857
V O L U M E 2 4 , NO. 5, M A Y 1 9 5 2 Table 11. Calibration Data for Determination of Dieldrin Concn.,
%
0,094 0,090 0.080 0.060 0 050 0.045 0,030
Av. Av. No. of Reading, Concn.. K O . of Reading, Readings Ratio A I R Readings Ratio A / B 3 1.234 0,025 3 1,058 3 1,223 0.0228 3 1.053 3 1,196 0 016 3 1.038 3 1.144 0.010 3 1 023 10 1.119 0 005 6 1.012 3 1.107 0.000 19 1,000 3 1.070 Standard deviation = 0.0037
In general, when a number of analyses are carried out in routine fashion three readings are taken per analysis. The probable error should be within &O.OOl% under routine conditions of analysis. ACKNOWLEDGMENT
The authors wish to thank John Wirth, John Streich, and
W. R. Miller for their assistance in collecting the expeGmental data. LITERATURE CITED
trations were prepared and a number of readings taken for each concentration. The accumulated data were then treated by the method of least squares and the probable error was calculated from the standard deviation. Aldrin. The calibration data pertinent to aldrin are tabulated in Table I, including the number of readings and the average value for each concentration. The individual readings rather than the averages were used in the actual statistical treatment. The standard deviation was 0.0016. The sensitivity was 0.000570 aldrin. Dieldrin. The (data collected for the calibration of dieldrin are presented in Table 11. Only the number of readings and the averages are listed. The standard deviation was 0.0037. The sensitivity was 0.0007c7, dieldrin.
Colthup et al., Rev. Sci Instruments, 18, 931 (1947). Danish, A. A,, Koenig, N., and Kuderna, J., “Treatment for Photometric Analysis of Biological Materials Containing Micro Quantities of Aldrin and Dieldrin,” Division of Agricultural and Food Chemistry, Symposium on Methods of Analysis for Micro Quantities of Pesticides, 119th Meeting of AMERICAN CHEMICAL SOCIETY, Boston, April 4, 1951. (3) Danish, A. A., and Lidov, R. E., Advances in Chem Series, No. 1, 190 (1950). (4) Heigl, J. J., Bell, >!I.F., and White, J. U.,ANAL. CHEM.,19, 292 (1947). (5) Lidov, R E., Bluestone, H., Soloway, 8.B., and Kearns, C. W , Advances in C h m . Series, KO.1, 175 (1950). ( 6 ) Wright, N., IND. ENG.CHEW., ANAL.ED.,13, 1 (1941). (1) (3)
RECEIVED for review April 26, 1951. -4coepted January 29, 1952. Presented before t h e Division of Agricultural a n d Food Chemistry, Symposium on Methods of Analysis for Micro Quantities of Peaticides, a t the 119th Meeting of t h e AMERICAN CHEMICAL SOCIETY, Boston, Mass.
Colorimetric Microdiffusion Determination of Chloride Application to Chlorinated Insecticides H. T. GORDON Division of En tornology and Parasitology, University of California, Berkeley 4, Calif.
.
This method is a simpler, faster, and more sensitive colorimetric modification of the Conw-ayniicrodiffusion analysis for chloride (0.3 to 3 micrograms of chloride, in 0.1 to 0.4 ml. of solution) and bromide. It is applicable to many organic compounds containing chlorine or bromine. Chlorine is liberated from organic compounds by direct permanganate oxidation, or by reaction with sodium-n-propoxide to form chloride. Chloride is oxidized to chlorine by permanganate. The chlorine formed diffuses into and quantitatively decolorizes a solution of the dye, Fast Green. Residues of 1 to 10 p.p.m. of chlorinated insecticides (such as chlordan, heptachlor, lindane, toxaphene, methoxychlor, and DDT) in biological material can be determined as alkali-labile chlorine.
08T micromethods for chloride ion are adaptations of argentometric macrotitration ( 13, 24), potentiometry (&?.),or turbidimetry (11). The Conway microdiffusion cell has also been used for chloride determination (4.). The chloride reacts with acid permanganate in the outer chamber of the cell, and chlorine diffuses into the inner chamber containing iodide solution; for 1 to 7 micrograms of chlorine, the method is colorimetric (starch-iodine complex.). X nioie sensitive colorimptiic micromethod for chlorine or bromine is now available, using the tiiphenylmethane dye, Fast Green (8). This paper describes the adaptation of Fast Green colorimetry to the Conway diffusion procedures for chloride and bromide, and the application of this new chloride method to the microanalysis of insecticides having alknli-labile or oxidant-labile chlorine. REAGE3TS
Fast Green FCF, 0.0167% in 0.05 -TI sulfuric acid. The voncentration is not critical, but designed so that 0.15 ml. will contnin 25 micrograms of dye. These are convenient values for the Connay cells and colorimeter tubes used in this n o r k .
