1253
V O L U M E 25, N O . 8, A U G U S T 1 9 5 3 pipets, they reported values of 25, 22, 28, 24, 34, 26, 26, 36, 26, 23, and 25 millimoles of alanine, respectively; the correct value was 25. DISCUSSION
R-hen maximal densities are used, the standard curves break down at the higher concentrations, as can be seen in Figure 1, D. Therefore, the standard curves cannot be extrapolated. Sample concentrations must be adjusted, usually by dilution, to fall within the limits of the standard series. One- or 2-11]. spots of solution give the best results. Application of larger volumes often results in hazy color spots that are large and difficult to read satisfactorily. In such cases the maximal color density may be found at the periphery rather than in the interior. Such a spot is useless for a quantitative measurement by optical scanning. Block ( I ) obtained good iesults with a 5 4 . pipet; however, in subsequent work ( 2 ) 2.5-pl. pipets have been employed and the use of 1-11]. pipets is being considered. If spots larger than 1 or 2 111. are to be used, the concentration of the solution and the standard series must be reduced accordingly. Figure 1, C and D.shows that the top concentration that can be used in a standard series when a 1 4 . pipet is employed is 0.05 M or a total concentration in the spot of 0.05 micromole. Very often the 5-pL spot exhibits excess diffusion in the paper: however, if this size of pipet is employed, the highest value in the standard series would be 0.01 h ' or 0.01 micromole. The lower concentrations of the standard series would then be serial dilutions of this 0.01 11.1 amino acid and would be extremely dilute and susceptible to dilution errors. This standard series would probably be much too dilute for satisfactory results. Block ( I ) recommends drying the paper at 30" C.; while this is not excessively hot or much above room temperature, the
authors' chromatograms have been dried a t room temperature (20" to 22" C.). The present work confirms that of Brush and coworkers (4). They showed amino acid destruction on chromatograms which were heated wet with solvent. If chromatograms wet with phenol or lutidine are sprayed with ninhydrin, the amino acid color spots are red and pink rather than the normal purple-blue. I t is therefore important to remove as much of the solvent as possible before spraying. A period of 18 to 24 hours of solvent evaporation has proved sufficient to permit proper color development. Several grades of paper have been tested and Whatman No. 1 filter paper appears to be a very satisfactory stratum for the color spots. By setting the Densichron to read 1.43 and recording several hundred readings in areas chosen a t random, the standard deviation in absorbance was found to be 0.013. Quantitative paper chromatography in its present state of development compares favorably with older methods of amino acid analysis (microbiological and chemical) as to both accuracy and reproducibility. Furthermore, it can be used to determine certain amino acids for which there was no previous suitable method. LITERATURE CITED
(1) Block, R. J., ASAL. CHEM.,22, 137 (1950). (2) Block, R. J., personal communication, 1952. (3) Block, R. J., and Balling, D., "Amino Acid Composition of Proteins and Foods," 2nd ed., Springfield, Ill., Charles C Thomas, 1951. (4) Brush, M.K., Boutwell, R. K., Bartin, B . P., and Heidelberger, C., Science, 113, 4 (1951). ( 5 ) Patton, A. R., J. Chem. Educ., 28, 629 (1951). ( 6 ) Patton, A. R., and Chism, P., - 4 ~ 4 CHEM., ~ . 23, 1683 (1951). RECEIVED November 3, 1952. Accepted M a y 21, 1953. Published with the approval of the director, Colorado Agricultural Experiment Station, as Scientific Series Paper 396. Supported in part b y a grant from The Nutrition Foundation.
