V O L U M E 2 1 , NO. 7, J U L Y 1 9 4 9
815
qecimen is diluted by the techniques mentioned. When the composition lies in the less distinguishable 2 t o 6% nitrate nitrogen range, the composition may be more closely approximated by quantitatively diluting the unknown specimen t o the lower nitrate region before comparing it with the known standards
this work was performed, and to acknowledge the assistance of Honora A. Mattare in the experimental work. LITERATURE CITED
Army-Navy Aeronautical Specification AN-LC-181, Assoc. Offic. Agr. Chemists, “Official and Tentative MettlodP of Analysis,” 6th ed., p. 136, 1945. Harvey, E. M., J . Am. Chem. Soc., 42, 1245 (1920). Hodgman, C. D., ed., “Handbook of Chemistry and Physics,” p. 1343, Cleveland, Ohio, Chemical Rubber Publishing Co..
SUMMARY
Speed and strength oi color development were used ab criteria to evaluate the utility of a variety of diphenylamine indicator bolutions with a wide range of nitrate compositions in the form of films cast from mixtures of cellulose esters. The plot of time for color development versus nitrogen content has a minimum value in the 2 to 6% nitrogen region. Water content is shown to be a critical factor, and diphenylamine concentration is found to exert a minor influence. Although no single indicator solution is best for all nitrate contents, a suitable reagent for general applicability in qualitative tests over a wide nitrate range consists of 0.1 gram of diphenylamine, 100 ml. of concentrated sulfuric acid, and 30 ml. of water. The method can be used for estimating nitrate nitrogen content by comparing samples with a series ol film standards of known nitrate composition.
1947.
Kolthoff, I. M., and Noponen. G. E. disillation and oxidized to formaldehyde, after n hich it is determined colorimetrically, using a sensitive fuchsin-sulfurous acid reagent. The method is applicable to the determination of small amounts of glycosidic methox5l. If ether-linked methoxql is present, as in highly methylated glycosides and cellulose derivatives, it is necessary to plot yield of methoxyl against time and extrapolate to zero time to obtain glycosidic methoxyl values. in the presence of ester-linked methoxyl or if the The method is not applicable _. sample steam-distills.
HEX the glucose-glucose bonds in cellulose are ruptured by methyl alcohol in the presence of hydrochlorir acid, tnethoxyl groups are introduced into the cellulose ( 7 ) . In applying tho conventional microadaptation of the Zeisel methosyl method ( 2 ) to the analysis of such modified celluloses it Kas noted that glucose or purified cotton cellulosr gave blanks that amount to approximately o.3y0. Similar result,s have been observed with polyhydric alcohols ( 1 ) . These blanks would account for more than half of t,he met’hoxyl found by the Zeisel procedure in fully methanolyzed native cotton cellulose and over a third of that on niethanolyzed mercerized cellulose; hence the Zeisel method is not suited to the accurate determination of small amounts of methoxyl in these materials. Frcudenberg and Soff (4)have suggested a modification of the Zeisel apparatus for measuring acid-labile, glycosidic methoxyl. hut their method is not easily adapted to the determination of the very small amounts encountered in methanolyzed cellulose. Inasmuch as the methoxyl in methanolyzed cellulose is hydrolyzed by aqueous acid, consideration was given to the possibility of determining the methyl alcohol so produced. The technique of von Fellenberg (8) for the determination of methoxyl in pectin appeared more promising. By substituting acid for alkaline h>-drolysis and refining the Schiff colorimet,ric procedure it was surressfully adapted t o the present purpose.
.
The methanol is separated by distillation, arid oxidized to formaldehyde by means of an acid solution of potassium permanganate ( S ) , and the formaldehyde is determined by a modified colorimetric method ( 6 , 6 ) , After drastic methanolysis native cotton celluloses yielded 0.20 to 0.25% and mercerized celluloses about O.5y0 methoxyl by the present method. Closely agreeing values are also obtained by the Zeisel procedure, if they are corrected for the large blank. With simple methyl glycosides the two methods give values which are in agreement. But with methylated methyl glycosides, such as methyl 2,3,4,6-tetramethyl-@-~-glucoside and methylheptamethyl-P-cellobioside, the Zeisel method measured all the methoxyl groups whereas the new procedure gave erratic results Tvhich in some cases were slightly greater than one methoxyl per molecule. In the latter cases it was found that, in addition to the acid-labile glycosidic methoxyl, a trace of etherlinked methoxyl was reacting. Because the two types of linkages are cleaved at vastly different rates, a modified method was developed for glycosidic methoxyl in the presence of ether methoxy1 groups. The methods described here are not intended to replace the Zeisel method for the determination of total methoxyl in materials containing appreciable methoxyl, but have proved useful in measuring small amounts of acid-labile methoxyl in materials surh as methanolyzed nelliilose.
