Quantitative Determination of Isomers of Q,O-Diethyl Ethylmercaptoethyl Thiophosphate KENNETH GARDNER
AND
D. F. HEATH
Pest Control, Ltd., Harston, Cambridge, England Pure 0,O-diethyl 0-ethylmercaptoethyl thiophosphate (I) prepared in the laboratory showed different physical and toxicological characteristics from those of the active constituent of the insecticide Systox, which is stated to be this compound. In view of the typefact that many compounds of the S=P-0 e.g., parathion-are known to be thermally unstable, it was presumed that the discrepancies were due to partial isomerization of the commercial product. Concurrent partition chromatography of a mixture of radioactive preparations (32P)of pure 0,O-diethyl 0-ethylmercaptoethyl thiophosphate and 0,O-diethyl S-ethylmercaptoethyl thiophosphate (11), with the active ingredient isolated from Systox demonstrates the presence of both I and I1 in Systox. It is also shown that I isomerizes to I1 on heating. A chromatographic method of analysis is described for determination of the two active ingredients in 0,O-diethyl (0,s)-ethylmercaptoethyl thiophosphate preparations. The analytical identification technique described-concurrent partition chromatography of the unknown sample with radioactive components of established structure and puritymay find more general application, particularly to and O=P-Stypes. preparations of the S=P-0
T""
active constituent of the insecticide Systox (3, 6, 20) is stated to he 0,O-diethyl 0-ethylmercaptoethyl thiophosphate: 1
Systox, hoxever, is virtually insoluble in water (21), which is inconsistent (9) with its systemic properties ( 2 , 11); and its toxicity to insects and mammals (5, 17, 20, 21) was greater than the toxicity of 0,O-diethyl 0-ethylmercaptoethgl thiophosphate synthesized in thesc laboratories. Parathion (0,O-diethyl O-pnitrophenyl thiophosphate) is known to isomerize to 0,s-diethyl 0-p-nit.ropheny1 thiophosphate on heating ( B 4 ) , and the reaction S=P-0-+ O=l'-Sappears to be fairly general (6, 20). Parathion normally cont,ains a few per cent of the S-ethyl isomcr, which is more toxic to mammals (24) than the parent, compound and is a much more potent inhibitor of cholinesterase. Consequently, as a reliallle method of analyzing parathion had not been worked out, much of the early work on the toxicity and cholinesterase-inliibitiag poiver of parathion has been proved incorrect ( I , 4 ) . By analogy it was natural to suspect that Systox contained either or both of the two isomers
0,O-Diethyl S-ethylmercaptoethyl thiophosphate
0,s-Diethyl O-ethylmercaptoethyl thiophosphate
or even that the active constituents might consist wholly of these isomers, for the patents describing the methods of preparation ( 7 ) contain no proof of constitution, and the isomerization may go
to completion under the reaction conditions used in manufacture.
-4method for determining the various isomers of Systox was therefore prerequisite of further work; the method should also distinguish between the methyl and ethyl homologs, for example, as a mixture of 0,O-diethyl- and 0,O-dimethyl 0-p-nitrophenyl thiophosphates was for a time distributed by one concern as parathion (14),the name normally given only to the diethyl analog. This paper describes the development of an analytical technique for determining the insecticidal constituents of Systox on lines which are of general application to the analysis of such mixtures. 0,O-DIETHYL S-ETHYLIMERCAPTOETHY L THIOPHOSPHATE
Preparation. Diethoxychlorophosphine oxide, (EtO),POCl, was prepared from phosphorus trichloride containing the radioactive isotope of phosphorus, alp, by a modification of the method of McCombie, Saunders, and Stacey (12). 2-Hydrosyethylethyl sulfide, HOCZH4SCzH6,was prepared by the method of Meyer (15) from ethylene chlorohydrin and sodium mercaptide. The fractionated product was a typical alcohol, with a sulfur content, determined after sodium peroxide fusion, of 30.0%, against 30.2% theoretical. This was converted t o 2-mercaptoethylethy1 sulfide, HS.C2H,SC2H,, by the method of Frank and Smith (8). The -SH groups in the product after fractionation were determined by iodine titration ( 2 2 ) ,and gave a titer 98% of theory. The sodium salt of the mercaptan was reacted with diethosychlorophosphine oxide in toluene a t 60' C. for 1 hour, (Et,0)2POCI
+ NaSC2H4SC2H5+
+ NaCl
(EtO)2PO(SC2H4SC2Hi)
The product, washed tmice nith i n t e r , was fractionated; 107110" C. a t about 0.5 mm. Twenty grams of phosphorus trichloride yielded 10 grams of fractionated product. This contained a few per cent of phosphorus compounds, extractable by water from iso-octane, which seem to be produced by oxidation. These were removed by dissolving in iso-octane, washing twice with wdcer. and sucking off as much iso-actane as possible a t 100" C. under I-mm. pressure. This product was used for further work. The photometric method of Sumner (43) showed it to contain 70% product. the remainder being iso-octane.