Microdetermination of DDT in River Water and Suspended Solids BEN BERCK Dirision of Entomology, Department of Agriculture, Winnipeg, Man., Canada ICRO amounts
of D D T are effective in controlling mosquitoes and black flies ( 2 ) . In studies in which D D T was used as a black fly larvicide ( 6 ) , information was sought on the D D T content of the river water at various distances downstream and on the role of suspended solids in affecting the amount and effectiveness of the DDT. This report deals with methods found useful in that regard. For determining small amounts of D D T in the South Saskatchewan River, where amounts as low as 1 microgram per liter were anticipated, three colorimetric methods ( 7 , 12, I S ) were considered. On the basis of satisfactory accuracy, precision, and specificity in exploratory tests, the Schechter-Haller method (12) \+asselected as the foundation for the methods described below. The following auxiliary aspects were given attention. In a limited side study, with recovery data as criteria of suitability, extraction technique Jyas developed empirically. Prospects of meeting the anticipated sensitivity limits through increasing the size of sample were investigated. It was found that samples between 1.50 and 1.75 liters could be handled in 2-liter, Squibb separatory funnels. The problem posed by suboptimal amounts of D D T \vas met by analyzing only the suspended solids for DDT. This was based on the finding that D D T is adsorbed to an appreciable extent by the suspended solids fraction (6). Thus, larger samples of n-ater-e.g., 30 liters-may be used, resulting in a corresponding increase in sensitivity. Since the amount of D D T adsorbed appears to vary xith kind, particle size, and the total amount of suspended solids, such analyses were
used mainly to provide an approximation of the D D T present in suboptimal samples. Interferences present in the water and suspended solids produced a yellow to amber color at the colorimetric stage. Davidow's ( 5 ) modification of the method of Schechter, Pogorelskin, and Haller (11) was effectivein eliminating the interferences, but the recovery of D D T was lowered. From recovery data so obtained, a correction curve was constructed. To increase the absorbancy of the final solution, the tetranitroDDT obtained from the sample was dissolved in a decreased volume of benzene and sodium methoside. Lowry-Bessey microcells (@, which have a capacity of less than 1 ml., were used, and the final solution was prepared with 0.7 ml. of benzene and 1.4 ml. of 2.40 A; sodium methoxide. Further lowering of percentage transmittance by dissolving the tetranitro-DDT in less than 0.7 ml. of benzene and 1.4 ml. of sodium methoxide was impractical owing to increased absorption of the blank. Figure 1 shows that these modifications result in a lowering of the slope of the curve, with resulting greater photometric accuracy. The data for Figure 1 were obtained 15-ith acetone solutions of D D T of a grade similar to that used in the field tests (6). Applying Ringbom's method ( I O ) for determining the inaccurate region of the standard curve (C, Figure l), it was found that 4 to 30 micrograms of D D T was the most accurate range under conditions described herein. Residues containing less than 4 micrograms were considered suboptimal, and where these were encountered, 10 micrograms of D D T were added (stand-
ANALYTICAL CHEMISTRY
1254
ard addition) to bring the concentration into the optimal range. Note should be taken, as pointed out recently by McBryde ( 9 ) , that when standard addition is used in the foregoing manner, no improvement in analytical precision is achieved, and that relative analysis error may be increased. REAGENTS AND APPARATUS
Use the reagents specified in the papers by Schechter et al. ( 1 2 ) and by Davidow ( 6 ) . Instead of 1.86 N sodium methoxide ( l a ) ,use 2.40 S to compensate for the reduction in volumes employed, and instead of 2% sodium hydroxide ( 1 2 ) use 10% potassium hydroxide (4)to wash the ether extracts
D DT,micrograms
Figure 1.
