Determination of Maleic Hydrazide Residues in Plant and Animal

V O L U M E 2 5 , NO. 1 2 , D E C E M B E R 1 9 5 3 LITERATURE CITED

(1) Cady, G. H., “Preparation of Fluorine,” Chap. 8 in “Fluorine Chemistry,” Vol. I, J. H. Simons, ed., New York, Academic Press, Inc., 1950. (2) Cromer, S., ”The Electronic Pressure Transmitter and SelfBalancing Relay,” Oak Ridge, Tenn., U. S. Atomic Energy Commission, MDDC-803 (1944). (3) Guerin, H., ”Trait6 de Manipulation e t d’dnalyse des Gas,” p. 319, Paris, lllasson and Co., 1952. (4) Katz, S., and Burr, J. T., ASAL. CHEM.,25, 619 (1953).

1879 (5) Kimble, R. H., and Tufts, L. E., “Complete Analysis of Fluorine Gas,” Oak Ridge, Tenn., U. S. Atomic Energy Commission, MDDC-195 (1945). (6) McKenna, F. E., Nucleonics, 9, 51 (August, 1951). (7) Miller, W. T., and Bigelow, L. A., J. Am. Chem. SOC., 58, 1585 (1936). (8) Schildknecht, C. E., “Vinyl and Related Polymers,” pp. 47583, New York, John Wiley & Sons, 1952. (9) Stein, L., Chemical Engineering Division, Argonne National Laboratory, private communication. RECEIVED for review July 11, 1953. Accepted October 23, 1953.

Determination of Maleic Hydrazide Residues in Plant and Animal Tissue PAUL R . WOOD Naugatuck Chemical, Division of United States Rubber Co., Naugatuck, Conn. 31aleic hydrazide, l,Z-dihydropyridazine-3,6-dione,is a growth regulant or retardant. Because it is useful on certain food crops, it is important to determine the residues in a large number of plant and animal tissues as a prerequisite to registration of the chemical for agricultural use. A method is presented whereby the hydrazide is reduced and hydrolyzed in water, alkali, and zinc, to split off hydrazine, which is then distilled and determined colorimetrically using p dimethylaminobenzaldehyde. Residue determinations of below 1 p.p.m. are possible. The procedure can probably be used without major modifications for other compounds that will form hydrazine under the conditions of this method.

ALEIC hydrazide ( 8 )(1,2-dihydropyridazine-3,6-dione) is a growth regulant or retardant. Because i t is useful on certain food crops, it is important to determine the residues in a large number of plant and animal tissues as a prerequisite to registration of the chemical for agricultural use. Maleic hydrazide(1) probably exists in tautomeric equilibrium. OH I

c1

/-\

HC

’C‘

/-\

HC

NH

S

red (yellow in dilute solution), Solutions of these water-soluble salts obey Beer’s lam and are stable after maximum color is developed in 10 to 15 minutes. The p-dimethylaminobenzalazine formed in alkaline solution gives canary yellox crystals, insoluble in water and slightly soluble in alcohol, melting point 264-6”. I n an acid, HX, isomerization to a p-quinone structure(I1) occurs, giving dark red crystals, melting point 224”; it is a reversible reaction. This method is sensitive to 0 . 1 ~ per ml. Based on this information, absorption and concentration curves n w e made from purified hydrazine monosulfate and p-dimethylaminobenzaldehyde. ( C H , ) , N~H=N-NH-CH=

/I

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0

OH

I It is stable to both acidic and basic hydrolysis, as evidenced by the fact that heating for several hours a t temperatures up to