Proof of Structure. The product can be hydrolyzed in alkaline solution to give a high yield of 2-mercaptoethylethyl sulfide. Neither the 0-S-diethyl 0-ethylmercaptoethyl thiophosphate nor O,O-diethylO-ethylmercaptoethy1 thiophosphate can liberate this compound, whereas 0,O-diethyl S-ethylmercaptoethyl thiophosphate can in principle yield either 2-mercapto- or 2-hydroxyethylethyl sulfide, according to 11hich of the two bonds in the P-S-C system is broken during hydrolysis. The liberation of the mercapto compound is thus positive proof of structure, and incidentally shows which bond is broken. The test was so designed that ethyl mercaptan (ethanethiol) could be determined also, were it liberated, as this similarly would indicate the presence of the 0-8 diethyl 0-ethylmercaptoethyl thiophosphate. The purity of the product was also tested chromatographically. Liberation and Determination of Mercaptans. A weighed sample, approximately 1 gram, was hydrolyzed in 100 ml. of IN sodium hydroxide overnight. The hydrolysis rate is rapid, 0.81 (OH-) min.-' a t 25" C., but was slowed somewhat in this instance by the resence of iso-octane, which rovided a second phase in which 8,O-diethyl S-ethylmercaptoe%yl thiophosphste
1849
ANALYTICAL CHEMISTRY
1850
is markedly more soluble than in 1.V sodium hydroxide The solution was acidified to about pH 3 with concentrated hydrochloric acid, and extracted three times with 30 ml. of chloroform. Any mercaptans liberated by hydrolysis were now in chloroform solution. This was carefully fractionated, and the first 10 ml. collected in methanol. As ethyl mercaptan is much more volatile than chloroform, any ethyl mercaptan would appear in this fraction, n-hile any 2-mercaptoethylethyl sulfide, boiling point 188" C., would remain in the residue. The residue was further concentrated to 10 ml.
W
30,000
25,000
E
E z 3
20,000
8
-
5
4
dn E
25
3 P0.000 -
50
75
ML. ELUTED
s+*
Figure 2. Chromatogram of 0-0-Diethyl 0Ethylmercaptoethyl Thiophosphate
t 3 W
I 0
PO
40
60
70
ML. ELUTED
Figure 1. Chromatogram of 0-0-Diethyl SEthylmercaptoethl-l Thiophosphate
I t was shown in a preliminary experiment that less than 5% of the 2-mercapto-compound would evaporate in this process. The -S-H groups in the distillate and residue were then determined by the iodometric method described by Siggia ( g 2 ) . with the modification that sufficient acetone-free methanol was introduced to homogenize the two-phase system produced by running standard iodine into chloroform. This modification was shown not to affect the results with pure thiol. The distillate contained no mercaptan, but the residue contained high boiling point mercaptan equivalent to 94% of the thiophosphate present in the original sample, The discrepancy of 6% is doubtless partly accounted for by some evaporation during the concentration of the chloroform residues, but may also indicate that the S-C bond is ruptured to some extent during the hydrolysis. Chromatographic Test of Purity. h small quantity of product in iso-octane was subjected to partition chromatography, using methanol-iso-octane (18) on kieselguhr ( I S ) . The iso-octane eluent vas assayed for radioactivity in a Geiger-Muller liquid counting tube with the usual electronic high tension ond scalar setup. All the phosphorus appeared in one sharp band, nith no sign that more than one compound was present. The chromatogram is reproduced in Figure 1. These tests, therefore, proved the compound to be 0,O-diethyl 8-ethylmercaptoethyl thiophosphate. 0,O-DIETHY L 0-ETHY LMERC4PTOETHY L THIOPHOSPHATE
This was prepared from thiophosphoryl chloride containing 32P by the reactions:
+ +
+
PSCIS ZNaOEt +(Et0)2PSCI 2NaCI (Et0)2PSC1 NaOC2H4SGHs + (Et0)2PS(OC2H4SC2Hj) NaCl
+
(1)
(2)
The second reaction u-as performed by refluxing in xylene for 4 hours. After two mater washings the product was fractionated:
(boiling point 92" C. under a!iout 0.5 mm. of mercury pressure) yield approximately 60%; on thiophosphoryl chloride. Proof of Structure. The product was very much less soluble in water, by a factor of about 100, than 0,O-diethyl S-ethylmercaptoethyl thiophosphate. The S-ethyl isomer is likely to have physical properties similar to 0,O-diethyl S-ethylmercaptoethyl thiophosphate, especially as regards solubility in water. An infrared spectrograph shorred absorption in the wave-length band expected for P=S. Chromatographic Test of Purity. A partition chroma togram. performed as above, shoved only one major band, with a small subsidiarj- band, corresponding to about 5% of the total phosphorus, which was due to 0,O-diethyl S-ethylmercsptoethyl thiophosphate. The chromatogram is reproduced in Figure 2 All the evidence therefore pointed to the product of Reaction 2 being 0,O-diethyl 0-ethylmercaptoethyl thiophosphate. Isomerization of 0,O-Diethyl 0-Ethylmercaptoethyl Thiophosphate. It appeared likely that 0,O-diethyl O-ethylmercaptoethyl thiophosphate would isomerize on heating. FThen some 0,O-diethyl 0-ethj-lmercaptoethyl thiophosphate was heated to 130' to 145" C. for 2.5 hours, and the product was hydrolyzed, it was found by the methods described earlier, that high boiling point mercaptan equivalent to 68% of the original sample was liberated, but no ethyl mercaptan could be detected. Only 78% of the sample after heating and prior to hydrolysis could be extracted from water by iso-octane, so under these conditions other reactions besides isomerization must occur. The production of high boiling-point mercaptan is not in itself a proof of the isomerization of 0,O-diethyl 0-ethylmercaptoethyl thiophosphate to 0,O-diethyl S-ethylmereaptoethyl thiophosphate, as it may have been formed after the thermal decomposition of the parent substance. It was therefore necessary t o confirm the result by some other method. At this stage of the investigation a suitable method of chromatographic analysis had not been devised. A heated sample of 0,O-diethyl 0-ethylmercaptoethyl thiophosphate was therefore subjected to a sequence of successive partitioning (IO) between iso-octane and 90% methanol-10% water (by volume), the partition ratios of the radioactive phosphorus being determined after each extraction. If only one compound is present, all the ratios will be the same; if two or more of different partition coefficients are present, the observed over-all partition ratios will change in a systematic fashion. From the changes observed, the quantities and partition coefficients of the individual compounds present can be estimated by arithmetical trial and error. I t was found that the partition ratios observed could be accounted for on the assumption that two compounds were present, 29.2% with an iso-octane-aqueous methanol partition ratio of
V O L U M E 25, N O . 1 2 , D E C E M B E R 1 9 5 3 1.06, and 70.8% with one of 0.101. These compare with 1.08 and 0.101 determined independently for 0,O-diethyl O-ethylmercaptoethyl thiophosphate and 0,O-diethyl S-ethylmercaptoethyl thiophosphate, respectively. The small difference between 1.08 and 1.06 is not significant, as the 0,O-diethyl O-ethylmercaptoethyl thiophosphate coefficient is highly sensitive t o the proportion of water used in the methanol, and it is difficult t o keep this precisely constant during the numerous transfers. It is therefore established that 0,O-diethyl O-ethylmercaptoethyl thiophosphate on heating is converted substantially to 0,Odiethyl S-ethylmercaptoethyl thiophosphate. The errors in the observed partition coefficients should not exceed 2.5%. Some of the calculated values differ from the observed ones by more than this, and in a systematic fashion, suggesting that a small proportion of the activity, not eweeding 5%. is present in other compounds. Experimental. A sample of pure radioactive 0,O-diethyl 0ethylmercaptoethyl thiophosphate was heated to 125' to 130" C. for 3 hours. The product was dissolved in iso-octane, and the isooctane extracted successively with an equal volume of 90% methanol-10% water (v./v.) mixture. The SZP count in both layers was determined as before after each extraction. The first methanol extract was similarly extracted with iso-octane. The observed partition ratios, and those calculated for a mixture of 29.2% 0,O-diethyl 0-ethylmercaptoethyl thiophosphate and 70.8% 0,O-diethyl S-ethylmercaptoethyl thiophosphate, are compared in Table I. The percentages and the iso-octaneaqueous methanol partition ratio of 1.06 for 0,O-diethyl 0ethylmercaptoethyl thiophosphate were chosen as best fitting the observed results.