Standard Curves for DDT
Beckman Model DU spectrophotometer A = standard cells, 600 my, 0.01-mm. slit width, 3 ml. of CsHe f 6 ml. of CHaOh-a 1.86 .V. B = standard cells 600 m*, 0.01-mm. slit width, 2 ml.’of CsHe 4 ml. oi CH30iXa 1.86 N . and C = microcells, 600 my, 0.12 mm. 1.4 ml. CHaONa, 2.40 N slit width, 0.7 ml: C6HO -i
nels twice with 15-ml. portions of additional solvent, and transfer the washings to the 1000-ml. collector funnel. After draining off water that separates in the collector funnel, add about 10 grams of anhydrous sodium sulfate. Shake gently until the sodium sulfate cakes, then more vigorously for 1 minute, and then allow to rest in the funnel rack for 2 minutes. Using a Gooch crucible holder fitted with a plug of ether-washed cotton batting, filter about half of the contents of the collector funnel into a 500-ml. Erlenmeyer flask containing a few glass beads. Evaporate to near dryness on a steam bath. Transfer the balance of the contents of the collector funnel to the Erlenmeyer flask, wish the collector funnel with two 15-ml. portions of solvent, and transfer the washings to the flask via the filter. Again evaporate to near dryness on the steam bath and remove the last traces of solvent with moderate vacuum, using a water aspirator. At this point, take only the blank (sample of untreated river water) through the complete Schechter-Haller procedure ( 1 2 ) . If, at the spectrophotometric stage, the water blank exhibits a small absorption value a t 600 mpc,with no brown-yellow color as compared with a reagent blank, then substances that interfere with proper measurement of tetranitro-DDT are, for practical purposes, absent. On the other hand, if the water blank gives an appreciable absorption value (optical density >0.02, for instance, where microcells as herein used are employed), then Davidow’s ( 5 )chromatographic column procedure can be interposed t o eliminate the interferences present in the residue as follows: Using a new extract, boil off the solvent as directed previously. Redissolve the residue in carbon tetrachloride and pass through chromatographic columns, prepared according to Davidow (6). Evaporate the carbon tetrachloride (moderate vacuum), redissolve the residue in acetone, and transfer to large test tubes (25 X 200 mm.) via a filter of ether-washed glass wool. If pilot tests indicate that the sample contains less than 4 micrograms of DDT, add a standard amount of 10 micrograms of D D T dissolved in acetone to each tube. Evaporate the acetone with the aid of an air jet; then nitrate, etc., according to the regular SchechterHaller ( 1 2 ) method. Use 10% potassium hydroxide (4)instead of 2% sodium hydroxide ( 1 2 ) . Read the color intensity in microcells ( 8 ) , using 2.40 N sodium methoxide to develop the blue color. By means of the standard curve, read the DDT concentration, correcting for the blank. Using a correction curve, such as illustrated in Figure 2, correct the D D T loads actually found to a theoretical 100% recovery. Suspended solids. Solids suspended in river water may be separated from the water in a number of ways. Of two methods which were used ( 6 ) , the method considered more useful consisted of allowing the suspended solids to settle from samples of river water in wide-mouth gallon jars. To assist flocculation of clay and other material in colloidal suspension, the water was acidified. Acidify each gallon of water with 5 ml. of 2 N sulfuric acid and mix for about half a minute. Set each jar a t a 45’ angle so that RECOVERY, % I
loot
+
’Mixed solvent. Mix peroxide-free ether and n-hexane 3 to 1 by volume. Sample jars. Glass, 1-gallon size, wide mouth. Separatory funnels. Squibb type, in 2000-, 1000-, and 125-ml. capacities, with three funnel racks to accommodate six funnels of each size. Wire the glass stoppers with No. 18 nichrome wire. Flasks, Erlenmeyer or Florence, 500-ml. capacity. Microcells. Lowry-Bessey type ( 8 ) , 3 X 10 X 25 mm., plus cell carriage, carriage holder, and diaphragm plate (Pyrocell Manufacturing Co., 207 East 84th St., New York, N: Y.). Extraction apparatus. Either intermittent or continuous type. PROCEDURE
River Water. Acidify the water with dilute sulfuric acid to a pH of 4.0 to 5.0, and store in a cool place protected from light, until required. Place a 1500-ml. aliquot of a well-shaken water sample into a 2000-ml. Squibb separatory funnel and add mixed solvent. Extract each sample four successive times by shaking for 10,8,6, and 4 minutes, using 250,200,150,and 150 ml. of mixed solvent, respectively, for each extraction. At the end of each shaking period transfer the water phase to another 2000-ml. separatory funnel for re-extraction, and deposit the solvent phase in a 1000-ml. separatory funnel, which serves as a collector. Discard the water after the fourth extraction. Wash the extracting fun-
40 PUB.
2ol
2
Figure 2.
