1662
ANALYTICAL CHEMISTRY
another portion of butyl bromide equal to three times the volume of the previous addition, and with the aid of heat and agitation, run the reaction to completion as for sodium metal (IO). Keep the vessel covered whenever possible and continue the flow of helium until the reaction has been completed.
ACKNOWLEDGMENT
The two sets of pliers Tvere partly designed and constructed by William Taylor. LITERATURE CITED
DISCUSSIOIV OF RESULTS
Eutectic sodium-potassium alloy is Xa:2K in mole ratio or 22 and 78% by weight, respectively. The alloy ( 1 ) is liquid a t room temperature. While sodium metal requires a 40% butyl bromide mixture, eutectic sodium-potassium alloy reacts rapidly a t lower temperatures, with 10 to 25% butyl bromide mixture. When the reaction slows at 35" C. with only a 10% butyl bromide mix, it is to be assumed that the remaining unreacted metal is sodium and that the potassium has been consumed. Aside from the necessary cooling precautions, eutectic yodium-potassium alloy is dissolved more smoothly than sodium alone. The composition of the sodium-potassium alloy must be known before the oxygen content is calculated. If a weighed sample is obtainable, the percentage composition may be calculated from the total bromide content and the m-eight of sample, by titration with acid, by conversion to salts, or by similar methods.
Chem. Eng. .Yews, 13, 64s (1955). Fischer, K., A n y e w . Chem., 48, 394-6 (1937).
Koenig, R. F., arid Tandenberg, S. R., Metal P T O Q ~61, . , 3, 71 (1 952).
Mitchell, J., Jr., and Smith, D. 31., "Aquametry," p. 103, Interscience, Kew York. 1948. Pepkowitz, L. P., and Judd, IF'. C., A12.4~.CHEM.,22, 1283 (1950).
Pepkowitz, L. P., Judd, W. C., and Downer, R. J., Ihid., 26, 246 (1954).
Silverman. L., Rec. Sci. Instr., 2 4 , 80 (1953). Silverman, L., and Bradshaw, W.,Sorth hmerican Aviation, Special Rept. 892. Silrernian. L., and Trego, IC.,Analust, 78, i17-22 (1953). White. J. C., Ross, IF'. J., and Rowan, R., Jr., AKAL.CHEW, 2 6 , 2 1 0 - 3 (1954).
Williams, D. D.. and Miller, R. R., I b i d . , 23, 1865 (1951). RECEIVED for review November 1, 1954. Accepted July 5 , 1955.
Spectrophotometric Determination of M e thoxyI A. P. MATHERS and M. J. PRO ATTD Laboratory, lnternal Revenue Service, Washington, D. C. Rlethoxyl is hydrolytically cleaved to methanol, the latter oxidized to formaldehyde, and the formaldehyde determined colorimetrically after condensation with chromotropic acid. This method is specific for the methoxy group in comparison with the Zeisel reaction. Applications to several types of compounds are tabulated and comparisons with theoretical values indicate the method is accurate. A modification is shown for application on a micro scale.