200” C. in 18N sulfuric acid or concentrated sodium hydroxide causes no breakdown. Some decomposition does occur in concentrated sodium hydroxide a t higher temperatures. However, after reduction, ring opening by hydrolysis readily occurs, yielding a quantitative amount of hydrazine which can be distilled out under favorable conditions. Many reduction-hydrolysis systems were evaluated, resulting in the present selection of zinc plus alkali in water. Of the possible quantitative methods for determining small amounts of hydrazine, the colorimetric method of Pesez and Petit ( 5 ) was selected because of minimum interference from naturally occurring substances. A recent publication by Watt and Chrisp ( 7 ) also describes use of this method. According to Pesez and Petit, hydrazine reacts with p-dimethylaminobenzaldehyde to give an azine, the acid salts of which are an intense

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I1 REAGENTS

Reagent grade p-dimethylaminobenzaldehyde was further purified by the procedure described by Adams and Coleman ( 1 ) . The purified product was washed free of chloride and dried t o a constant weight in a vacuum desiccator over calcium chloride. If passed through a 60-mesh screen prior to final drying, the resulting product will dissolve more rapidly. The dried compound or dissolved reagent should be stored in the dark. The reagent should be prepared fresh daily by dissolving 0.20 gram in 5 ml. of 2N sulfuric acid. Purified maleic hydrazide available from Naugatuck Chemical, Division of U. S. Rubber Co., Naugatuck, Conn. Hydrazine monosulfate, c.P., freshly recrystallized from water. p-Dimethylaminobenzaldehyde, practical grade, purified according to (1). Sodium hydroxide, analytical reagent grade pellets. Sulfuric acid, analytical reagent grade. Nitrogen, oil pumped. Benzene, analytical reagent grade. Oxygen-free water, freshly boiled distilled water, used while still hot. Zinc, analytical reagent grade, 30-mesh granular.

ANALYTICAL CHEMISTRY

1850 An excellent discussion ( 6 ) on the stability of p-dimethylaminobenzaldehyde in analytical work is of interest. CHOICE O F WAVE LENGTH

Curves of wave length us. per cent transmittance are shown in Figure 1. All readings were taken on a Model D U Beckman quartz spectrophotometer using I-cm. Corex cells. Curve A was obtained by developing maximum color under optimum conditions. using a solution of 0.5 ml. of reagent plus 5.5 ml. of distilled water which contained 6 7 of hydrazine monosulfate. Distilled water was used as a blank. The solution for curve B contained 0.5 ml. of reagent plus 5.5 ml. of distilled water, using distilled water as a blank. Curve C was obtained from the solution for curve A using the solution for curve B as a blank. The preferred wave length of 455 mb was thus chosen.

5.5 ml. with water and the azine color was developed with 0.5niL of reagent. A curve plotting hydrazine monosulfate concentration against per cent transmittance a t 455 mp is shown in Figure 4,A . Excellent conformity to Beer's law is evident. A recovery curve obtained by using aliquots of a standard solution of maleic hydrazide (107 per ml.) put through the entire analytical procedure is shown in Figure 4, B. This curve lies

EFFECT OF ACID NORMALITY ON COLOR INTEYSITY

The effect of acid normality on degree of color obtained with a fixed quantity of hydrazine monosulfate (7.57)and reagent was determined uaing both hydrochloric and sulfuric acids. A blank containing an equal acid concentration and 0.5 ml. of reagent was used as a reference source. The curves in Figure 2 were obtained by plotting acid normality against per cent transmittance a t 455 mp. For a given increase in normality, a greater deviation in per cent transmittance occurs with hydrochloric acid than with sulfuric acid. The normal working normality used is about 0.17, obtained by adding 0.5 ml. of reagent to 5.5 ml. of approximately neutral distillate sample. Decreasing the normality much below 0.17 precipitates the reagent and the azine. EFFECT O F REAGENT CONCENTRATION ON COLOR DENSITY

To determine the optimum concentration of reagent for maximum color development, samples containing 7.57 of hydrazine monosulfate with varying amounts of reagent were run against blanks containing equal amounts of reagent. From the curve in Figure 3, 0.02 gram of reagent in the 6-ml. sample was chosen as the most desirable concentration for normal use.