Correction Curves for Microdetermination of DDT in River Water
D4D T IN 6D D T - 5 K) 0 STANDARD 20
3 0 4
1255
V O L U M E 25, NO. 8, A U G U S T 1 9 5 3 the suspended material will settle in a V-channel. (Two boards nailed a t 90' to form a V-trough can he used to support a series of jars in this manner.) When it appears that no further sedimentation will occur, siphon out most of the supernatant water, leaving about 2 inches of water above the V. Combine the samples that lend themselves to compositing (optional, and depending on preliminary results), and let them stand as before for further settling and further siphoning, this time to within l / q inch from the top of the solids layer. Shake the jar samples well and transfer to evaporating dishes or large Petri plates. Rinse each sample jar several times with reagent grade acetone, using about 25 ml. for each rinse. U s e an all-glass pressurized wash bottle to dispense the acetone while the jar is being rotated on a vertical axis. When the samples are sufficiently dry, weigh a suitable amount into an extraction thimble or filter paper envelope, and extract overnight in a Soshlet or Goldfisch extractor, using Skellysolve F (Skelly Oil Co., Tulsa, Okla.) as extracting solvent. Transfer the extract to large test tubes and, after adding a glass bead, boil off the solvent. Redissolve the residue in reagent-grade carbon tetrachloride and process as indicated in the latter part of the water analysis section discussed previously.
Table I.
DDT Recovered from DDT-Water Standards
DDT-H10 Standard Alean Standard DDT:H 0 D D T added, Recovery, Deviation, ratio ( X 106) micrograms % hIicrograms Interferences absent (1949) 1:25 1:50 1:lOO 1:150 1:200
93.W 93.3Q 85.0" 85.OU 83.3= i) Interferences present (1950)
40 20 10 6.7
2.6 1.6 0.6
0.58 0.29
1:50 30 74.3: 1:75 20 65.8 1:54 1:150 10 62.sc 1.14 1:250 6 54.2c 0.66 1:3i5 4 50.0b .. a Bverage of triplicate determinations of 1-liter samples. b .4verage of duplicate determinations of 1.5-liter samples. C Average of triplicate determinations of 1.5-liter samples.
Coefficient of Variation,
% 6.5 8.0 5.0 8.6 5.8
7:i 11.4 11.0
Table 11. Efficiency of Recovery- of DDT from Suspended Solids Containing Interfering Compounds D D T .4dded, Micrograms
Standard Deviation, Micrograms 0.29 0.86 0.29 2.0
Mean Recovery, 70"
i o .8 75.0 Sl.? 86. J
4 6 10 20
Coefficient of Variation, % 7.25
14.5 2.9 10.0
Average of triplicate determinations.
For standard curve B , Figure 2, the optimal or most accurate portion, as determined by Ringbom's (10) method, is the 4 to 30 microgram range. The D D T load of a number of field samples was below 4 micrograms, and in their case standard addition was employed to augment with known magnitude the relatively weakabsorbing D D T solutions. The subsequent finding (6) that solids suspended in river water (clay, silt, very fine sand) can adsorb D D T explained how it could be transported for long distances under favorable conditions, and also suggested that determination of D D T directly from the suspended solids fraction would enable analysis of field samples containing suboptimal amounts of DDT. In the latter case, considerably larger samples of water could be processed as described previously, with substantial gain in sensitivity. To test the efficiency of recovery of D D T from suspended solids, known amounts of D D T in acetone solution were impregnated into 2.5-gram samples of suspended solids containing interferences. The recovery of D D T from samples so treated is shown in Table 11. Standard addition was used to assist measurement of the 4- and 6-microgram levels.
..
RE IOC
NEW, % 1
, RESULTS ARD DISCUSSIOR-
Table I presents data on efficiency of recovery of D D T added to untreated river water obtained at different times. Although the test waters were obtained from the same location, interferences in the first sample were negligible, whereas in the second sample, they were present in considerable amount. In Figure 2, data of Table I are plotted, with percentage recovery of D D T as a linear function of log concentration of D D T in DDT-lmter standards. Curve B was used in most of the held determinations (6). Figure 2 shows how the mean recoveries were plotted as correction curves. The vertical lines represent the range in the values obtained. (As an example of use of correction curve B, for instance, the mean recovery value for a load of 6 micrograms of D D T is 54.27,; thus the correction factor for that load is 100/54.2 = 1.85.) Figure 2 shows that the interference removal procedure loL5ei-s recovery values. On the basis of a limited number of observations, the level of substances that interfere with determination of D D T in water, as herein described, varies both between and n-ithin different years. I t is necessary to do pilot tests for the individual case. Such tests indicate whether elimination of interferences ie necessary and, if so, the influence of interference counteraction on recovery of D D T in the expected n-orking range. A correction curve can then be constructed whereby the D D T loads as f o n d in a given effluent are corrected to a theoretical 100% recovery and thus are equated to the presumed D D T concentrations. Table I shows that there can be appreciable relative error in this particular method of estimating presumed concentrations. However, it is reasonable to suppose that agreement of & l o to 15y0 might be acceptable for field purposes, in view of the small D D T to water ratios encountered in practical situations.