T
HIS investigation was undertaken to develop a methoxyl determination applicable to research problems concerned with the study of some alkaloids and compounds isolated from alcoholic beverages. I n general, the determination of methoxyl utilizes the Zeisel reaction in which the methoxyl is converted to methyl iodide. The methyl iodide is quantitatively absorbed and converted to silver iodide, ionized iodide, or iodate. Then, the iodide or iodate is determined by a standard method. The use of sulfuric acid for hydrolytically cleaving the methoxy group, as employed in this investigation is not new; alkalies and mineral acids have been used in hydrolysis and fusion processes to convert methoxyl to methanol in many natural products such as wood ( 8 ) , pectin ( I I ) , and lignin (9). The methanol produced under these conditions has been determined by specific gravity, colorimetry, and several other means. Pavolini and Malatesta (12) employed satisfactorily to nine alkaloids and six phenols this same type of hydrolysis in their determination of methylenedioxyl and methoxyl, utilizing warm SO% phosphoric acid to convert methylenedioxyl to formaldehyde and warm concentrated sulfuric acid for the hydrolysis of methoxy groups to methanol. The methanol was oxidized to formaldehyde with potassium dichromate, and the formaldehyde produced from both reactions was determined with Nessler's or Tollen's reagent. The value of chromotropic acid as a reagent for the detection of formaldehyde was demonstrated by Eegriwe ( 7 ) . Bricker and
Johnson ( 4 ) developed the test into a quantitative procedure and discussed a number of the factors affecting color development. I n addition Bricker and Vail(6) extended the use of chromotropic acid to the microdetermination of formaldehyde, in the presence of large concentrations of various organic compounds. Further, Bricker and Roberts (6) described the determination of end unsaturation in organic compounds by converting the end carbons to formaldehyde and using the chromotropic acid condensation product for a quantitative measure of the doubly bonded carbon. Boos (3)utilized permanganate solution to oxidize methanol to formaldehyde and determined the latter colorimetrically after condensation with chromotropic acid. Beyer ( 2 ) adapted the procedures of Bricker and Johnson ( 4 ) and Boos f3) to the determination ot methanol in distilled spirits. Based on a review of methods for the colorimetric determination of methanol via formaldehyde, Mathers (IO) specified the conditions necessary, in the various stages of the proceduw, to determine methanol with reproducibility from the formaldehyde-chromotropic acid condensation product. EXPERIMEiYTAL
Apparatus. Volumetric flasks and pipets.
Simple distillation apparatus. Beckman Model DU spectrophotometer with cuvettes, 1-cm. square. For microdeterminations. Total condensation, variable takeoff type distillation column with a packed section 1 X 20 em. lagged with a silvered vacuum jacket. The packing material consists of single turn glass helices. Reagents. Standard methanol solution (conforming to A.C.S. specifications), 20 mg. per 100 ml. in 5.5y0v./v. ethyl alcohol. Ethyl alcohol (U.S.P.), 5.5 to 6770 v./v. Chromotropic acid (Eastman Kodak Co. Xo. P. 230) solution (4,5-dihydroxy-2,7-naphthalenedisulfonicacid), 1 gram per 25 ml. of water. Prepare fresh daily. Potassium permanganate (-4.C.S.) solution, 3 grams of potassium permanganate plus 15 ml. of 85y0phosphoric acid (.4.C.S.) diluted to 100 ml. with distilled water. Sodium bisulfite, C.P. Sulfuric acid (A.C.S.) concentrated.
V O L U M E 2 7 , N O . 10, O C T O B E R 1 9 5 5 Procedure. Introduce a weighed sample (approximately 0.1 gram) into a 250-ml. flask and attach to an efficient reflux condenser. Through the condenser add about 10 ml. of concentrated sulfuric acid (double the quantity of sulfuric acid for larger samples) and heat t.0 fumes of sulfur trioxide for 5 minutes. Cool the reaction mixture and dilute with 75 ml. of water, added through the condenser. Again cool the solution, remove the condenser, attach a simple distilling head, and distill about 45 ml. of the liquid into a W m l . volumetric flask containing 3 nil. of 95y0 ethyl alcohol and make to the mark with water. Pipet 1 ml. of this d u t i o n into a 50-nil. volumetric flask, set in an ice bath, and add 2 ml. of chilled permanganate solution. A%llow oxidation to t,ake place for 30 minutes a t ice bath temperature, then destroy the excess oxidant with approximat,ely 0.2 to 0.3 gram of sodium bisulfite. To the clear solution add 1 ml. of chromotropic acid solution followed by the slow addition of 15 ml. of concentrated sulfuric acid with swirling. Set the open flask in a 55' to 65' C. water bath for 30 minutes. Dilute the solution with water, cool to room temperature, and dilute to volume with water. Prepare a reference blank and a standard methanol color by treating, respectively, 1 ml. of 5.5 to 6% ethyl alcohol and 1 ml. of the standard methanol solution in the above manner beginning with the oxidation step. Read the absorbance of the sample and standard methanol a t 570 mp versus the referenre ethyl alcohol blank. The absorbance is directly proportional to the quantity of formaldehyde, which in turn is proportional to the methosyl and methanol, respectivel! , However, the color intensity is also a function of temperature, and thus, it is necessary to read the absorbances of both sample and standard methanol solution a t nearly identical temperatures. Calculations. The quantity of methoxyl is calculated by the following formula:
.I 8 X F X JI X R = wt. % of methoxyl A, A , is absorbance of sample. A , is absorbance of methanol standard. F is dilution factor of sample. ,If is per cent by weight of methanol in standard. R is molecular weight ratio of methoxyl to methanol. MICRODETERMINATION
1663 sample and permanganate a t approximately ice bath temperature. This tends to prevent the oxidation of methanol to formic acid or carbon dioxide even though a large excess of oxidant is present. In Table I are shown results of the determination of methoxyl, both with and without the addition of ethyl alcohol to some of the samples. More accurate values were obtained by oxidations in the presence of ethyl alcohol; however, in work not reported there was some indication that the absence of ethyl alcohol might be preferable when the content of methanol in the distillate was high, about 4 to 5%> and again in samples where only a few micrograms of methanol were present.