%

PREPARATION OF STANDARD CONCENTRATION CURVES

Aliquots of a standard solution (IO7 per ml.), representing from 1 to 127 of hydrazine monosulfate, were brought to a volume of

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0.8 1.2 1.6 2.0 ACID NORMALITY

0.4

Effect of Acid Normality on Color Intensity

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RAQENT IN 5.5 ML OF He0 CONTAININQ 6 Y # . O F HYDRAZINE MONO SULFATE, USlNe %O AS A BLANK

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ORAMS Of DAB 1N:FLOR

Figure 3.

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Standard Concentration Curves

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1881

V O L U M E 25, NO. 12, D E C E M B E R 1 9 5 3

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and 1drop of concentrated sulfuric acid in the distillate receiverand immerse the condenser tube. Place 30 grams of sodium hydroxide in the 125-ml. flask and add the maleic hydrazide standard sample (I? and u ) and water to make a total volume of 15 ml. of water added. s e a t on a hot plate until a well temperature of 180' C. is reached. Silicone oil has proved a safe fluid for the temperature well. Remove the flask from the hot plate, xipe the ball joint dry, and apply silicone grease, being careful t o keep it out of the neck of the flask. Add through a funnel 15 grams of 30-mesh granular zinc metal. Swirl the flask once to distribute the zinc evenly and quickly clamp the flask in place. Start the flow of nitrogen to remove oxygen from the system, which might cause autooxidation of the hydrazine formed. A bubble rate of 100 to 150 per minute as counted through the condenser tip is sufficient. Reduction of maleic hydrazide and hydrolysis tc~hydrazine begins a t low temperatures. However, hydrazine does not begin to distill from concentrated sodium hydroxide until a temperature of approximately 140" C. is reached. Therefore, to avoid low results due to side reactions of the liberated hydrazine, it is important to reach the distillation temperature at quickly as possible. This is best accomplished by heating with a free flame, played over the bottom of the flask. Throughout the distillation, adjust

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%OF MALEIC HYDRAZIDEACTUAL RECOVERY CURVE

n 10 cm. FUNNEL

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4 6 6 7 8 9 10 I 1 9 3 MICROGRAMS IN 6 ML.COLOR DEVELOPMENT SAMPLE

Figure 4. Effect of p-Dimethylaminobenzaldehyde Concentration on Color Density

slightly above the theoretical recovery curve, C. The actual recovery curve might vary slightly with changes in the distillation cycle but is usually a t least 95% of the theoretical curve. APPARATUS

From early developnient \Fork on this analytical method it was evident that a special reduction-distillation apparatus would be useful. A detailed draning of the apparatus in use in this laboratory a t the present time is shown in Figure 5 . A complete apparatus or any part may be purchased from Macalaster Bicknell, 181 Henry St., S e w Haven. Conn. The main feature is the thick~ a l l e dflask made necessary by the corrosive action of concentrated sodium hydroxide a t high temperaturei. The x-apor line of the apparatus should be mapped with asbestos tape. A metal valve from an ordinary rubber suction bulb placed in the tube connecting the funnel and flask inlet tube is helpful in preventing steam from blowing back into the inlet tube. A free flame was found to be most satisfactory for heating. HoLTever, a 200-ml. heating mantle may be used. -4smaller mantle does not have the required heating capacity. Tap 11-ater for condensing may be adequate in winter months. averaging below 10" C., but ice water should be used when water temperature is above this value. PROCEDURE FOR OBTAINIh-G STANDARD MALEIC HYDRAZIDE CURVE

Place approximately I ml. of oxygen-free water

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DOUBLE T H I C K N E S S FLASK 125 ml.