ANALYTICAL CHEMISTRY
816 REAGENTS
Potassium permanganate solution, 3.0 grams of potassium permanganate and 15 ml. of phosphoric acid (85%) dissolved in 85 ml. of water. Oxalic acid solution, 5.0 grams of oxalic acid dissolved in 100 ml. of 18 4 sulfuric acid. Standard Methanol Solution. Approximately 2 ml. of absolute methanol are accurately weighed and diluted to 1 liter in a volumetric flask. -420-ml. aliquot of this solution is diluted to 250 ml. in a volumetric flask. The exact weight of methoxyl in each milliliter of this solution is calculated from the weight of methanol used. Schiff's Reagent (6). Basic fuchsin (rosaniline hydrochloride, 0.5 gram) is dissolved in 500 ml. of water, 5.15 grams of sodium acid sulfite are added, and allowed to stand 15 minutes. Then 17 ml. of 6 N hydrochloric acid are added and allowed to stand for a t least 3 hours before using. The reagent should be practically decolorized. Some batches of rosaniline hydrochloride give a reagent with appreciable red color. If this is the case, a satisfactory reagent may be obtained by adding 1 gram of decolorizing carbon and filtering before use. Sulfuric Acid, 72%. Three volumes of concentrated analytical reagent grade sulfuric acid are chilled and diluted with 2 volumes of water. Acetone, C.P. grade.
straight line, which is extrapolated through these points back t o the ordinate, as illustrated by Figure 1. The point of the intercept with the ordinate is regarded as a measure of the glycosidic methoxyl present in the original sample. DISCUSSIOIV O F R E S U L T S
That purified cellulose and glucose give essentially a zero blank by the procedure described is shown in Table I, which also illustrates satisfactory recovery of small known amounts of methoxyl added in the form of methanol or a-methylglucoside. In Table I1 are shown the blank values obtained on various purified celluloses by the Zeisel method. I t is doubtful that these values represent true methoxyl, in view of similar values obtained with pure dextrose (0.08, 0.09, 0.10, and 0.12%) and the polyhydric alcohols. Furthermore, the values slowly increase when the time of distillation with hydriodic acid is prolonged.
SAMPLE MATERIALS
The n-glucose (National Bureau of Standards dextrose), a-n-methylglycopyanoside, a-n-methylmannopyranoside, 6methylcellobioside, 2,3,6-trimethyl-n-glucose, methyl 2,3,4,6tetramethyl-p-=glucoside, and methyl heptamethyl-p-cellobioside used in this investigation were crystalline preparations having physical constants in agreement with those recorded in the literature. The methyl 2,3,6-trimethyl-a,p-n-glucosidewas a sirup prepared from the crystalline 2,3,6-trimethylglucose by refluxing with methanolic hydrochloric acid. Its total methoxyl content (Zeisel) agreed m t h that calculated from the formula. A commercially purified low viscosity sample of cotton linters was used. The purified linters were methanolyeed by heating for 1 hour a t 120' C. in an autoclave with 50 parts of methanolic hydrochloric acid. Initial concentrations of both 1 and 3.7% hydrochloric acid were employed, but both concentrations appeared to yield identical products. The methanolyzed linters were methylated with methyl sulfate and sodium hydroxide in an inert atmosphere. Where mercerized linters are indicated, the mercerization was accomplished by treating purified linters a t room temperature with 30 pafts of 18% sodium hydroxide solution for 0.5 hour, followed by rinsing, souring, rinsing, and drying. PROCEDURE
In Absence of Ether-Linked Methoxyl,. A sample containing 0.3 to 2.0 m . of methoxyl is weighed mto a 100-ml. Kjeldahl flask, 5 ml. 72% sulfuric acid and 3 glass beads, are added and the sample is allowed to stand with occasional shaking until dissolved. It is then allowed to stand overnight at approximately 27" C., 55 ml. of distilled water are added and distilled slowly into a 50-ml. volumetric flask until a t least 36 ml. of distillate have collected. The condenser is rinsed with water into the flask and the solution made to volume. A 10-ml. aliquot is transferred to a 25-ml. volumetric flask, 1 ml. of potassium permanganate solution is added and allowed to stand for exactly 10 minutes, Excess permanganate is destroyed by adding 1.5 ml. of oxalic acid and shaking well. Then 2 ml. of acetone (6) and 10 ml. of Schiff's reagent are added and the solution is made to volume and mixed well. After 3 to 3.5 hours it is read in a photoelectric colorimeter equipped with a filter with maximum transmission a t approximately 580 mp, and compared with standard methanol solutions developed in the same way. In the present work an Evelyn colorimeter equipped with a 565 mp 6lter was used. In Presence of Ether-Linked Methoxyl. Eight or more samples are weighed out and treated with 72% sulfuric acid exactly as in the first step in method 1. The solutions are kept at a temperature of 27" C. and a t various intervals from 24 to 240 hours samales are diluted and distilled to isolate the methanol. Alternatively *a large sample may be treated with 50 ml. of sulfuric acid and 5-ml. aliquots removed a t the various time intervals. When all the distillations have been completed, methanol is determined exactly as in the first procedure and the results are plotted on coordinate paper. The point a t which the curve of methoxyl vs. time in 72% sulfuric acid becomes a straight line is noted (usually 24 to 48 hours at 27") and all subsequent points are employed for determination of the best
07
40
80
120
200
160
240
HOURS Figure 1. Yield of Methoxyl us. Time
Table I.