Potassium permanganate, 67, in water. Sulfuric acid, c P. conrentrated, low in chloride. This is prepared by adding about 1 gram of C.P. chromic acid to 200 ml. of concentrated sulfuric acid and slon-ly heating to the boiling point in a fume hood, Sulfuric acid, 1 JI, prepared from the low-chloride concentrated acid by diluting approximately 5.4 ml. in water to 100 ml. Standard solutions roiitaining 5, 10, 15, and 20 micrograms of sodium chloride per nil in water. Standard solutions caontaining 10, 20, 30, and 40 micrograms of sodium bromide per nil. in water. Chromic acid, c.P., 20% (m./v.) in 30% (v./v.) sulfuric acid (low-chloride). The chloride is removed by slowly heating to the boiling point in a fume hood. Sodium n-propoxide, approximately 2 M in 1-propanol, is prepared by dissolving clean sodium metal in anhydrous 1-propanol (redistilled over sodium from Eastman Kodak No. 848). The solution contains chloride; most of this ill settle out on standing. The clear supernatant is siphoned off; this has a very low chloride blank. The solution gradually turns yellow, and becomes cloudy after repeated exposure to air, but this does not interfere with its analytical use. Perchloric acid, c.P., 72%. Toxaphene (chlorinated camphene), technical, 67 to 69% chlorine, from the Hercules Yon-der Co., JVilmington, Del.
858
ANALYTICAL CHEMrSTRY
Lindane (1,2,3,4,5,6hexachIarocycloh~ane),99% gamma isamer, from the California Spray Chemical Carp., Richmond, Calif. Chlordan (octaehloro-4,7-met.hanotetrahydroindane), technical, 63 to 65% chorine, from the Velsicol Corp., Chicago, Ill. This is a mixture of a t least five chemically similar components (iRi
Utes. The chloride prescrrt will have been oxidized to chlorine during the interval. and the evacuation ensures a verv ION rcagent-blank. 6 . Pipet 0.2 ml. of the acid-potsssium permanganate reagent quickly into the outer chamber of each Conway oell,pressimg its Lucite cover on firmly immediately. The volumetric memurement need not be nrecise; a 1-ml. Mohr Dinet. with a fine tin.
chlord%d DDT [2,2bis(p-chlorophenyl)-l,l,l-trichloroethan~], melting point 107' C. DDE [2,2bis(p-chlorophenyl)-l,l-dichloroethyl~ne],prepared from pure D D T hy refluxing with potassium hydroxide m ethanal, and recrystallieed from ethanol. TDE [2,2-bis(pehlorophenyl)-l,ldichlo~o~thane],purified sample, from Rohm & Haas Co., Philadelphia, Pa. Methoxychlor [2,2-his(p-methoxyphenyl)-l,l,l-trichlo~o~thanel, purified sample, from E. I. du Pant de Nemours & Co., Inc., Wilmington, Del. Aramite ( 8-ohloroethyl-8'-(p-tert-hutylphenoxy)-a'-methylethyl sulfite), distilled sample, from Naugatuck Chemical Division, U. S. Rubber Co., Naugatuck, Conn.
high lters tine
...the
in+""
C n
ganate reagent'dready pkiared, so that littlk time is Idst.
Figure 1. Automatic Koclting Shake:r with TwelveCell Holder on Four-Drawer Stoi age Cabinet Driven by synchronous electric motor a t irequen, 30 oscillations per minute. Used to aooelerat chlorine and!- bromine . " ~ in ~"Conyay ~" ,~i oells~ and ~for s ~~
~
_ ~ . ~ ~ .
~
~
~
8. Stop the shaker. The cells may he left overnight a t this stage, if necessary. Remove the covers; they need not be
into a coldmeter tube; rinse the inner chamber twice with &er. ANALYTICAL PROCEDURE FOR CHLORIDE
Method A. In Solutions Containing Negligible Quantities of Other Substances Reacting with Potassium PermanganateSulfuric Acid Reagent. 1. Pipet 0.2 ml. of the chloride solution into the outer chamber of each Conway cell. A volume of 0.1 to 0.4 ml. may he used. The quantity of chlorine in the sample should he from 0.5 to 2.5 micrograms, if possible, each analysis should he set up in duplicate. For each series of analyses, a blank of 0.2 ml. of distilled water should he set up in du licate. 2. Pipet 3 ml. of the potassium permanganate solution into a 50-ml. Erlenmeyer flask and slowly add 1 ml. of concentrated sulfuric acid while gently shaking the flask and cooling i t in running water. If the mixture is not kept cool, it evolves oxygen and is useless; the reagent must he freshly prepared, as it is st,ahle only 2 to 3 hours a t room temperature. The reagent preferred by Conway ( 4 )is a mixture of equal volumes of saturated otassium permanganate and 75% sulfuric acid (other workers gave used 50% sulfuric acid): this is more convenient to orepTe, a8 no o o o h g is needed.'' 3 . Apply fixative to the outer edge of the Conway cells. 4. Pipet 0.15 ml. of the 0.0167% Fast Green reagent into the inner chamber of each cell. 5. Evacuate the acid-potassium permanganate from step 2 by conneoting to a water pump, while shaking gently, for 5 min-
GZ
readins, if necessary.