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Reduction-Distillation .4pparatus for Determination of Maleic Hydrazide

1882

ANALYTICAL CHEMISTRY

the flow of nitrogen for a bubble rate of 100 to 150 per minute. During a typical run, the caustic solution starts to boil within 1 or 2 minutes. Within approximately 5 minutes the temperature rises to 180" C. When this temperature is reached, remove the flame and add approximately 10 ml. of oxygen-free water slowly through the dropping funnel. Apply heat and continue distilling until a temperature of 180" C. is again reached. The whole operation should not take more than 15 minutes. B preheater heating mantle can be used if desired, but the distillation cycle should correspond closely to the cycle using a free flame. Applying about 65 to 80 volts to a 200-ml. mantle is usually sufficient. After the distillation, disconnect and wash the condenser by sucking the distillate up into the condenser several times, using a rubber suction bulb with an 18/9 male ball joint attached. Rinse with only a fen milliliters of water to keep the distillate volume a t a minimum. For initial work it may be well to connect another condenser in place to collect an additional 5 ml. to run separately to be sure that all the hydrazine has been collected. At times, the one drop of concentrated sulfuric acid added to the distillate receiver may not be sufficient to keep the distillate acid. Until the proper amount to add has been determined, check the pH of the distillate with pH paper several times during the distillation. Use the minimum amount of sulfuric acid to keep the distillate acid, to prevent a serious normality change in the final 6-ml. color development sample. Sometimes it may be more convenient to add an appropriate amount of reagent (0.5 ml. for every 5 ml. of distillate) in place of the water and acid. The reagent serves as a pH indicator, in that it will begin to precipitate before the distillate becomes neutral. The 125-ml. flask should be ~ a s h e das soon as possible after each distillation or the caustic mass will solidifv. Wash the flask by slowly adding a small stream of water directed to run down the side of the flask. When the spattering stops, rinse with tap miter, Before the flask is re-used, rinse with concentrated sulfuric acid to be sure that no zinc remains. To a 5.5-ml. or smaller aliquot of the distillate add 0.5 ml. of reagent and adjust the total volume to 6 ml. A 10-ml. precision mixing cylinder is useful for this work. After 15 minutes determine the per cent transmittance a t 455 mp, reading against a blank prepared by adding 0.5 ml. of reagent to 5.5 ml. of distilled water. Plot per cent transmittance figures, using varying amounts of maleic hydrazide, against concentration to obtain the standard maleic hydrazide curve. In case the reagent is added to the distillate receiver, merely adjust the volume accordingly prior to determining the per cent transmittance.

'

of the analysis. Adding the sample through a funnel will aid in directing it to the bottom. Wash the sample through the funnel with 15 ml. of miter, add 30 grams of sodium hydroxide, and heat on a hot plate until a well temperature of 180" C. is reached. This caustic precooking step removes most of the interference encountered from plant and animal tissues. When working with a new type of tissue, a recovery curve should be obtained by adding known amounts of maleic hydrazide to an untreated check sample in the concentration range expected. Recoveries from 90 to 100% are usually obtained. If recoveries with certain samples are not in this range, vary the amounts of water and caustic used, as well as the temperatures involved. INTERFERENCE

When plant and animal tissues are carried through the reduction-hydrolysis procedure, the distillate sometimes contains materials TThich interfere with the colorimetric test for hydrazine by developing a pink color in contrast to the yellolT azine. This interference is additive; however, it can be compensated for or eliminated where precise or low concentrations of maleic hydrazide are encountered. For approximate data it may only be necessary to run several untreated blank samples of the particular tissue to be analyzed to determine the average error due to interference, which can then be subtracted from the total reading. In most cases, the absolute value of interference is independent of sample size. Therefore, a 1-gram sample n.ill show approximately one third the interference encountered from a 0.3-gram sample, as in the former case the interference is divided by 3 to obtain parts per million.

Table I.