1851 evidence that the radioactive and nonradioactive compounds were the same.
Experimental. A column was prepared from 20 grams of kieselguhr (Hyflo Super-cel, Johns-Manville & Co., Artillery Row, London E. 13) mixed with 12 ml. of methanol, packed in 1.2-cm. tubing according t o the method of Martin (16). Standard solutions of radioactive 0,O-diethyl S-ethylmercaptoethyl thiophosphate, 0,O-diethyl 0-ethylmercaptoethyl thiophosphate, and the constituents of Systox freed from wetting agent were prepared in iso-octane, and mixed in known proportions. Then 1 ml. of the composite iso-octane solution was placed on the column. This aliquot contained 1.16 mg. of radioactive C,Odiethyl 8-ethylmercaptoethyl thiophosphate, 0.26 mg. of radioactive 0,O-diethyl 0-ethylmercaptoethyl thiophosphate, and extracts from 20.36 mg. Systox. The last contained phosphorus equivalent t o 48% 0,O-diethyl 0-ethylmercaptoethyl thiophosphate according to the authors' analysis, against the 507 stated on the label.
Table 11. Properties of 0,O-Diethyl 0-Ethylniercaptoethyl Thiophosphate (a) and 0,O-Diethyl S-Ethj-lmercaptoethyl Thiophosphate ( b ) Very low
3Iisc.
(bj Circ. 2000 p.p.m. Misc.
Veri laree VPI; large 1 63 (18" C . )
AQDrOX 1.5' V&Y large 0.161 (18' C.)
1 . 0 8 (18' C . )
0.101 (18" C . )
(a)
Solubility in water Solubility in methanol Partitioii ratios Iso-octane-water Chloroform-water Cyclohexane-90% l I e O H , 10% H20
. ., . .
Iso-octane-gO% AIeOH. 10% Hz0 v./v.
Half life 1S S a O H
75 inin. (25' C. in 0 85 min. (29" C. 50% EtOH) 550 min. (82.3' C.) 330 min. (84 5 O C. in 50% EtOII) a The partition ratio of 0,O-diethyl S-ethylmercaptoethyl thiophosphate between iso-octane and water proved difficult to measure, because the compound in the aqueous layer tended to stick to the vessel. Values ranging from 8 to 20 have been obtained by radiotracer assay. the lower values being found for very dilute solutions. I t is crobable that the partition coefficient is highly sensitive t o concentration.
I S HCl
Table I. Partition Ratios for Heated 0,O-Diethyl 0Ethylmercaptoethgl Thiophosphate between Aqueous Methanol and Iso-octane s o . of Extract
Partition Ratio, Iso-octane-Methanol Iso-octane Extracted with First Methanol Extract 90 MeOH/10 H10 v./v. Extracted with Iso-octane Obsd. Calcd. Obsd. Calcd.
PHYSICOCHEMICAL PROPERTIES OF 0,O-DIETHYL 0-ETHY LMERCAPTOETHY L AND 0,O-DIETHYL S-ETHYLMERCAPTOETHYL THIOPHOSPHATE
Some typical physicochemical properties of the compounds were determined to aid later identification and in a search for methods of anal? sis. They are given in Table 11.