Recovery of Added Methoxyl Methoxyl
Sample
Added Substance
Found
Mn.
Mg.
D-GluCose blank, 0.5 gram Purified cellulose blank, 0.1-gram samples
Purified cellulose blank, 0.5-gram samples
Methanolyzed, mercerized linters, 0.2-gram samples
None None 0.66, methanol 1.25, a-niethylglucoside None 0.66, methanol 1.25, a-methylglucoside None 0.66, methanol 3.12. a-methylglucoside
0 0 0 0 0.63 0.65 0.21 0.21
ReTheory covery
Mn.
%
0
...
... ...
0 0.64
...
0.20
98 102 105 105
0 0
0
0.62 0.60 0.20 0.20 0.51% 0.527, 1.39 1.40 0.96 1.08
0.64
... ..
0.20
.. . . . .. .
1.65 1.65 1.03 1.12
97 94 100 100
... ...
85 85 92
97
Table 11. Zeisel Blank Values on Purified Celluloses Substance
Methoxyl Content"
% 1. Purified cotton linters
2. Purified cotton linters refluxed with methanol, then exhaustively extracted with water 3. Mercerized purified cotton linters 4. Mercerized purified cotton linters after treatment as ho. 2 5. a-Cellulose from purified wood pulp mercerized Average Zeisel value for purified cellulose 5 Analyses calculated t o anhydrous basis.
0 30.32, 0.34, 0.32, 0.33, 0.310.34.
0.34, 0.29, 0.28, 0.32 0.23, 0.28 0.41, 0.43 0.29, 0.33 0.32
817
V O L U M E 21, NO. 7, J U L Y 1 9 4 9 I t is probable that these Zeisel blanks reflect the low volatility of some higher alkyl iodide produced during the distillation process. The agreement between glycosidic methoxyl values obtained by the present method and Zeisel methoxyl values after the latter are corrected by subtraction of the blank of 0.32%, shown in Table 111, indicates that all the methoxyl entering cellulose on methanolysis becomes the acid-labile glycosidic (or acetal) type. In Table IV is shown the agreement between analytical values and theory for several simple methyl glycosides. The methoxyl contents of these compounds are, however, considerably higher than the optimum range for the method. In order to explore the limits of the method, several types of organic compounds containing methoxyl groups in glycosidic and other configurations Tere analyzed by method 1. Results obtained are summarized in Table V. Two of the compounds distilled to some extent, and when saturated aqueous solutions of these compounds were oxidized with permanganate and treated with Schiff’s reagent, considerable color was developed. Consequently, the results found in these cases were probably due to interference by the oxidation products of the original compound rather than the release of methanol in the method. Thus guiacol, which distills, gave an apparent yield of methoxyl, whereas guiacol acetate, which does not distill, gave no methoxyl. Similarly, anisaldehyde, which distills, gave an apparent yield of methoxyl, whereas p-anisidine gave no methoxyl. The value obtained for methyl galacturonide methyl ester is considerably higher than that corresponding to one glycosidic methoxyl, though less than the total methoxyl in the compound; this indicates that ester methoxyl reacts somewhat, but not completely, under the conditions of the determination. This is also shown by the value obtained for citrus pectin, the methoxyl of which is ester-linked.
Table 111. Comparison between Glycosidic and Zeisel Methoxyl Analyses on Methanolyzed Celluloses Treatment Given Purified Cotton Linters 1 hour a t 120° with methanol-HC1
Mercerized, then 1 hour a t 120’ with methanol-HCI
b
Methoxyl Contentu Glycosidic Zeiselb method I
%
%
0.21 0.22 0.53 0.55
0.21 0.21 0.52 0.54 0.56 0.57
Calculated on basis of anhydrous fiber. Zeisel values after subtracting 0.32% blank.
Table IV.
Table VI.
