Determination of 2,4=Dichlorophenoxyacetic Acid and Its Compounds in Commercial Herbicides HERBERT A. ROONEY S t a t e of California, Department of Agriculture, Bureau of Chemistry, Sacramento, Calif.
I
PROCEDURE
IiCREASISG use is being made of 2,4-dichlorophenouyacetic
acid (popularly known as 2,4-D) and its salts, esters, and amides as growth-regulating substances for various purposes and also as selective herbicides. Information on the chemical structure of many of these new compounds and their effects on plants has been published (4). The analytical methods described in this paper have been developed for use in the Bureau of Chemistry Laboratory, California State Department of Agriculture, in connection with the enforcement of the provisions of the AgriculturaI Code relating to the sale of rconomic poisons (pest control materials) in California. FORMS OF 2,4-DICH LOROPHENOXYACETIC ACID ENCOUNTERED IN REGULATORY SERVICE
Since the free acid is only slightly soluble in water, 2,4-D is usually applied as a solution of one of its salts. Although it is occasionally sold as the free acid dissolved in polyethylene glycol, it is more frequently distributed in powdered form mixed with sodium carbonate or sodium bicarbonate. Addition of water to the latter miuture, as directed, results in a solution of the sodium salt suitablr for application. Dilution of the polyethylene glycol solution with tvater results in a dispersion suitable for use. Other salts such as those of ammonium, diethanolamine, morpholine, and triethanolamine have been developed for experimental and commercial purposes. The esters such as butyl, ethyl, and isopropyl are frequently dissolved in dispersing agents such as alcohols and glycols with or without soaps and neutral oils. Dilution of these products with water results in emulsions suitable for application.
1 . Salts of 2,4-D. Dissolve a weight of sample equivalent to about 1.0 gram of the pure material in 100 nil. of water, transfer to a 250-nil. separatory funnel, acidify with 1 to 1 sulfuric acid, and extract) the aqueous phase twice with 75-m1. portions of ethyl ether. Filter and wash samples containing insoluble carriers before transferring them t o the separatory funnel. \Vash the combined ether extracts free of mineral acid with 10-ml. portions of water, until the washings remain alkaline with the addition of 1 drop of 0.1 S sodiuin hydroxide and phenolphthalein, and evaporate the ether extract containing a few Carborundum chips with a stream of air on the stcam bath until the volume is reduced to about, 25 ml. Reinove the sample from the steam bath and complete the evaporation of the ether at room temperature with the stream of air. This precaution is necessary to prevent volatilization of 2,4-D which n-ould occur if evaporation were completed on the bath. Dissolve the residue in 75 nil. of ethyl alcohol and titrate with 0.1 S sodium hydroxide, using thymolphthalein as an indicator. Calculate the percentage of 2,4-D salt, using the appropriate equivalent given in Table I.
Table I.
Comparison of Results by Titration and b y Total Chlorine Determination TT-eig h t Equivalent t u 1111,
a
B u t y l 2,4-dichlorophenoxyacetate E t h y l 2,4-dichlorophenoxyacetate Isopropyl 2,4-dichlorophenoxyncetate Alorpholinium 2,4-dichlorophenoxyacetate M e t h y l 2,4-dichlorophenoxyacetate Ammonium 2,4-dichlorophenoxyace. tate 2,4-Dichlorophenoxyacetaniide 2.4-Dichloroohenoxvacetic acid
CORIPARISOY OF METHODS OF AYALYSIS
Essentially, the methods described herein consist in the determination of 2,4-D by titration of the acid group, or in the case of 2,1-D salts and esters, the conversion to the free acid followed by separation and titration in alcoholic solution. Any method used for the determination of 2,4-D or its salts and esters involving a total chlorine analysis will give erroneous results if other chlorine compounds are present. Likewise the titration method will give high results if the sample contains other organic acids not separated in the procedure indicated. I n cases \\here results obtained by titration are apparently too high, a check analysis involving a total chlorine determination should be made. The equivalent percentage of 2,i-D or the appropriate compound can be calculated from total chlorine determined by oxidation with a mixture of sodium peroxide, sugar, and potassium nitrate in a Parr bomb (1). This method is rapid but its accuracy is limited because of the small sample that can be used. I n samples containing petroleum oils and soap acids, combustion is often inromplctr. The dc3composition of organic halogen compounds by the sodium-alcohol method (b,3)is he-consuming and necessitates a sample free of water. The following methods have been found suitable for the quantitative determination of 2,i-dichlorophenoxyacetic acid, its salts, esters, and amides in various commercial preparations that contain these compounds.