0
30
PROOF OF CONSTITUTION OF SYSTOX
To show that the active ingredients of Systox are 0,O-diethyl S-ethylmercaptoethyl thiophosphate and 0,O-diethyl O-ethylmercaptoethyl thiophosphate, pure radioactive samples of 0,Odiethyl S-ethylmercaptoethyl thiophosphate and 0,O-diethyl 0ethylmercaptoethyl thiophosphate were chromatographed on a kieselguhr met hanol-iso-octane column concurrently with a sample of the active ingredients of Systox separated from wetting agent. The radioactive phosphorus and total phosphorus in the eluent fractions were estimated. The method of separation from wetting agent and the method of estimating total phosphorus are given in detail below. As the quantities of the radioactive samples placed on the column were much smaller than the Systox sample, the total phosphorus was used to estimate the latter and the radioactivity the former. TROchromatograms were thus run simultaneouslv on the same column, and the identity in shape and position of the total phosphorus and radiophosphorus bands was
60 ML. ELUTED
90
Figure 3. Chromatogram of Systox Plus Radioactive 0-0-Diethyl 0-Ethylmercaptoethyl Thiophosphate and 0-0-Diethyl S-Ethylmercaptoethyl Thiophosphate
--- -.-I
Total phosphorus azp Both indistinguishable
Total phosphorus and radioactive phosphorus were determined in 3-ml. eluent fractions. The errors on the former were about &1.570 and of the latter less than =k3% (1000 counts total) except near the feet of the peaks, ivhere the errors were greater, as the quantities m-ere too small for accurate analysis. The chromatogram is reproduced in Figure 3. The concentration units are arbitrary, for it has been arranged that the radioactive and total phosphorus bands are of equal height a t the points of highest concentration to bring out the similarities most clearly. Interpretation of Chromatogram. The phosphorus recovered
ANALYTICAL CHEMISTRY
1852 from the column totaled 1.27 mg., while 1.34 mg. were introduced. The peaks occur in the same position, and their shape is in fairly good agreement, especially for 0,O-diethyl O-ethylmercaptoethyl thiophosphate. In both bands the 3*Pfalls off faster than the total phosphorus, owing t o traces of impurity in the commercial product. This agreement is strong evidence, but not proof, that Systox is predominantly 0,O-diethyl O-ethylmercaptoethyl thiophosphate and 0,O-diethyl S-ethylmercaptoethyl thiophosphate. The solubilities of isomers containing P-S-C bonding are likely to be very similar, so that the chromatograph may not distinguish between 0,O-diethyl S-ethylmercaptoethyl thiophosphate and the 0-8-diethyl O-ethylmercaptoethyl thiophosphate. The substitution of methyl for ethyl groups changes the solubility heavily in favor of the polar phase, so, while a single chromatogram is not sufficient to prove the absence of the Sethyl isomer, it is sufficient to show that the radioactive and commercial compounds do not differ in any other way. The subsidiary band appearing only on the total phosphorus chromatogram may be due to other such compounds, but the small proportion of total phosphorus this band represents suggests that it is due to an impurity accidental from the method of manufacture. I n the study of partition coefficients of Systox between several pairs of solvents, no pair suitable for partition chromatography was found dissimilar enough from methanol-iso-octane to lend any hope that the radioactive and nonradioactive peaks would be separated, even were they of different compounds. Further evidence had therefore to be adduced from other sources. The Bayer patents ( 7 ) associated with Systox describe reactions which are formally identical with those used by the authors to produce 0,O-diethyl S-ethylmercaptoethyl thiophosphate and 0,Odiethyl 0-ethylmercaptoethyl thiophosphate, which have been shown to produce the stated products. This, combined with the chromatographic findings above, establishes with reasonable certainty the constitution of the insecticide. ASALYSIS OF 0,O-DIETHYL S-ETHYLMERCAPTOETHYL A S D 0,O-DIETHY L 0-ETHY LMERCAPTOETHYL THIOPHOSPHATE
The analysis may be performed by a slight modification of the chromatographic technique. used for the identificstion cf the phosphorus-containing compounds in Systox. T t e active ingredients we frst separated from wvtting agent and chromatogrsmed. and tlie eluent iractioni anslyzed for phosphorus. Separation from Wetting Agent. The active ingredients of B816Y cou1J he vstractrd b!. tlit.< t-srr:ictioiis with isopropJ.1 ether, t h e ;ictive compounds appe:,ri:>g3viriuslly ircc of wetting agerrt, i n thc eth1.r. . KtJllo\\3: 1 5
