduced this problem, especially if each cap was used only once. Any sample tube which leaked, as evidenced by a black residue on the outside of the tube, was discarded. Uniform addition of reagents and improved precision were achieved by dissolving all chemicals in one of two solutions. The catalyst solution was prepared by the standard method ( 4 ) . The oxidizing solution was prepared by combining potassium dichromate with mercuric sulfate and making the solution 6 N with sulfuric acid to solubilize the mercury salt. However, the mercuric sulfate was not completely soluble in the cooled, combined reaction mixture. The height of the sampler probe was carefully adjusted so as to avoid aspirating the precipitate. Otherwise, aspiration of the particulate matter caused severe noise.
Rei std
of
\o.
detns
'ilea?
1
11
40
2 3
10 11
4
10
230 26 270
Ranq,
4 90 4 12
Std d i \
1.4
de\,
1.3
3.5 12.2 5 .O
4.6
1.7
28.0
Spectrophotometric Analysis. The COD of wastewater samples has been determined spectrophotometrically, after digestion, by measuring the decrease in Cr(V1) concentration a t 352 (32) or 440 nm (19). Alternatively, the increase in Cr(II1) concentration has been measured at 600 (27) or 6 5 0 nm (25).All of these authors found the spectrophotometric procedure to be easier to perform than the manual titration. Molov and Zaleiko showed that better sensitivity could be achieved by measuring the decrease in Cr(V1) concentration than the increase in Cr(II1) concentration (28). However, the precision of a method based on the measurement of the decrease in Cr(V1) absorbance is very dependent on the reproducibility of reagent addition. This problem was avoided and adequate sensitivity was achieved by measuring the appearance of Cr(II1) a t 600 nm, using a 50-mm flowcell, and the scale expansion capability of the Technicon colorimeter. T o increase the sensitivity of the standard method, two different concentrations of oxidizing reagent are commonly used. These correspond to two levels of COD measurement, 5-50 mg/l. and 50-800 mg/l. (11). Moore and Walker found that the working range of the low level modification was limited by the diminished oxidation potential of the digestion solution after 50% of the dichromate was consumed (36). Because of the adequate sensitivity of the spectrophotometric semi-automated method, only one set of reagent concentrations was necessary to cover both levels of the standard method. T o test the linearity of the semi-automated method, standards between 500 and 1000 mg/l. COD were analyzed in increments of 25 mg/l. Potassium hydrogen phthalate was chosen for use as a standard because of its stability in solution and its complete oxidation under the conditions of the COD test ( 4 ) . The results, presented in Figure 3, show the colorimetric method to be linear up to 900 mg/l. COD. The entire system was cleaned for about $!2 hour before first use with 50% sulfuric acid to prevent the occurrence of severe base-line drift due to leachable organic matter. Also a 1:l dilution loop was added to the system to reduce the viscosity of the sample stream so that proper debubbling occurred in the flowcell. The recorder trace in Figure 2 was undamped. Precision, Accuracy, a n d Detection Limit. Since it was difficult to correct the semi-automated results for the small base-line drift, the working detection limit was defined as the mean bias of the blank plus two standard deviations. Eleven blank samples were analyzed to determine the detection limit. The mean value obtained was 1 mg/l. with a standard deviation of 0.8 mg/l. COD. These values were used to define the detection limit a t 3 mg/l. COD. This number compares quite favorably with the detection ANALYTICALCHEMISTRY, VOL. 47, NO. 8, JULY 1975
1399
Table 111. Comparison of Semi-Automated and Standard Chemical Oxygen Demand Methods
Table 11.Recoveries of Potassium Hydrogen Phthalate Added to Water Samples with Semi-Automated Method
[ C O D ! , n y , 1.