(If
Saiiiple
By Titration
B y Total Chlorinea
n
5%
0.0277 0 0249 0.0263
13.94 99,58 99 60
14.06 99,33 99.20
0 0308 0.0235
99.82 99.29
99.96 99.64
0.0238 0.0220 0.0221 0.0221 0.0221 0.0221 0 (1221
89.58 98.16 10 83 50.35 60.84 7i.79 9.77
88.96 98.10 10 45 50.00 60.21 77.08 46
0 0221
58.35
58.72
0.0200
99.25
99.96
n 2-Alethyl-4-chlorophenoxyacetic acid a B y P a r r boifib
0.1 s
SaOH Gram
(i’i’ii
I _ _ _
-.
7. , A7
.
I
.5 1
2. Esters and Amides of 2,4-D. Reflux a sample weight, equivalent, to 0.8 gram of the ester or amide with about 1 gram of potassium hydroxide and 90 nil. of 95% ethyl alcohol for 1 hour in a 250-nil. S/T Erlenmeyer flask. Transfer the contents of the flask to a 250-1111. beaker, add 50 nil. of water, and evaporate on the steam bath t o about 50 ml. to remove the alcohol. Cool the remaining aqueous solution, transfer to a 250-ml. sepaas in ( l ) ,beginning with “acidify ratory funnel, and proc:ed with 1 to 1 sulfuric acid. 3. Esters of 2,4-D in Presence of Soap, Acids, Alcohols, and Oils. Saponify a m i g h t of sample equivalent to 0.7 gram of the ester by the method in (2) and after evaporating the alcohol on the steam bath, transfer the aqueous solution to a 250-ml. separatory funnel, and extract with 75 ml. of petroleum ether to renibve unsaponifiablc oils. Draw off the aqueous phasc into a 200ml. volumetric flask, add a few drops of a 17?phenolphthalein solution and a few drops of 1 to 1 hydrochloric acid to the disappearance of the pink color, and then add 1 to 1 ammonium hydroxide solution until slightly alkaline. A4ddsufficient water to give a volunic of about 150 ml. Akldslo~vlysufficient loc; barium chloridc solution t o precipitate fatty acids, make t o volume, shake, and filter. The. solution must) be alkaline after the addition of barium chloride; otherwise the 2,4-D will pre-
V O L U M E 19, NO. 7
476 ~~
~
Table 11. Recopery of 2,4-D from Prepared Rlixtures by Titration Method Sample
Oleic Acid
A
% 4 09
B
8.20
Petroleum 011
% 44.48 22.60
dr%d
2,4-D Added
2,4-D Found
%
%
70
46 55 59;lO
4 88 10 10
4 78 10 33
cipitate. Place a 100-ml. aliquot in a 250-ml. separatory funnel, acidify with hydrochloric acid, and proceed as in (1) with the ether extraction, evaporation, and titration. 4. 2,4-D in Presence of Oil, Soap, and Alcohol. Dilute a sample weight equivalent t o 0.5 gram of 2,4-D t o 75 ml. with petroleum ether and transfer to a 250-ml. separatory funnel. Extract with six consecutive 10-nil. portions of a 50% alcohol-water solution, t,o each portion of which have been added a few drops of 407, potassium hydroxide solution and a fcm drops of phenolphthalein indicator. Draw off the combined alcohol-water extracts into a 250-ml. beaker ahd place on a steam bath to reniove the alcohol. Transfer the aqueous residue to a 200-ml. volumetric flask and cool t o slightly below room temperature. Proceed as in (3), beginning with “add 1 t o 1 hydrochloric acid to the disappearance of the pink color.”
5. 2,4-D in Alcohol or Glycol Carriers. Dilute a sample nTeight equivalent to 0.8 to 1.0 gram of 2,4-D to 75 ml. with 9570 ethyl alcohol and titrate directly with 0.1 N sodium hydroxide solution. AYALYTICAL DATA
In Table I are results obtained on some relatively pure compounds and samples of commercial herbicides containing 2,4-D by the titration and total chlorine methods. Differences in results obtained by these methods may be attributed t o experimental error in the analyses performed by the Parr bomb where 0.1 ml. of 0 1 S silver nitrate is equivalent to an error of about 1% on 100-mg. samples containing 2,4-D 99%. .4nalytical data on samples prepared in the laboratory containing 2,4-D dissolved in varying amounts of oleic acid, petroleum oil, and amyl alcohol are prcbr)ntcti in Table 11. LITERATCRE CITED
Instiumeiit Co., Moline, Ill., Diwction Booklet 116, (2) Stepanow, A., Ber., 39, 4066 (1906). (3) Umhoefer, R., ISD I?%. CHEY.,A N < LED..15, 383 (1943). (4) Zimmerman, P. IT., Iiad. Eng. Chem., 35 596 (1943). (1) Parr
.