Dilute with 10 volumes oi w>irer, 8h:ike the sample with 30 volumes oi iso-octone, and centriiuge. Remove the iso-octane with a pipet, and eitr:>ct successivrly n i t h five more 30-volumc quantities oi iso-ocriine. L\.npor:ltc’ the iw-wtaiie under vacuum to LI volume suitalilt. l o r chroinatograph!.. The isooctane extract seems to be free oi wetting agent. This stage is omittcd \\hell the niaterial to br nnal>-zcdconwins 110 w t t i n g agent or other d d i r i v e . Chromatographic Analysis. l’ack a column of 1.2-cni. diameter \villi 30 gt’ams o i kieselguhr nii\ed \\it11 12 nil. of nietlianol under iso-octane. R u n n.ith iso-octanc approximately satura;ed uith nictliai~oluirl\.. II’ the iso-ocraiie is fully saiuquecwcl iron1 :he colutrin, and the rated, methanol tends l o IJC bands are not so clear. Figure 3 shows thnt, ior an approximate analysie, it is necessary to take only three fractions: one t o contain the 0,O-diethyl 0-ethyliiie~c~ptoetli~l thiophosphate, one between the lmids to check the,procedure, and n large final fraction. If on accurarr nnol!.sis 15 required, it is neccseary to take scveral eluent frLictioiis on the descending side of the 0,O-diethyl S-cthylnierc:cptoetli~~l thiophosphate band. to obtain separation from the niiiior ingredients \vhich follow 0,O-diethyl S-ethylnierwptoethyl thiophosphate through the column. The eluent fractions shoulJ be concentrated carefully to about 10 ml. and treRtcd as brlon. brfore proceeding to the anolysis for phosphorus (19, 23,.
Method of Estimating Fractions. REAGENTS. Bromine water; sulfuric acid, 7.5-V; hydrogen peroxide, 100 volume; saturated sulfur dioxide water; ammonium molybdate, 66 grams per liter; ferrous sulfate, 5 grams of ferrous sulfate heptahydrate plus 1 ml. of 7.5N sulfuric acid to 50 ml., freshly prepared. To the eluent fraction add 3 to 4 ml. of bromine water and 5 ml. of 7.5N sulfuric acid. Remove the iso-octane by careful evaporation on the hot plate, adding bromine water dropwir;e to maintain an excem. Continue the evaporation until fumes of sulfuric acid appear, cool, and add 2 to 3 drops of hydrogen peroxide to destroy any organic matter. Boil to decompose most of the hydrogen peroxide. Cool, dilute with water to about 10 ml., add a few drops of sulfur dioxide water, and boil to remove excess sulfur dioxide. Transfer the cooled solution to a standard 50-ml. flask, dilute to 40 ml. and add 5 ml of ammonium molybdate with shaking, followed by 4 ml. of ferrous sulfate. ilfter shaking, make up the solution to 50 ml., and measure absorption against a reagent blank on a spectrophotometer a t 720 mp. (-4potassium dihydrogen phosphate calibration curve showed only slight deviation from linearity up to 80y of phosphorus in 50 ml.) DISCUSSION
The isomers of 0,O-diethyl (0,s)-ethylmereaptoethyl thiophosphate have been separated and determined using partition chromatography. This type of technique is very useful for the analysis of preparations of the S=P-0type, which are often thermally unstable and therrfore extremely difficult to prepare in a state of high purity. In particular, the technique of concurrent partition chromatography of the “unknon-u” sample with radioactive compounds of established structure and purity may find more general application in the identification of homers of the S=P-0and 0=P-Stypes. Discussion. I n analyses on commercial products Bayer 8169 and Systox were found to contain approximately 15% 0,Odiethyl 0-ethylmercaptoethyl thiophosphate, 15% 0,O-diethyl S-ethylmercaptoethyl thiophosphate, and 18% 0,O-diethyl 0ethylmercaptoethyl thiophosphate, 30% 0,O-diethyl S-ethylmercaptoethyl thiophosphate, respectively. The presence of such high proportions of 0,O-diethyl 8-ethylmercaptoethyl thiophosphate, which is about ten times more toxic to mammals (20) and insects (17) than 0,O-diethyl 0-ethylmereaptoethyl thiophosphate, adequately explains the insecticidal and toxicological properties of these formulations. ACKNOWLEDGMENT
The authors wish to thank L. J. Bellamy (Ministry of Supply) for the infrared spectroscopy. D. W. J. Lane prepared the radicactive samples by methods first used in these laboratories by D. W. Pound. Thanks are also due to F. M. Freeman and P. 0. Park for technical assktance, and to the directors of Peat Control, Ltd., for permission to publish this pape1. LITERATURE CITED
Mdridge, W. N., and Davison, A. N., Biochem. J., 52, 663 (1952).