Table I.
Methoxyl, Weight Calculated Found 22.8 22.8
rc
Compound Anisaldehyde, CsH8Oz (p-methoxybenzaldehyde) a-Chloroisobutyraldehyde dimethyl acetal, CeHiaOzCl 40 (i a-Methoxyisobutyraldehyde 2,4-dinitrophenylhydrazone, CiiHlnOsN~ 411.0 0.7 Methyl Cellosolve, CsHsOz (2-Methoxyethanol) Cocaine hydrochloride, CiiHzzOIKCl (benzoyl methyl ecogonine) Codeine sulfate hydrated, C86H6&0isKzS (methyl morphine) Codeine sulfate hydrated, CasHsaO~aXzS(microdetermination)
DISCUSSIOY
The oxidation of methanol by acid permanganate solution is an equilibrium type reaction in which less than a 50% yield of formaldehyde is present under equilibrium conditions. For this reason it is one of the most critical steps in the procedure (IO). Methanol and ethyl alcohol are oxidized to the respective aldehydes and acids with the possibility of the former giving some carbon dioxide. The acetaldehyde produced from the ethyl alcohol interferes with the determination of formaldehyde to some extent by giving a yellow colored solution, which has some absorption a t 570 mp. Interference due to acetaldehyde is minimized bv the use of a reference blank in which an identical quantitv of ethyl alcohol is treated in the same manner as the sample. The oxidation of the methanol can be carried out in the absence of ethyl alcohol, but especial care must be taken to have both
9 1
7.9 7.9
40,2 41 00 .. 48
-in. (j
8.9 8 8
7 . Za 7.8 7 . sa 8 0
, .o
-
0
Methyl e-chloroisobutyrate, CaHsOzCl Methylcellulose, ClHnOs (monomethyl ether) Methyl methacrylate, CsH90zC1 Pectin
22.7 17.6 31.0
22.9 17.5 30.6
Vanillin, CsHsOs (Phydroxy-3-methoxybenzaldehyde)
20 4
20.2 20 2
Glycine Glycerol Ethylene glycol p D i m e t hylaminobenzaldehyde Lactic acid Tartaric acid Methyl iodide Glycolic acid (hydroxyacetic acid) a
Introduce a weighed sample of approximately 1 mg. of sample into a 100-ml. round-bottomed flask, attached to a reflux condenser. Add 10 ml. of concentrated sulfuric acid and heat to fumes of sulfuric acid for 5 minutes. Then cool and add about 40 ml. of urater through the reflux condenser. Remove the condenser, attach the flask to the distilling column, and place the solution under total reflux for 20 minutes. Maintaining a reflux ratio of 30 to 1, collect 2 ml. of distillate. Pipet 1 ml. of distillate into a 10-ml. volumetric flask and add 1 drop of 95% ethyl alcohol. Cool the flask in an ice bath, add 2 ml. of chilled permanganate solution, and allow oxidation to proceed for 30 minutes. Destroy the excess permanganate with sodium bisulfite, add 1 ml. of chi-omotropic acid solution, and 6 ml. of concentrated sulfuric acid, and heat the mixture for 30 minutes in 55' to 65' C. water. Then cool the flask, fill to the mark with sulfuric acid, and read the absorbances a t 570 mfi versus a reference blank prepared with 1 drop of ethyl alcohol. Treat 0.2 ml. of standard methanol solution in the same manner as the sample. Calculate the methoxyl content of the sample according to the preceding equation.