ICOD1,mg 1. S3r,ple Samplc
SOILTCI’
O r g a n i c s industry effluent Raw sewage R i v e r water Harbor w a t e r Industrial cooling water River water River water Channel w a t e r n e a r dredging Industrial d i s c h a r g e Industrial discharge T r e a t e d sewage Receiving w a t e r of sewage T r e a t e d sewage Steel mill effluent Mean Standard deviation
Rc:;oi ~ q ,
I\TNP ndded
I\ilP
13
200
217
102
164 31 16 65
200 100 200 200
370 122 224 262
103 91 104 99
15 28 52
100 100 100
116 124 152
101 96 100
30 16 40 25
100 100 100 100
131 116 144 127
101 100 104 102
14 14
100 100
124 112
110 98 101 4
innplc
S n m p l e soircL
limit of 5 mg/l. COD reported by Moore and Walker for the low level standard COD method (36). The relative precision of both modifications of the standard method and the semi-automated COD method was determined by performing replicate analyzes on four wastewater samples. Since the standard and semi-automated determinations were performed at different times, two similar sets of water samples were chosen so that the relative standard deviations could be meaningfully compared. The relative standard deviations of both methods for low COD concentrations, as shown in Table I, compare very closely. However, the precision of the semi-automated method a t high COD concentrations was approximately seven times better than for the standard method. Adelman pointed out that one of the factors contributing to the poor precision of the standard method was the potential loss of volatile components of the wastewater samples (30). This can be caused by the heat generated by the mixing of the sample with the concentrated acid prior to or during the reflux step. This problem was eliminated in the semi-automated procedure by avoiding the mixing of the sample and acid layers until the tube is capped so that sample digestion occurs in a complete closed system. The authors found that the ability to take a representative aliquot of nonhomogeneous sample was the limiting component of analysis variability regardless of method. Precision data for the standard COD method determined from interlaboratory analyses of standard-like solutions must be regarded only as a lower limit (5,37). The accuracy of the semi-automated method was determined by measuring the recovery of standards of potassium hydrogen phthalate added to several types of water samples. The mean recovery from 14 water and wastewater samples, shown in Table 11, was 101% with a standard deviation of 4%. Comparison of Semi-Automated a n d S t a n d a r d Methods. A variety of surface and wastewater samples were analyzed by both the standard and the semi-automat1400
ANALYTICAL CHEMISTRY, VOL. 47,
NO. 8,
JULY 1975
Raw sewage P a p e r mill cooling water Steel mill w a s t e T r e a t e d sewage P r i m a r v treated sewage B o i l e r bloivdown P o t t e r y shop w a s t e C r e e k downstr e a m f r o m pottery shop Primary treated sewage P a p e r mill w a s t e Raw sewage T r e a t e d sewage T r e a t e d sewage T r e a t e d sewage Organic chemical plant w a s t e Steel mill w a s t e C h e m i c a l plant w a s t e Raw sewage Chemical plant effluent Distillery Raw sewage Mean Standard deviation 0
Standard
SL‘1T‘L-
mctllad
iuto’noted
(SI
metliod
(‘XI
5 ‘li x 100
4 20 39
421 46
99.8 84.8
2 70 50 63
273 51 51
98.9 98.0 123.5O
180 140 94
183 156 99
98.4 89.7 94.9
90
87
103.4
4 50 170 36 27 21 270
464 164 35 27 22 275
97.0 103.7 102.9 100.0 95.5 98.2
90 9800 150 1500
89 9200 160 1500
101.1 106.5 93.8 100.0
83 4 50
90 4 50
92.2 100.0 98.0 5.2
Result rejected for calculation of mean and standard deviation.
ed COD methods to determine the comparability of data. These samples included raw and treated sewage, industrial, chemical, and food process wastes. Results comparing the two methods are shown in Table 111. Initially the largest discrepancies between methods occurred in samples which contained large quantities of particulate matter, e.g., raw sewage. Homogenizing samples of this type greatly improved the comparability of the data. Within the standard deviation of the mean ratio of results, no significant bias in COD values exists between methods. In addition, several pure organic compounds were analyzed to determine whether the semi-automated method achieved a more complete digestion than the standard method. The experimental results and calculated maximum theoretical COD values are shown in Table IV. The semi-automated values were slightly higher than the standard method results indicating that the former method achieved a more complete sample digestion. The difference could also be due to the fact that no volatile material can escape from the sealed tubes during oxidation in the semiautomated method, while in the standard method, volatile material may escape before sample oxidation is complete. Interferences. One of the major problems encountered in other automated COD methods is the inability of the techniques to compensate for the positive interference caused by the oxidation of chloride present in wastewater samples (28-32). The standard procedure for eliminating
~
Table IV. Comparison of Chemical Oxygen Demand Methods on Organic Compounds [