Ashdown, D., and Cordner, H. B., J . Econ. Entomol., 45, 326 (1952). Deichmann, W. B., Brown, T., and Downing, C., Science, 116, 221 (1952). Diggle, W. H., and Gage, J. C., Biochem. J . , 49, 491 (1952). Dubois, K. P., and Coon, J. M., Arch. Ind. Hug. Occupational Med., 6 , 1 (1952). Emmett, W. A,, and Jones, H. O., J . Chem. SOC.,99, 713 (1911). Farbenfabriken Bayer, German Patent Applications 451, L57; 120, P41994D (1949); 120, F1406; 120, F1498; 120, F1499 (1950). Frank, R. L., and Smith, P. V., J . Am. Chem. Soc., 68, 2103 (1946).
Hartley, G. S., International Entomological Congress, Amsterdam, 1951. Heath, D. F.. Lane, D. W. J., and Llewellyn, bf., J . Sci. Food & Ag., 2, 60-73 (1952). Jeppson, L. R., Jener, M. J., and Complin, J. O., b. Econ. Entomol., 45, 669 (1952).
1853
V O L U M E 25, NO. 1 2 , D E C E M B E R 1 9 5 3 (12) McCombie, H., Saunders, B. C., and Stacey, G. J., J . Chern.
Sac., 1945, 380.
(13) Martin, A. J. P., “Biochemical Society Symposium on Partition Chromatography,” Cambridge, England, Cambridge University Press, 1949. (14) Martin, Hubert, J. Sci. Food & Ag., 1, 165, Col. 2 (1950). (16) Meyer, V.,Ann., 240,310 (1887). (16) Patterson, W. I., Food & Drug Administration, Washington, D. C., personal communication, 1962. (17) Pest Control, Ltd., unpublished work. (18) Ramsey, L. L.,and Patterson, W. I., Assoc. Ofic.Agr. Chemists, 31, 139 (1945).
(19) Rockstein, M.,and Herron. D. W., ANAL.CHEM.,23, 1500 (1951). (20) Sohrader, G., “Die Entwicklung. neuer Insektizide usw.,” Berlin, Verlag Chemie, 1952. (21) Scott, R. C., and Wagner, W. E., Annual meeting, American Society of Economic Entomologists, Cincinnati, 1951. (22) Siggia, S., “Quantitative Organic Analysis via Functional Groups,” New York, John Wiley & Sons, 1949. (23) Sumner, J. B.,Science, 100,413 (1944). (24) Topley, B.,Chemistry & Industry, 1950, 5559. RECEIVED for review December 16, 1952.
.4ccected July 31. 1953.