Results of Rlethoxyl Determinations
..
0 0
0 0
n 0 21.8 40.8
3.55 3.5 0
0 0
n
n
0 7.6 24.7
Oxidized in absence of ethyl alcohol.
Although no attempt was made to apply the microdetermination to samples other than codeine sulfate, it is believed the method is applicable to any compound containing methoxyl. The esters and acetal were readily hydrolyzed by distillation from 10% sulfuric acid solution, other compounds were hydrolytically cleaved by heating with concentrated sulfuric acid on a steam bath for 1 to 2 hours, while a few compounds required heating to fumes of sulfur trioxide to ensure complete hydrolysis. Therefore, the latter step was required to make the procedure general. Beroza ( 1 ) has demonstrated that methylenedioxyl or other labile methylene groups can be converted to formaldehyde by hydrolytic cleavage of the molecule; therefore, compounds containing this group interfere with methoxyl determinations. In this work two compounds shown to interfere with the test were glycolic acid and methyl iodide. This x-as not unexpected, because any compound which can be decarboxylated or hydrolyzed to methanol will yield positive tests by this reaction. From the data presented in Table I it is apparent that the methoxy group in a rather n-ide variety of compounds can be determined accurately. Methoxyl n-as determined on several different types of materials and some of these substances were specifically selected, because of their difficult solability in the solvents ordinarily used in the Zeisel reaction. The results show that hydrolytic cleavagc was accomplished in concentrated sulfuric acid. The simplicity of the apparatus and the speed with which the determination can bc made are added advantages of this method. N o interference is offered by other alkoxy or alkimide groups in the determination of
1664
ANALYTICAL CHEMISTRY
the methoxy group by this method, whereas the Zeisel reaction is not specific for the methoxy group. LITERATURE CITED
Beroaa, bl., AKAL.CHEM.,26, 1970 (1954). Beyer, G., J . Assoc. Ofic. A g r . Chemists, 34, i 4 5 (1951). Boos, R. N., A s . 4 ~ CHEM., . 20, 964 (1948). Bricker, C E., and Johnson, H. R . , ISD. E m . CHEM.,ANAL. ED., 17, 400 (1945). ( 5 ) Bricker, C. E., and Roberts, K. H., AKAL.CHEM.,21, 1331
(1) (2) (3) (4)
(1949).
Bricker, C. E., and Vail, A4.H., Ibid., 22, 720 (1950). Eegriwe, E., 2. anal. Chem., 110, 22 (1337). Hawley, L. F., and Wise, L. E., “Chemistry of Wood,” p. 263, Chemical Catalog, New York, 1926. (9) Manning, K. R., and DeLong, W. A,, Sei. A g r . , 22, GU (1341). (10) Mathers, A. P., “Report on Methanol Determination,” .innus1 Meeting of Assoc. Offic. Agr. Chemists, Opt. 12, 1954. (11) Xanju, D. R., and Norman, A. G., J. SOC.C‘hem. I n d . , 45, 337 (6) (7) (8)
(1926). (12)
Pavolini, T., and llalatesta, A.. Ann. chim. up&, 37,495 (194;).
RECEIVED for review February 18, 1955. Accepted July 26, 1955.
Spectrophotometric Method for Determining Hydroxylamine Reductase Activity in Higher Plants D. S. F R E A R
and R. C. BURRELL
D e p a r t m e n t o f Agricultural Biochemistry, The O h i o State University, Columbus, O h i o
A rapid, simple color test for hydroxylamine has been adapted for the quantitative determination of micromolar amountsof hydrovylaminein biological materials. The hydroxylamine reacts quantitatively w-ith an excess of 8-quinolinol to form the stable 5,8-quinolinequinone5-(8-hydroxy-5-quinolylimide). When measured spectrophotometrically at its absorption peak, 705 mp, this compound obeys Beer’s law over the range of 0 to 5 X millimole of hydroxylamine per nil. of solution. The procedure has been applied successfully for the determination of hydroxylamine reductase activity in soybean leaves.