analytical seriis is several units low&, as the diitilled water and reagents are not absolutely chloride-free. Readings of duplicates
volumetric technique. In the ii-ark reportid here Mis&Amicropipets were used as delivery pipets, without riding; this is a faster but less accurate procedure than that recommended by Kirk (14). The final volumetric adjustment to 5 ml., in oalibrated Klett colorimeter tuhes, is also relatively inaccurate. though very convenient.
A standard curve, for known sodium chloride solutions, is shown in Figure 3. The colorimeter readings are on the ordinate, the "total" micrograms of sodium chloride on the abscissa This total value is ohtained l y adding, to the known micrograms of sodium chloride added, thc calculated micrograms of sodium chloridc in the reagent blnnli. The calculation assumes that the
V O L U M E 24, N O . 5, M A Y 1 9 5 2
859
ANALYTICAL PROCEDURE FOR ALKALI-LABILE CHLORINE
Figure 2. Quantitative Transfer of Dye Reagent Solution from Inner Chamber of Conway Cell to Colorimeter Tube
It is possible, by reaction with sodium in %propanol, to convert total organic chlorine to chloride (SS),but the high chloride blank of commercial sodium metal makes this procedure unsuitable for accurate microanalysis. However, many chlorinated organic compounds react with sodium or potassium hydroxide or alkylate, part or all of the organic chlorine being liberated as chloride. The reaction rate varies for different compounds (6,251, and can be used to distinguish the components of 8. mixture (16). The procedure described here is designed to liherate all alkali-labile chlorine, by reaction in very strong alkali a t 95"; but i t can be modified, where desirable, to a reaction under milder conditions. Method C. (1) Pipet into a 10-ml.borosilicate glass beaker 1 ml. of a solution of ehlorinated'compound in C.P. benzene (or other chlorine-free organic solvent). The alkali-labile
colorimeter reading (in the range fsom 0 to 0.5 microgram) is proportional to the micrograms of sodium chloride. As the reading of the Faat Green (0.15ml. diluted t o 5 ml. in water) is 148, while the reagent blank reading is 145, and the reading for 0.5 microgram of added sodium chloride is 135, the micrograms of sodium chloride in the reagent blank is 3 (0.5)/10 01.0.15. The standard curve so obtained is used t o determine the micrograms of sodium chloride in both reagent blanks and unknowns of subsequent analytical rung. The true micrograms of sodium chloride in the unknowns is obtained by .subtracting the micrograms of sodium chloride in the reagent blank from the micrograms of sodium chloride in the unknowns. The result does not vary even when the reagent blank is high. The fact that the standard cnrvr is not linear makes this procedure necessary, for the reagent blank does vary in more complex procedures. It is desirable to check the reading of the "tNe" blank (0.15 ml. of stock Fast Green reagent in 5 ml. of water dilution) at interv&. There is mme color fading in the first few days after preparing the reagent, but the color is very stable for several months thereafter. Perfect cleanliness of the distilled water blank, against which all tubes are read, i s ementid for accurate work; distilled water that has been standing far a long time may become sufficiently cloudy to l o u w readings by several units. The range can be easily extended up to 8 micrograms of sodium ohloride by using B 0.03370 Fast Green solution in 0.1 M sulfuric acid, and B final dilution to the 10-ml. mark on the Klett tubes. It is also possible to extend the range by using a less effioient color filter, suoh as the Klett No. 54 green, which gives readings about one sixth of those obtained u,ith the special red filter used in this work. This would $low using a 0.2% Fast Green solution, and extend the range up to 50 micrograms of sodium chloride. Metbod B. In Solutions Containing Substances Reacthg with Potassium Permanganate-Sulfuric Acid Reagent. Method A is not applioabk to solutions containing high concentrations of bicarbonate or carbonate, because the carbon dioxide formed will raim the internal pressure and blow off the cover of the Conway cells. Oxidizable inorganic or organic substanoes also interfere, both by reducing the permanganate to manganese dioxide or to manganous ion, and by forming excessive quantities of carbon dioside. Such interference can usually be eliminated by adding the following to step 1 of Method A.
the beakers' vigor6usly. R h s e down dry sohinm propoxide on the walls of beakers from Step 3-a, and add 1 ml. of benzene.