80
60
40
lo
*O I
2
Figure 3.
Correction Curve for Microdetermination of DDT in Suspended Solids
4
6
20
3040
Data of Table I1 are plotted in Figure 3 with percentage recovery of D D T as a linear function of log concentration of D D T in DDT-suspended solids standards. Vertical lines represent ranges of recoveries obtained. This log-linear plot of average recovery as a function of D D T concentration is shown as a correction curve. Using Figure 3, the D D T to water ratio calculated for a given sample on the basis of the suspended solids content was corrected for loss of recovery. This indirect calculation of D D T to water ratios from BUSpended solids of suboptimal samples (DDT concentration less
1256
ANALYTICAL CHEMISTRY
than 0.003 p.p.m.) is predicated on the assumption that D D T is quantitatively sorbed by the solids. In exploratory tests (6) of the assumption, quantitative sorption of DDT by suspended solids was not obtained. The quantity sorbed was influenced by the amount and the nature of the solids-i.e., different physical properties resulting from different amounts of clay, silt, and very fine sand, different sedimentation rates, etc. Ability of sui+ pended solids to adsorb microquantities of D D T from river water will no doubt vary with the individual case. Although the experiments on synthetic samples failed to show quantitative sorption by the silt, the procedure described may nevertheless be valuable in obtaining some approximate data for D D T concentrations in samples taken a t long distances-e.g., 68 miles ( 6 ) from the point of application of the DDT. In such cases the solvent extraction procedure is not practicable because of the very minute amounts of D D T in the water, and the photometric error becomes large.
ACKNOWLEDGMENT
The author wishes to thank C. R. Twinn, H. E. Gray, and -4. P. Arnason, Division of Entomology, for cooperation; B. N. Smallman, now of the Science Service Laboratory, London, Ont., for completing the arrangements whereby this aspect of the black fly problem was explored; J. W. T. Spinks, Department of Chemistry, University of Saskatchewan, and members of his staff, for advice and excellent working conditions in the laboratory supplied for the investigation; and W. A. E. McBryde, Department of Chemistry, University of Toronto, for very helpful criticism of the manuscript. REFERENCES
(1) Archibald, R. h l . , ANAL.CHEW,22,639 (1950). (2) Arnason, -1.P., Brown, A. W. d.,Fredeen, F. J. H., Hopewell, W. W., and Rempel, J. G., Sci. Agr., 29, 527 (1949). (3) ;lyres, G. H . ,AK.\L. CHEM.,21, 652 (1949). (4) Clifford, P. A . , J . Assoc. Ofic.Agr. Chemists, 28,152 (1945). (5) Davidorv, E., Ibid., 33, 130 (1950). (6) Fredeen, F. J. H., Arnason, A. P., Berck, B., and Rempel,
SUMMARY
J. G., Can. J . Agr. Sci., in press.
D D T in river water (as low as 0.003 p.p.m. of DDT) and in suspended solids wag determined, using solvent extraction and the Schechter-Haller procedure. Interferences in the extract were removed by Davidow's chromatographic method. I t was found that solids suspended in river water (clay, silt, very fine sand) adsorbed DDT. In samples containing suboptimal amounts of DDT, for whirh the regular solvent extraction of water was not feasible, DDT was determined directly from the suspended solids fraction. Larger samples of water may thus be processed with gain in sensitivity.