Manganese Chloride Solution. Manganese chloride, C.P. 0.001iM solution. Reduced Diphosphopyridine Sucleotide Solution. Reduced diphosphopyridine nucleotide (Sigma Chemical Co.), 3 X 10-4M solution. Keep refrigerated. Phosphate Buffer Solution, p H 6.8. Adjust the p H of a 0.05M monobasic sodium phosphate solution to pH 6.8 by the addition of 0.05M dibasic sodium ohosnhate solution. Keea refrigerated. Hydroxylamine Standard Solution. Dissolve 0:0695 gram of dry, recrystallized hydroxylamine hydrochloride, c.P., in water and dilute to 1 liter. Take a 250-ml. aliquot of this solution, adjust to p H 3.0 with 0,0lLVhydrochloric acid, and dilute to 1 liter. This solution contains 0.25 micromole of hydroxylamine per milliliter, and is stable for several days. ~
PROCEDURE
P
REVIOUS procedures for the determination of hydroxyl-
amine have been reported by Blom (W), Endres and IZaufniann ( 5 ) ,and Csaky ( 4 ) . These procedures involve the oxidation of hydroxylamine to nitrous acid, which is then determined colorimetrically by the Rider and bIellon (7) or S h i m (8) procedures. Although these methods are sensitive for the resulting nitrous acid formed, the oxidation of the hydroxylamine is neither specific nor simple. Xason and others (6) have demonstrated the presence and the requirements of hydroxylamine reductase in soybean leaves using Csaky’s method for the determination of hydroxylamine. Colter and Quastel ( 3 ) have recently reported a manometric procedure for the determination of hydroxylamine, which depends upon the oxidation of hydroxylamine by manganese dioxide t o produce nitrous oxide. This method, however, is not very sensitive and is also rather involved. Berg and Becker ( 1 ) have reported a very sensitive and specific qualitative color test for hydroxylamine. This test has now been applied quantitatively to biological materials for the determination of micromolar quantities of hydroxylamine. The hydroxylamine reacts quantitatively with an excess of 8-quinolinol in the presence of ethyl alcohol and sodium carbonate to form the stable 5,8-quinolinequinone-5-( 8-hydroxy-5-quinolylimide) designated as Indooxine. This compound exhibits a very prominent adsorption peak at 705 mp (Figure 1).
(e)
Hydroxylamine Standard Curve. I n a 15 X 125 mni. teat tube place up to 1.0 ml. of the hydroxylamine standard solution (0.00 to 0.25 micromole of hydroxylamine), 1.0 ml. of the 0.0511/1 phosphate buffer, pH 6.8, and water to bring the volume to 2.8 ml. Add 0.2 ml. of the trichloroacetic acid solution. Follow with 1.0 ml. of the 8-quinolinol solution and sn-irl gently. Neut, add 1.0 ml. of the 1.OM sodium carbonate solution, shake vigorously, and stopper before placing in a boiling m-ater bath for 1 minute to develop the green color. On removal from the water bath, cool for 15 minutes, and then read in the Beckman D G spectrophotometer a t 705 mH using matched 1-em. Corex cuvettes. Carry out simultaneously a blank determination which contains everything but hydroxylamine and set a t 100% 2‘: Within this concentration range (0.00 to 0.25 micromole of hydroxylamine), the Beer-Lambert law is obeyed, giving R
REAGENTS
8-Quinolinol Solution. Dissolve 1.O gram of 8-quinolinol (Eastman Kodak Co.) in 100 ml. of absolute ethyl alcohol. Keep tightly stoppered. Sodium Carbonate Solution. Sodium carbonate, c.P., 1.OM solution. Trichloroacetic .kcid Solution. Water solution, 12% by weight.
600
Figure 1.
700 WAVELENGTH I N MILLIMICRONS
800
Absorption spectrum of Indooxine