Pipet 0.2 to 0.4 ml. of the 20% chromic-30% sulfuric reagent into the outer chamber of each Conway cell. Let stand 15 minutes to 1 hour. The color changes from orange to red-brown as the chromic acid oxidizes the organic matter prcsent; if it clianges to green, more chromic acid is needed.
microsepara&xy funnels. Add benzene to the beakers to near the brim, and let stand until the supernatant benzene layer is fairly clear, The subnat,ant aqueous layer contains sodium
labiie chlorine, &-om 3 to 50 micrograms will .be an adeauate
M a r pipet griduated in 0.5-ml.uu6ts, can be used to make a series of additions. Mix the solutions in the beakers by swirling gently.
I
I
I 0
0.5
1.0
TOTAL
Figure 3.
1.5
2.0
2.5
MICROGRAMS
3.0
3.5
4.0
NACI
Calibration Curve for Known Sodium Chloride Solutions
3-a. For reactions a t 95", set the beakers on a metal plate over a water bath (a 1- or N i t e r beaker, half full of water boiling
ventil&io& and pFeferably a fume hood, are desirabre. After 30 to 40 minutes, and before the beakers go completely . dry, .. remove them and allow t o cool. 3-b. , For reactions at room temperature, set the beakers under a bell iar for 60 t o 90 minutes. Other reaction times and temperatui.es may also prove useful. ~
ANALYTICAL CHEMISTRY
860 sulfate, some sodium bisulfate, and the sodium chloride formed in the reaction. 5 . Siphon off most of the benzene laver from the beakers, using a vacuum suction system similar t o ihat in Figure 2, but with a large stopper fitting a large Erlenmeyer flask. Refill the beakers with benzene, and again siphon off most of the supernatant layer. This process removes 1-propanol and any other benzene-extractable organic matter. Steps 4 and 5 are more convenient than extraction in a separatory funnel, and there is no transfer loss of the aqueous layer. The wash benzene may be collected and redistilled. 6. Set the beakers on the water bath for a few minutes to evaporate the residual benzene and some of the water (to a residual volume of 0.4 t o 0.45 ml., preferably, but evaporation nearly to dryness does no harm). Remove and allow to cool. 7 . If necessary, add distilled water t o redissolve any crystals of salt that may have formed in any of the beakers. Using thcx pipet control, draw all the solution from a beaker into a 0.5-nil. precision transfer pipet. Add a drop of distilled water to the beaker, rinse, and draw the rinsings into the pipet, finally adjusting to the 0.5-ml. mark. Transfer to a clean, dry, 1-dram (ca. 2-ml.) vial. Mix by drawing the solution back into the pipet, then driving i t back into the vial, and bubbling air from the pipet through the solution in the vial. Cork the vial. If desired, the solution can be stored for weeks before determining chloride. 8. Determine chloride by Method -4 or B. A preliminary trial on a single 0.05-ml. aliquot should be run first, if the chloride content of the solution is likely to exceed the upper limit of the method. This trial will indicate the necessary dilution. Data from analyses of known solutions of various chlorinated insecticides are summarized in Table I. ANALYTICAL PROCEDURE FOR OXIDANT-LABILE CHLORINE
Total chlorine can be liberated from an organic compound by oxidation with sodium peroxide in a Parr bomb (19), or by oxidation with silver-dichromate in concentrated sulfuric acid a t
Table I. Analyses of Known Amounts of Chlorinated Insecticides by Method C (All NaOPr reactions, except as otherwise indicated, a t 9 5 O , 30 minutes)
Compound Aramite
Chlordan
Heptachlor Lindane
NaCI, y per CYonway from Cella Total C1 Foundb
of Total ?! 1Labile Alkali-
8
12 16d 16 20 3 2 4.8 6 4e 6.4d 8 0 6d 6 3.6d 3 6 4 8
Theoretical o/o .4lkaliLabileC 100
3.4 5.1 6.8
1.5
44 41,47
S'.k
2 1.2 4 0.3 0.3 3.6
7 6
0 0
1 6
0 21
4 3
2 1 2 1 2 8
48 48 48
5 8
..
4 4
42
14
50
..