Typical Data on Residues

Maleic Anhydride. P.P.M. Added t o blank Recovered (including onion sample interference) 0,24-O. 26 0 0,75-0,80 0.50 1.00 1.30-1.26 5.25-5,27 5.00

EFFECT O F ADDED SLBSTANCES

Compounds which before or after zinc-alkali reduction give the Ehrlich test (4)give a characteristic pink interference which is an additive error. Such compounds as pyrrole, pyrogallol, resorcinol, pyrazoles, and piperidine give a positive test without reduction, while tryptophan and nicotine do so only after reduction. Compounds such as diethanolamine, urea, ethylamine, and formaldehyde give no interference when added to the zincalkali reduction mass, unless several grams are added. Ammonium hydroxide and sodium cyanide have no effect. Several milligrams of copper sulfate had no effect, but large quantities should probably be avoided ( 2 , 3 ) . Sodium bisulfite and other compounds which break doxn to sulfur dioxide interfere with the color development. DETERMINATION OF MALEIC HYDRAZIDE IN PLANT AND ANIMAL TISSUES

All samples should be reduced by conventional methods to approximately 0.5 pint (0.3 liter) in volume. This laboratory uses a heavy-duty potato French-fry cutter for this work. The reduced sample is then finely ground and blended. An Osterizer made by the John Oster Manufacturing Co., Racine. Wis., has roved very satisfactory for this final grinding and blending. kome samples require an addition of 1/2 part or more of water to facilitate grinding. An aliquot of the final blend representing from 0.5 to 3 grams of original sample is then placed in the 125-m1. reaction flask. Samples having high starch content, such as potatoes and sugar beets, present a serious foaming problem unless a sample weight of 1 gram or less is used. The sensitivity of the method is such that 1 gram of sample is usually sufficient for an analysis. I n some cases a larger sample weight only increases the complexity

The situation may arise in which it is not known whether maleic hydrazide is actually present in a sample, and any color obtained could all be due to interference, or mostly to interference and only a small amount t o maleic hydrazide. I n such cases, an absorption curve should be run to detect the presence of the characteristic dip a t 455 mp. INTERFERENCE REMOVAL BY BENZENE WASHING

When the caustic precook does not remove essentially all of the interference, i t may be further reduced by washing with benzene. Increase the acidity of the distillate by adding 1 drop of concentrated sulfuric acid for every 5 ml. collected, and then vigorously wash two or three times with approximately IO-ml. portions of benzene. After each wash allow the two layers to separate by centrifuging and remove the benzene layer with a vacuum line. After the last washing, bubble nitrogen through the distillate for several minutes to remove the last traces of benzene (heating to remove the benzene causes low results). Add 0.5 ml. of reagent to an aliquot of the distillate. Add potassium hydroxide dropwise until the precipitated reagent is just persistent. Add just enough 1-V sulfuric acid to redissolve and adjust the total volume to 6 ml. I n this way free hydrazine in a basic solution never occurs. LOW RESIDUE WORK

If the maleic hydrazide residue in a sample is of such a level that the distillate collected contains much less than 17 of hydrazine monosulfate in a 5-ml. aliquot, the per cent transmittance will be above 90% on the concentration curve. Therefore, for the

V O L U M E 2 5 , NO. 1 2 , D E C E M B E R 1 9 5 3 most accurate work the hydrazine in the distillate should be concentrated. This cannot be accomplished by heat evaporation of the nater, because this method produces low results. A satisfactory method, consisting of precipitating the hydrazine as p-dimethylaminobenzalazine and redissolving in a smaller volume of water, has been Tvorked out. Add 0.5 ml. of reagent for each 5 ml. of distillate to be concentrated. After complete color development, add dropwise a saturated solution of potassium hydroxide until precipitation is complete. Filter through a 15-ml. medium or fine fritted-glass funnel and aash the precipitate and funnel with a fen milliliters of natcr. Also wash inside the funnel stem. Place the funnel in a micro bell jar vacuum filtration apparatus, using a 10-ml. beaker as a receiver. .4dd 6 ml. of water containing only a sufficient quantity of concentrated sulfuric acid (5 to 10 drops) to dissolve the precipitate completely. Because of the slope of the curve in Figure 3, i t is necessary to make a standard curve similar to curve B in Figure 4,using the same reagent concentration in the blank. TYPICAL DATA ON RESIDUES

The data in Table I were obtained using 1 gram of onion sample throughout. All samples were run using the standard procedure and the distillate concentrating method.