Determination of Flavanones in Citrus Fruits W. B. DAVIS Laboratory of Fruit and Vegetable Chemistry, c‘. S. Department of Agriculture, Los .4rigeles, Calif. This paper describes a new colorimetric method using alkaline diethylene glycol for the determination of the bitter rhamnoglycoside naringin and other fla\anones that may be present in grapefruit in particular, as well as in other citrus fruits (6). .Although not specific for naringin, it is a rapid procedure of practical value, which is particularly applicable to the assay of naringin in the jhice and colored flavedo of grapefruit, and of hesperidin in other citrus fruits. The possibility of other substances interfering w-ith the method is discussed. Citral, furfural, and geraniol produced color with
alkaline diethylene glycol but did not interfere with the method when added to grapefruit juice in quantities larger than those ordinarily found in that juicc. A n interesting difference between the behavior of flavanones afid flabones in the extracts of certain other plants is pointed out and the suggestion made that the method may be useful in the determination of flavones. The method has been used to determine the distribution of flavanones in the various tissues of citrus fruits and the recovery of pure naringin added to various mixtures, and to follow the course of naringin hydrolysis.
ETfIODS already- described for estimating naringin are either SIOK or not specific. Poore ( 9 ) ,who used a crystalli-
alkaline, but are less suitable and effective than dietkiylene glycol as a reagent. Color intensity appears to be specifically related to the flavanones present when the reaction is carried out in the manner described below. This specificity is indicated by the fact that, when extra flavanone glucosides are added to various extracts of citrus fruits, not only niay the addition be accurately determined, but the results may also be extrapolated to give highly probable values for the original flavanone content of the extract.
zation procedure, found 0.0ec6 naringin in grapefruit juice; however, this method is not adaptable to rapid determinations. The ferric chloride method (4,10) used for the determination of naringin in the comparatively colorless albedo tissues of grapefruit cannot be applied to the juice, because citric acid and other hydroxy compounds present develop a strong yellow color with ferric chloride. This color masks the weak reddish-broFn color produced by such small quantities of naringin as occur in grapefruit juice. Although the boric acid method (11) for hesperidin is applicable to naringin and is sensitive to small quantities, it is slow and inconvenient. Gibbs’ test ( 3 ) gives no distinctive color with naringin, while the procedure of Folin and Ciocalteu ( 2 ) produccs rcsults that are clearly too high (0.3 to 0.8%). A pink color is produced when a solution of naringin in alcohol is boiled with hydrochloric acid and magnesium. However, attempts t o use the color formed as a basis for a colorimetric test have been unsuccessful. An investigation of the addition of alkali to solutions of naringin and hesperidin led to the observation that a stable yellon color n-as formed on the addition of strong sodium hydiovide to a solution of the flavanonr in diethylenr glycol. Fairly concentrated sugar solutions, glycerols, other glycols, and even ~ a t e develop r color with naringin when made strongly
PROCEDURE
Ten milliliters of 9 0 7 diethylene glycol are placed in a IilettSummerson colorimeter tube, and 0.2 ml. of an unknown extract,, or of the solution to be tested, is added and mixed. The colorimeter is then adjusted to its zero reading (using a blue filter) to give a starting point that allows for the natural color of the extract. Spectral transmittance curves for the color developed by naringin and naringenin with the reagent show a narrow minimum near 420 mp, thus necessitating the use of the blue filter. Thereafter 0.2 ml. of approximately 4 N sodium hydroxide is added and the increase in color is read after 5 minutes. The observed color increases are compared with standard curves prepared from the pure flavanone, or its glucoside, known to be dominantly present in the species of fruit used-e.g., naringin in grapefruit juicc, and hesperidin in orange juice. Readings of duplicate analyses in the Klett colorinictcr usually agree n-ithin 1 or 2 scale divisions. The development and reading of the color should take place at a definite temperature.