110" C. ( 1 2 ) . The latter mctliod is applicable to quantities of the order of 1 microgram of chlorine. Both methods require relatively complex apparatus and technique. Some chlorinated organic compounds are readily oxidized b y acid potassium permanganate a t room temperature, part or all of the organic chlorine being liberated as chlorine. The following procedure for this "oxidant-labile" chlorine is therefore sirnilar to llethod -4for inorganic chloride. Method D. (1) Pipet 0.05 to 0.2 mi. of a solution of chloi,inated compound in benzene (or other volatile solvent) into the outer chamber of a Conway ccll. Set on the shaker, uncovered, until the solvent has evaporated, leaving a thin, fairly uniforni solid deposit) of some 10 micrograms of the chlorinated conipound. A4ccclerateevaporation by blowing a gentle stream of air over the cells. Heating is usually undesirable because of evaporntive losses. 2-a. Identical wit,h step 2 of Method 2-b. Similar to step 2-a, but 2 ml. of concentrated sulfuric acid are added to the 3 ml. of 6% potassium permanganate. T h e permanganate decomposes, in 5 to 10 minutes, t o oxygen and lower manganese oxides. This reagent differs somewhat in oxidizing power from that of step 2-a; it reacts more slowly vcith methoxychlor, and more quickly with TDE. 2-c. Similar to 2-a, but 2 ml. of 72% perchloric acid are added to 2 ml. of 6% potassium permanganate. This reagent is similar in action to that of step 2-a, but reacts somewhat inore quickly. 3. From this point on, the procedure of Method A is follo\r-ed, except that 0.3 or 0.4 nil. of the acid potassium permanganate reage!it is used in etep 6. Thc time required for complete reaction varies. A \ .
Data from analyses of known solutions of various chlorinated insecticides are summarized in Table 11. I)ETER\IINATION
OF ORGANIC CHLORINE IN EXTRACTS OF BIOLOGICAL MATERIAL
Organic chlorine determination in plant extracts is considered a satisfactory, though unspecific, method for residues of rhlorinated insecticides (8, 9). As in all insecticide residue analyses, it is necessary to check possible interference by biological extractives; this is done by adding known amounts to an extract and measuring the % recovery. Interference and Losses in Analysis. These may arise in several ways: When a large volume of extract is evaporated to a small rolume, loss of insecticides of relatively high vapor pressure (such as lindane, aldrin, .4raniite) may occur. This can be prevented by using the Kuderna-Danish evaporative concentrator (10, 15). Inorganic chloride may be carried into the organic solvent during extraction. This can be corrected for by a blank extract from insecticide-free material, or removed by washing with water or sulfuric acid. The extractives maj- react with and neutralize the sodium Droaoxide reagent in Method C. To eliminate this, a larger ;ol;me of the-sodium propoxide reagent can be used, or a @eliminary treatment of the extract with concentrated sulfuric acid (6, 21), or chromatographic purification (15). Water-soluble compounds, such as glycerol, formed from the extractives by reaction with sodium propoxide, may interfere with chloride analysis by 3Iethod A, but can usually be eliminated by using Method
B.
DDT
6
20
9
12e
12 1.5 18
TDE Methoxychlor
10.W 10.8 3 6 9 12d
25
1 3 4 6
5 1 6 1
0 4 1 1
1 6
0 1
26 3,5 35 2 36 this
2 2 12 a Total micrograms in analytical salnple were 2 . 5 x duplication of chloride analyses. b Average of duplicate chloride analyses. C No theoretical value calculable for chlordan and toxaphene. d NaOPr reaction at 2 5 ' , 60 minutes. e h-aOPr reaction a t 2 5 O , 30 minutes.
33
to ailow
The extractives or their hydrolvsis products may act as einulsifiers and interfere with the benzene extraction of Method C, step 4. The acidity and high salt content of the aqueous phase counteract emulsification; but if necessary the beakers can he let stand overnight before drawing off the benzene layer. The extractives react with the acid potassium permanganate reagent in Method D, and also prevent contact between the' reagent and the chlorinated compounds to be oxidized. Therefore, i t is not possible to use Method D for residue analyses; but the more complex method of Judah ( 1 2 ) can be used, with Fast G re en colorimetrv. qrganiC chlorihe COlnpOUlldS may OCCUt' naturally in living orgiinisnis. However, this is extremely rare, except in fungi ( 1 , S , 20).
Application of Method C. If thcl quantity of plant, extractives in the 1-nil. aliquot, of bcnzene solution for analysis does not
V O L U M E 2 4 , NO. 5, M A Y 1 9 5 2 exceed 50 mg., Method C usually need not be altered, except for the use of Method B in the chloride determination. Data from analyses of known amounts of insecticides in benzene extracts of frrsh olive leaves comminuted in a TTaring blender, and solutions of olive oil in benzene, are summarized in Table 111. If more than 50 mg. of extractives is present in a sample, the following sulfuric acid treatment must be used: Method E.