1883 it may be well to investigate general precautions necessary for quantitative work with this compound. Two excellent references (%,3 ) are given for this reason. Solutions containing hydrazine or p-dimethylaminobenzalazine should not be filtered through filter paper or any other absorptive filters. Fritted-glass filters are satisfactory. Various plant samples handled by this laboratory include onions, white and sweet potatoes, sugar and table beets, turnips, parsnips, carrots, celery, spinach, apples, pears, tomatoes, tobacco, grasses, cottonseed oil, and various seeds. Animal samples include muscle, stomach, spleen, pancreas, gonad glands, liver, kidney, small and large intestine, feces, urine, and milk. Samples of soil have also been run. Duplicate determinations on final blended samples have checked to within 10% or better. Some work has been done on the possibility of extending this analytical method to other compounds. In general, this procedure can probably be used without major modifications for compounds which will hydrolyze, or be reduced and hydrolyzed under these conditions to give hydrazine. For example, bicyclodisuccinhydrazide (111), 0

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I11 yields hydrazine under these conditions, while phthalhydrazide

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60&0 .4b 440 450 460 4jO WAVE LENGTH my

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Figure 6.

48

Use of Absorption Curves to Estimate Residue

A . Blank 1-gram sample of onion B . Blank 1-gram sample of onion with 1 p.p.m. of maleic hydrazide C. Unknown with 0.5 p.p.m. of maleic hydrazide

II

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does not. Phthalhydrazide probably could be analyzed if i t were first reduced by other means, but this would be classed as a major modification. LITERATURE CITED

Adams, R. A, and Coleman, G. H., Org. Syntheses, 1 , 214 (1941 1. (2) Audrieth, L. F., and Mohr, P. H., Ind. Eng. Chem., 4 3 , 1774-9

(1)

Interference in the blank could be reduced by increasing the sample size or using the benzene washing method, thus permitting analysis below 0.5 p.p.m. When only an estimate of the residue in the 1 p.p.m. range is necessary-Le., 1% hether it is above or below 1 p.p.m.-a great saving in time is realized by omitting special concentrating and interference removal steps. This is illustrated graphically in Figure 6. The relative dip in the curves at 455 nip indicates that the unknown obviously contains less than 1 p.p.m. DISCUSSIOK

Because the determination of maleic hydrazide actually involves the determination of microgram quantities of hydrazine.

(1951).

Audrieth, L. F., and Ogg, B. A , , “Chemist.ry of Hydrazine,” Yew York, John Wiley &- Sons, 1951. (4) Morton, *4.A., “Chemistry of Heterocyclic Compounds,” 2nd ed., p. 68, New York, NcGraw-Hill Book Co., 1946. (5) Pesez, M., and Petit, -4., Bull. SOC. chim. Frunce, 1947, 122-3. (6) Spies, J. R., and Chambers, D. C., AXAL. CHEM., 20, 30-9

(3)

(1948). (7) (8)

Watt, G. W., and Chrisp, J. D., I b i d . , 24, 2006-8 (1952). Zukel, J. W., “Bibliography of Published Reports on Maleic Hydrazide,’’ Naugatuck Chemical, Division of United States Rubber Co., Bethany 15, Conn., February 1952.

RECEIVEDfor review March 20, 1953. Accepted September 26, 1953. Presented before the Division of Agricultural and Food Chemistry, PestiSOCIETY, cides Subdivision, a t the 122nd Meeting of the AhfmIcAx CHEMICAL Atlantic City. h’. J.