(7) Herriott, R. M., Science, 104, 228 (1946). (8) Lowry, 0. H., and Bessey, 0. S., J . Bid. Chem., 163,633 (1946). (9) MoBryde, W. A. E., . ~ N A L . CHEM.,24, 1639 (1952). (IO) Ringbom, A., Z . anal. Chem., 115, 332 (1939). (11) Schechter, M. S..Pogorelskin, M. A,, and Haller, H. L., ANAL. CHEX, 19,51 (1947). (12) Schechter, M. S., Soloway, S. B., Hayes, R. A., and Haller, H. L., IND.E m . CHEN.,ANAL.E D . , 17, 704 (1945). (13) Stiff, H. a., and Castillo, J. C., Science, 101, 440 (1945). RECEIVED for review October 17, 1952. hccepted March 9, 1953. Contribution 3021, Dix-ision of Entomology, Science Service, Department of Agriculture, Ottawa, Canada.
Quantitative Insolubility of Thorium Oxalate HAROLD L. KALL
AND
LOUIS GORDON
Departriient of C h e m i s t r y , Syracuse Cnicersity, SyraczLse 10, V. I-
u
THE
course of a study of coprecipitation with thorium
I-oxalate, some doubt arose as to the quantitative insolubility (2, 6, 9) of this salt, since the thorium content of a solution analyzed by an oxalate procedure ( I O ) was found to be consistently less than the value obtained by the hexamine method (3). This apparent solubility loss of thorium oxalate in dilute acid solutions was previously reported by Rider and Mellon in a paper describing a colorimetric method for thorium ( 5 ) . Although these authors did not completely describe the conditions under which the solubility loss was obtained, they reported that 0.1 mg. of thorium remained in 20 ml. of solution a t pH 0.7. They further reported that quantitative precipitation of thorium oxalate could be obtained in solutions of pH 0.7 to 3.0 without the use of a large excess of oxalate. Since oxalate is widely used for the determination of thorium, an investigation of the solubility losses of thorium oxalate in dilute acid media was undertaken. A recent sensitive and accurate method for the colorimetric determination of microgram quantities of thorium (8) was utilized in the analysis of filtrates. APPARATUS AND REAGENTS
Thorium Nitrate Solution Solutions were prepared by dissolving pure thorium nitrate (Lindsay Chemical c~.,. Code 103) in distilled water, adding 100 ml. of concentrated nitric acid, and then diluting to 1 liter. These solutions contained approximately 68 mg. of thorium oxide per 50 ml. l-(o-Arsonophenylazo)-2-naphthol-3,6-disulfonicacid. A 0.1 % reagent solution was prepared by dissolving 0.1 gram of this reagent (marketed as Naphtharson by the SmithaNew York Co.) in 100 ml. of distilled water. Lanthanum Perchlorate Solution. A solution was prepared by dissolving lanthanum oxalate (Lindsay Chemical Co., Code
518) in a mixture of nitric and perchloric acids, evaporating to fumes of perchlorate, and then diluting to 1 liter. The solution contained 20 mg. of lanthanum oxide per milliliter. Dimethyl Oxalate. The Matheson Go. product was used. Hexamine. Hexamethylenetetramine, The Matheson Co. product, was used. All other chemicals were C.P. .4 Beckman Model B spectrophotometer with 1-cm. Corex cells was used to measure the transmittancies of the thorium solutions. ANALYSIS O F FILTRATE FOR THORIUM CONTENT
In each case, except where otherwise noted, 68 mg. of thorium oxalate were precipitated as oxalate in a 250-ml. volume as described, and the precipitate was then filtered with S o . 42 Whatman filter paper. The filtrates were evaporated to dryness with nitric acid, the residue was dissolved with nitric acid, a suitable aliquot was evaporated to dryness with perchloric acid, and the residue was analyzed by the procedure of Thomason, Perry, and Byerly (8). RESULTS
Effect of pH on Solubility Loss of Thorium Oxalate Precipitated from Homogeneous Solution. Solutions containing 68 mg. of thorium oxide were diluted to approximately 225 ml., then adjusted to various pH values using ammonia or hydrochloric acid. The solutions xere diluted to 250 ml., and 1 gram of dimethyl oxalate was added. These solutions were heated for 45 minutes at 70" C. Subsequently, 7 grams of oxalic acid dihydrate were added, and the solutions were allowed to digest at room temperature for 12 hours. The results of these experiments are summarized in Table I. Effect of Variation of Oxalate and Thorium Concentrations on Solubility Loss. Precipitations were carried out, except for