The benzene solutions of olive leaf extractives and
of olive oil were shaken four times with concentrated sulfuric acid
in separatory funnels, then washed twice with water. The treated solutions were separated and evaporated to dryness. By weighing the residues, it was found that 92% of the leaf extractives and 96% of the oil had been removed by the sulfuric acid treatment. This treatment makes analysis by Method C possible. ANALYTICAL PROCEDURE FOR BROMIDE
Bromide conimonly occurs together with chloride, though in much smaller amount ( 4 ) . Organic bromine compounds, such as methyl bromide, are widely used as fumigants. Methods have been described for sampling air contaminated with volatile
Table 11. Analyses of Known Amounts of Chlorinated Insecticides, by Method D
Coinpound Aramite Chlordan
Heptachlor
per Conway Cell 30 16
I5
Lindane
15
Toxaphene DDT
15
DDE
8
6
Equivalent Reaction N ~ CReagent ~ D-2-c Reagent D-2-a Time, (from Y iVaC1 % of y NaCl % o f M n . Total C1 I founda max. founda max. 120 5.25 0.1 2 0 0 5 15 9 1.0 7 0.5 3 15 1.4 9 1.2 8 30 11 1.7 60 2 . 0 , 2 . 1 13 2 i . 2 . 4 iS 120 2.5 16 .. .. 5 18 9 0.2 1 0.06 0.3 15 0.4 2 0.15 0.8 30 1 6 8 .. 60 1.7 9 1'5 8 120 2 . 1 , 2 . 1 11 .. .. 120 18 0.4 2 0 0 120 16 8 0.2 1 0 0 il 3 0.9 18 0 2 4 15 1 . 5 29 1 0 20 30 28 1.4 60 2 . 8 , 2 . 3 47 2 . 3 , 2 . 8 k6 120 2 . 5 , 2 3 48 .. .. 5 4 5 1.7 38 1 3 34 15 2.6 59 2 2 49 30 3.1 70 .. .. 60 3 2 , 3 . 4 74 3 2 , Z . i 67 120 3 . 8 , 3 . 9 87 , . .. 5 4 4 0 . 9 5 21 15 1.5 33 1'9 ii 30 1.9 42 60 2 . 1 , 2 . 2 49 25,'2'.7 $9 120 2.6 59 .. .. 5 4 6 1 . 0 22 0.3 6 15 1 . 7 37 1.1 24 30 1.9 41 .. .. 60 2 . 3 , Z . Z 49 1 . 9 41 120 2 . 2 , z . j 51 ..
86 1 organic chlorine or bromine compounds, and for the analysis of bromide in fumigated plants ( 7 ) . Method F.
The analytical procedure, adapted from Conway
( 4 ) , is identical with that of Method B for chloride, except that
steps 2 and 5 are omitted, and 0.2 nil. of the 20% chromic-30% sulfuric reagent is used in step 6 . Data for known sodium bromide solutions are summarizedin Table IV. The colorimeter readings were converted into equivalent micrograms of sodium chloride by the standard curve of Figure 3. The micrograms of sodium bromide found were calculated by multiplying the equivalent micrograms of sodium chloride by 1.76 (the ratio of the molecular weights of sodium bromide and sodium chloride). Conway states that the acid potassium permanganate reagent does not oxidize bromide quantitatively to bromine, but that a marked loss as bromate occurs. Houever, using Method -4, about 85% of added bromide is recovered as bromine. The faster diffusion resulting from use of the automatic shaker, and the efficient irreversible absorption of bromine by the Fast Green solution, minimize the secondary oxidation of bromine to bromate. Connay also states that the chromic-sulfuric reagent has a "scarcely detectable influence on 1 ml. of 1% sodium chloride (10,000 micrograms) even after 20 hours' action, whereas all the bromide is oxidized and absorbed in 2 to 2.5 hours." The conversion of chloride t o chlorine in Method F is in fact very slight (0.01%)>but not negligible when a few micrograms of bromide are to be determined in the presence of several thousand micrograms of chloride.