Determination of Zinc with Special Reference to Separation from Cobalt and Copper A. E. MARTIN Division of Soils, Plant and Soils Laboratory, Commonwealth Scienti3c and Industrial Research Organization, Brisbane, .4ustralia The extraction of zinc from citrate solutions at pH 5 to 10 with di-2-naphthylthiocarbazone and sodium diethyldithiocarbamate is described. A t pH 9.8 negligible amounts of zinc are extractable with sodium diethyldithiocarbamate. In presence of both reagents, zinc is extracted practically entirely in the form of zinc di-2-naphthylthiocarbazone from pH 7 to 9.5. Extraction of copper as the complex of di-2-naphthylthiocarbazone or sodium diethyldithiocarbamate from pH 1 to 10 showed irregularities which were considered due to nonattainment of equilibrium conditions. Extraction of cobalt with di-2-naphthplthiocarbazone and sodium di-
T
HIS work was undertaken as part of a project designed t o
investigate the status of zinc in black soils on the Darling Downs, Queensland, as for some time certain pasture species have been showing symptoms characteristic of zinc deficiency ( 1 ) . An essential prerequisite of this program was the developmenf of a reliable method for the determination of total zinc in soils, based on a wet digestion procedure employing a sulfuric-perchloric acid mixture. Colorimetric methods for zinc are usually based on dithizone (Dz) and more recently on di-2-naphthylthiocarbazone (Dn) (5, 20).despite the fact that many other transition metals interfere. Turbidimetric methods using diethyldithiocarbamate ( 2 ) or ferrocyanide ( 4 ) show a greater lack of specificity. The use of the dropping mercury electrode affords the most specific and sensitive method for zinc over a wide range of concentrations. With careful choice of the basal solution most interferences can be eliminated. This is a great advantage when concentration of metals as dithizone or di-2-naphthylthiocarbazone complexes into organic solvents is undertaken, a procedure which is known to be only weakly selective. Cobalt is the most serious interference in the polarographic determination of zinc, since the half-wave potentials of these two metals are too close to permit accurate wave-height measurements. Although Prajzler (18) found that zinc could be determined polarographically in the presence of nickel, cobalt, and manganese by using a nearly saturated solution of ammonium oxalate as supporting electrolyte, trials conducted during the present study showed that the Rave form was ill defined and difficult t o measure with precision. Further work indicated that basal solutions composed of potassium citrate (acid or alkaline) plus fuchsin, neutral
ethyldithiocarbamate demonstrated that equilibrium is attained very slowly with this metal. The use of sodium diethyldithiocarbamate is unnecessary in preventing the interference of copper and cobalt in the estimation of zinc. The implications of these findings are discussed and a tentative method for determining zinc in dilute digests containing sulfuric acid is described. Satisfactory recoveries of zinc (97 to 102%) can be effected from citrate solutions at pH 9.8 in the presence of copper, cobalt, iron, phosphate, and titanium and in combinations of these in concentrations likely to be encountered in soils.
barium chloride plus gelatin, neutral calcium chloride plus methyl red, or the basal solution employed by Walkley (22) were unsatisfactory with regard to the successful resolution of the zinc and cobalt waves. I n other respects, Walkley’s solution is satisfactory as good separation of the zinc and nickel waves can be obtained. Furthermore, both zinc and cobalt are extracted from ammoniacal citrate solutions with either dithizone or di-2-naphthylthiocarbazone. Cobalt must therefore be separated chemically from zinc before the final polarographic determination. This is particularly important in analyses of soils or rocks in which the content of cobalt may be of the same order as the zinc content. [Kidson, Askew, and Dixon (12) report a range of 0.3 to 41 p.p.m. in New Zealand soils.] The ratio of zinc to cobalt in plants is normally large enough to preclude great errors in wave-height measurements. Holland and Ritchie (8) and Cowling and Miller ( 7 ) described procedures in which zinc could be determined in the presence of a number of other transition metals by the use of diethyldithiocarbamate (De) combined with dithizone. Cholak, Hubbard, and Burkey ( 5 ) modified the method of Cowling and Miller by employing di-2-naphthylthiocarbazone, which gives 100% recovery of zinc a t pH 9 to 10 in a single extraction compared with 70 to 90% using dithizone. Quantitative extraction a t high pH is important in the removal of zinc from silica (2%’). However, the results of Cholak et al. for extraction of the zincdithizone complex exaggerate this difference, probably because of the high concentration of citrate present (0.286211) in the solutions. The data of Walkley (21) refer to recoveries from 0.05M citrate solutions, a concentration more usually employed. A further