Analysis of Some Methylated Carbohydrates for Glycosidic Methoxyl . Substance
Glycosidic Methoxyl Content B y Method 2 Theory
%
%
2 3 6-Trimethyl-~-glucose 0.00 0.00 illdthyl 2,3,6-trimethyl-r,8-~-~ucoside 13.2 13.1 .Methyl 2 3 4 6-tetramethyl-8-~-glucoside 11 .O 12.4 Methylhe’p~~methyl-i3-cellobioside 6.5 6.8 Partially methylated, methanolyzed linters (total methoxyl 27.5%) 0.15 0.16a Partially methylated, methanolyzed linters (total methoxyl 40.9%) 0.16 0.15a Glycosidic methoxyl prior to methylation was 0.20%, from which it follows t h a t theoretical values are calculated to be those indicated. A s essentially complete sample recovery was achieved on methylation i t is assumed that no fractionation with change of glycosidic methoxyl occurred during methylation process.
The method of treatment applied to substances containing both ether and glycosidic methoxyl is illustrated in Figure 1. I t was observed that for compounds containing ether-linked methoxyl the yield of methoxyl varied with the length of time the compound was allowed to react with the 72% sulfuric acid. This suggested that ether-linked methoxyl reacts slowly under the conditions of the determination. The reactivity of ether-linked methoxyl was therefore investigated by analyzing 2,3,6trimethylglucose by method 2, which involved varying the time of standing with 72% sulfuric acid. When yield of methoxyl was plotted against time a straight line was obtained with a slope of 0.011% methoxyl per hour and an intercept of zero methoxyl. In a similar manner, points were obtained for methyl 2,3,6trimethylglucoside, methylheptamethylcellobioside, and methyl 2,3,4,6-tetramethylglucoside. For each of these samples a linear relationship between yield of methoxyl and time was obtained in the region between 48 and 240 hours. The best straight lmes through the points corresponding to times greater than 48 hours were calculated by the method of least squares, and extrapolated back to zero time. The intercepts which these lines make with the Y-axis correct for the small amount of hydrolysis of etherlinked methoxyl and show the content of glycosidic methoxyl. The satisfactory agreement of these extrapolated values with the theoretical values is shown in Table VI. When the method was applied to fully methylated Empire cotton fiber, previously purified by extractions with alcohol and 1% aqueous sodium hydroxide, the intercept on extrapolation This small negative value is probably a reflection was -0.04%. of the uncertainty of the analytical determinations and the data are interpreted as indicating the absence of acid-labile methoxyl in the sample.
Methoxyl Analysis on Methyl Glycosides Substance
a-D-Methylglucopyranoside a-D-Methylmannopyranoside 8-Methylcellobioside
Methoxyl Content Method 1 Theory
%
%
16.2 16.0 8.2
16.0 16.0 8.7
ACKNOWLEDGMENT
The authors are indebted to L. W. Mazzeno for the preparation of some of the materials used in this investigation. LITERATURE CITED
Table V.
Results Obtained by Glycosidic Methoxyl Method on Organic Compounds Compound
Methyl salicylate Guiacol Guiacol acetate p-Anisidine Anisaldehyde p-Nitroanisole p-Methoxybenryl alcohol Methyl galacturonide CHaOCHO(CH0H)aCHCOOCHa methyl esters I 2 Citrus pectin (9.57%) methoxyl) Aqueous solution of compound gives color.
Methoxyl Theory Found-
70
%
20.40 25.00 18.67 25.19 22.79 20.26 22.45
None 4.8U None None 13.Oa Xone Xone
27.93
17.9
9.57
6.2
(1) Araki, T., and Hasi, Y., J . Chem. SOC.Japan, 61, 99 (1940). (2) Assoc. Offic. Agr. Chemists, “Official and Tentative Methods of Analysis,” 6th ed., p. 762, 1945. (3) Dodge, B. F., IND. ENG.CHEM.,ANAL.ED., 4, 23 (1932). (4) Freudenberg, K., and Soff, K., Ann., 493, 68 (1932). (5) Hoffpauir, C. L., Buckaloo, G. W., and Guthrie, J. D., IND. ENG. CHEM.,ANAL.ED., 15, 605 (1943). (6) Hoffpauir, C. L., and O’Connor, R. T., ANAL.CHEM.,21, 420 (1949). (7) Reeves, R. E., Schwartz, W. M., and Giddens, J. E., J . Am. Chem. SOC., 68, 1383 (1946). (8) Snell, F. D., and Snell, C. T., “Colorimetric Methods of Analysis,” Vol. 2, p. 18, New York, D. Van Nostrand Co., 1936.
RECEIVED July 23, 1948. Presented before the Division of Sugar Chemistry and Technology, Symposium on Woods a n d Wood Sugars, a t the 114th Meeting of the AMERICAN CHEMICAL SOCIETY, Portland, Ore.