Table IV. Analyses of Standard Sodium Bromide Solutions NaBr Added, Y
NaCl Added, Y
Method Used
Lqulralent SaC1, Y
Calculated SaBr,
"0 of Added
Y
SaBr
1.8 3 3,3.5 1.8 3 3,3.7 5.4 7 2,7.6
89 82,87 89 82, 92 90 90, 9d
3:o
3.8,6.2
io0
97,108
The conversion of bromide to bromine in Method F is only 90 t o 95y0complete, and the yield is not increased even by 3 hours' reaction on the shaker. There is also more variation than in TDE 6 chloride analysis by Method A. I t is probable that the less than quantitative yield and the greater variability in the bromide analysis are due t o the slowing of the reaction rate as the hIethoxychlor 0 bromide concentration falls below 1 microgram per ml. In the presence of 2000 micrograms of sodium chloride, the liberation of a minute amount of chlorine seems to catalyze the conversion of .. bromide t,o bromine, so that "Equivalent" y of XaCI, from Figure 3. the yield rises near to 100% (Table 11'). Table 111. Analyses of Chlorinated Insecticides in Extracts of Plant Material Using DISCUSSION Methods C and B y NaCl S e n s i t i v i t y of C h l o r i d e Fresh Extractives y per Reaction y from Y R of Microdetermination. The limit Plant Wt., in Sample, Insecticide Conway T e p p . Time, NaCl InsectiTheor. NaCl Material hlg. Mg. Added Cell'" C. Min. Foundb cide Expected Yield of detection of chlorine is about Olive leaves 500 10 Lindane 3.6 95 30 2.9 2.1 2.16 97 0.1 microgram, by the colori0.0 0.8 2.5 60 2.1 1.95 2.16 90 3.6 metric procedure described in 0.0 0.15 Xlethod -4. If the Beckman 0 0 3.6 25 60 2.1 2.1 2.16 97 Olives (oil)c 250 50 Lindane 3.6 25 60 2.3 2.2 2.16 102 spectrophotometer, a t 625 mp, 0.0 0.1 500 100 TDE 10.8 25 60 1.2 1.0 2.0 50 is used for the colorimetry (10 0 0 10.8 25 60 1.1 1.1 2.0 55 micrograms of Fast Green, final 500 100 Lindane 3.6 Y5 30 2.4 1.7 2.16 80 0.0 0.7 dilution to 2 ml,), the limit is a Total micrograms in analytical sample were 2.5X this value, t o allow duplication of C1 analyses. lowered to about 0.02 micro* Average of duplicate chloride analyses. gram of chlorine. If the miSolutions of olive oil in benzene were used; fresh weight of ollves calculated assuming 20% oil content. c r o c e l l s of L o w r y a n d
862
ANALYTICAL CHEMISTRY
Bessey ( 1 7 ) are used with the Beckman spectrophotometer (1.25 microgram of Fast Green, final dilution t o 0.25 ml.) the limit should be less than 0.005 microgram of chlorine; however, this assumes that both the oxidation reaction and microdiffusion remain quantitative in this ultramicro range. In fact, preliminary trials show that, while 0.1 microgram of sodium chloride can be determined with these microcells, reagent blanks are high and the oxidation-diffusion reaction is slow and probably not quantitative. A modification of the Conway cell, allowing the use of volumes of the order of 0.01 ml., will be necessary for accurate analysis of quantities of the order of 0.1 microgram of chlorine. Specificity of Analytical Reactions for Organic Chlorine Compounds. It is not possible to identify an unknown chlorinated compound by a single chloride analysis, but two or more dechlorination reactions will make possible a calculation of reaction rates, and a partial identification. For example, the ratio of “chloride formed a t 95’ in 30 to 40 minutes” t o “chloride formed at 25’ in 60 minutes” can be calculated from Table I. It is near 1 for D D T and lindane, 2 for T D E , 5 for toxaphene, 10 for chlordan, and 15 for methoxychlor. Some chlorinated insecticides, such as aldrin and dieldrin, do not react with sodium n-propoxide, and \vi11 not interfere with analyses for other reactive insecticides. A mixture of 2 insecticides can be analyzed if their reaction rates with sodium n-propoxide differ. For example, a mixture of z micrograms of chlordan and y micrograms of D D T will yield 0.16 y) = A micrograms of sodium chloride in the 95”, (0.45 z 30-minute reaction, and (0.045 z 0.16 y) = B micrograms of sodium chloride in the 2 5 O , 60-minute reaction. Both z and y can be calculated by solving the simultaneous equations. The factors are derived by calculation from the data in Table I. Applicability to Insecticide Residues. Method C can be used to determine residues of the order of 1 to 10 p.p.m. in fresh plant leaves, and 10 t o 100 ).p.ni. in oils and fats. If preliminary treatment of the extracts TX ith concentrated sulfuric acid is included in the procedure, the sensitivity is increased by a factor of 10. Further purification of the extracts by chromatographv would not only increase t h e smsitivity. but might also make
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possible the separation of a mixture of insect’icides, or of an insecticide from its partial breakdown products. LITERATURE CITED
(1) Calam, C. T., Clutterbuck, P. W., Oxford, A. E., and Raistrick, H., Biochem. J . , 41,458-63 (1947). (2) Carter, R. H., Nelson, R. H., and Gersdorff, W. A., Advances in Chem. Series, No. 1, 271-3 (1950). (3) Clutterbuck, P. IT., hlukhopadhyay, S. L., Oxford, il.E., and Raistrick, H., Biochem. J . , 34, 664 (1940). (4) Conway, E. J., ” hlicro-Diffusion Analysis and Volumetric
Error,” 3rd ed., Chap. XXIV, XXIII, London, Crosby Lockwood, 1950. ( 5 ) Cristol, S.,J . Am. Chem. Soc., 67, 1494-8 (1945); 69, 338-42 (1947). (6) Davidom, B., J . Assoc. Ofic. Agr. Chemists, 33, 130-2 (1950). (7) Dudley, H. C., IND.ESG. CHEM.,ANAL.ED., 11, 259-61 (1939); Public Health Repts., 56 (19), 1021-7 (1941). (8) Gordon, H. T., AKAL.CHEM.,23, 1853 (1951). ENG.CHEM.,h . 4 ~ ED., . 17, 149-50 (1945). (9) Gunther, F. A , , IWD. (IO) Gunther, F. A., Harris, W.D., Blinn, R. C., Kolbeaen, M. J., Simon, H. S.,and Barkley, J. H., ANAL.CHEW,23, 1835 (1951). (11) Haslam, J., and Squirrell, D. C. IT.,Biochem. J . , 48, 48-50 (1951). (12) Judah, J. D., Ibid., 45,60-5 (1949). (13) King, E. J., and Bain, D. S., Ibid., 48,51-3 (1951). (14) Kirk, P. L., “Quantitative Ultramicroanalysis,” New York. John Wiley & Sons, 1950. (15) Koenig, N. H., Kuderna, J. G., and Danish, A. A , , ANAL.
CHEM.,submitted for publication. (16) LaClair, J. B., IND. ENG.CHEM.,ASAL. ED., 18, 763-6 (1946); ANAL.CHEM.,20, 241-5 (1948). (17) Lowry, 0. H., and Bessey, C. -i., J . Biol. Chem., 16, 633 (1946). (18) March, R. B., Ph.D. thesis, University of Illinois, 1949. (19) Peel, E. IV., Clark, R. H., and Xagner, E. C., IND.ENG. CHEDI.,ANAL.ED., 15, 149-51 (1943). (20) Raistrick, H., A’ature, 163, 553-4 (1949). (21) Schechter, M. S.,Pogorelskin, bI. .1.,and Haller, H. L.. ISD. ENG.CHEM.,ANAL. ED.. 19, 51-3 (1947). (22) Scott, B. A., J . SOC.Chem. Ind. (London). 67, 1-2 (1948). ED., 15, 383-4 (23) Lmhoefer, R. R., ISD. ESG. CHEW,-4s.~~. (1943). (24) yswanathan, R., Biochem. .J.. 48,239-40 (1951). (25) Ton Oettingen, W.,and Sharpleas. N., J . Pharmacol. Erptl. Therap., 88, 400 (1946). RECEIVED for review August 22. 1931.
.\ccepted Fehrilary 13, 1952.
Fluorometric Determinations of Traces of Fluoride H O R i R T H. WILLARD AND CHARLES A. HORTOK’ Zniversity of Michigan, Ann irbor, Mich.
I
S 1938 Goto (6) outlined three methods for the qualitative
spot-test detection of fluoride using fluorescence. Onemethod ,depended on the bleaching by fluoride of the strong yellow-green fluorescence of the zirconium-niorin complex in hydrochloric acid solution. The other fluorescence tests were based on the restoration of the violet fluorescence of salicyclic acid in acetate solution, which had been bleached by ferric or titanium ions, ,owing t o formation of fluoride complexes of these two metallic ions. Later, Bourstyn ( 1 ) proposed a quantitative method based on the effect of fluoride on the fluorescence of the aluminumPontachrome Blue-Black R sl-stems used in methods for aluminum published by him and by Reissler and White ( I I ) , but he ,did not investigate the method suggested. Okac ( 9 ) developed a viwal volumetric method for large amounts of fluoride, using aluminum chloride as the titrant and nioiin as the fluorescent indicator, but did not give the effects ,of variables in detail. l l o r e recently Feigl and Heisig ( 3 ) have published a test for fluoride depending on the quenching of the fluorescence of paper treated with aluminum 8-hydroxyquinolate. 1 Present addre-. -Ridge, T c n n
K-25 Plant, Carbide and Carbon Chemicals Co.. Oak
Many other fluorometric methods are known in which fluoride has a deleterious effect, but, none has been investigated as an approach t o the quantitative determination of traces of fluoride. This paper reports an investigation of several fluorometric systems as possible methods for the determination of traces of fluoride with greater sensitivity than is possible with the common colorinietric methods. Two satisfactory systems are discussed in detail and results on the unsatisfactory systems are reported briefly. REAGENTS AIVD APPARATUS
Water and alcohol were redistilled. -411 chemicals used were reagent grade. Commercial grade organic indicators were used unless otherwise noted. Fluoride solutions were stored in maxlined bottles. A Klett fluorometer was employed for most of the work. Corning, Lumetron, and Wratt,en filters were used in the fluorometer. ALUMIKUM-OXIKE SYSTEM
Gentry and Sherrington (4)suggested a fluorometric method for aluminum using the fluorescence of aluminum oxinate (8hydroxyquinolate) in chloroform solution. Preliminary experi-
