ANALYTICAL CHEMISTRY
182 Table V.
Variable Sealing Temperature
Initial Temp., Glass Plates, O F. 200 250 3 00
Table VI.
Sealing Strength Grams/Inch 2.6 2.8 3.3
Effect of Paper Variations Sealing Strength. Grams/Inch
Stub Roll
3.1
2.9
3.1
2.5 2.5 2.8
tures indicated no advantage over those run a t 200 F. Experimentally it is easier to work a t the lower temperature. Table VI demonstrates the change in sealing strength caused by variations in the paper. These variations can be corrected by the use of a control run with a standard wax. Each value was obtained using paper from a different stub roll. A11 the stub rolls were cut from one large roll. DISCUSSION
In most laboratories, the preparation of R axed paper for a sealing strength test does not duplicate the conditions attained by machine-waxed paper for commercial use. With this in mind a test method should eliminate or reduce to a minimum the effect of as many variables as possible. The results of experiments conducted have shown that waxing temperature and moisture content of the paper before waxing for normal laboratory conditions do not affect sealing strength values. Hand waxing of paper produces an uneven film of wax, which results in erratic sealing strength values. If the paper is placed in an oven to allow the excess wax to drain off, reproducible results are obtained. It was found that 7 to 10 minutes a t 200" F. resulted in a uniform wax film thickness. Oven treating also adjusts the humidity content of the paper to a common level, thus minimizing the effect of moisture content. Aging time after oven treatment results in a gradual increase but is within experimental error if the test is completed within 1 hour after oven treating. Although sealing
strength increases with sealing teniperature, the increase between 200" and 250" F. is only SYc. Therefore, sealing strength valuee are Re11 within the limits of experimental error when small changes of plate temperature occur at 200" F. S o method of known accuracy was available for comparisoD with this method. The mean deviation from the mean of 60 teste was found to be less than 5% after correcting for deviation in results due to paper differences. In addition to evaluating straight paraffins, this method has been used to compare the sealing ability of wax blends and to evaluate wax additives. S o t only can tests be made on laboratory-waxed samples, but this same instrument has been useful in comparing machine-waxed samples. In this use, commercial mavhine-waxed samples are sealed tugether bet\%een hot glass plates in the manner described for oven-treated laboratory-waxed bamples. The force required to separate the heat-sealed sherts is measured by means of the same sealing strength tester. An important use that has been made of this sealing strength tester is the investigation of customer complaints. CONCLUSIOhS
This test method permits the determination of sealing YtreIigth of different grades of paraffin waxes, and values can be correlated with results found in commercially waxed paper. With usage a minimum sealing strength can be established that will result in satisfactory seals for bread-wrap paper. ACKNOWLEDGMENT
The authors desire to express their appreciation to E. \V. Gardiner, California Research Corporation, San Francisco, Calif., for his technical assistance. The original design of this sealing strength tester should be credited to D. L. Shinn, Central Research and Technical Department, Crown-Zellerbach Corporation, Camas, Wash., where a similar instrument has been in use for over 12 years. LITERATURE CITED
(1) du Pont de Nemours & Co., E. I., Brit. Patent 529,399 (NOT.. 20, 1940). (2) Padgett, F. W., Oil & Gas J.,38,30 (1938). RECEIVED April 16, 1949.
Colorimetric Determination of Reducing Sugars with Triphenyltetrazolium Chloride A. M. MATTSON' AND C. 0. JENSEN The Pennsylcania State College, State College, Pa. W R I P H E N Y L T E T R A Z O L I U M chloride dissolves in water to form a colorless solution. Upon being reduced, this salt forms triphenylformazan, which is a red water-insoluble compound (4). Reducing sugars in an alkaline medium are capable of reducing triphenyltetrazolium chloride (6)and the quantity of formazan formed is proportional to the quantity of reducing If time. temDerature. and alkalinitv are consugars . , mesent. trolled, this reaction can be used as the basis of a convenient colorimetric method for the determination of reducing sugars.
1
REAGENTS
Sodium hydroxide solution, 1.00 'Y. 1
Present address, N. Y..4grioultural Experiment Station, Genela. K. Y
Acid-pyridine, 15 ml. of concentrated hydrochloric acid per 1 0 0 ml.ofpyridine. Tetrazolium reagent, 0.5Oj, solution of 2,3,5-triphenyltetrasensitive to zolium chloride in water. hi^ reagent is light, It is desirable to make it daily, although it may be kept for longer periods if stored in a dark cool place. (The tetrazolium Salt can be secured from Arapahoe Chemicals, Inc., Boulder, Colo., or Panone Chemical Co., Farmington, Conn.) APPARATUS
.Iconstant teniperature water bath maintained at 25' * 0.1' C. A photoelectric colorimeter with a 490 mp filter or a spectrophotometer. The data reported in this article were secured with a Coleman Model 14 spectrophotometer using square cuvettes. 13 x 13 x 100 mm.
V O L U M E 2 2 , NO. 1, J A N U A R Y 1 9 5 0
183
convenient colorimetric method has been developed for the determination of sugars by the reduction of triphenpltetrazolium chloride and found applicable to the analysis of lactose in milk. The percentages of glucose and of fructose may be determined in a mixture of these sugars by using the Quisumbing and Thomas method and the proposed method. Glucose and fructose have been determined in honey by this method. .J,
datisfactory results were also secured when round cuvettes, 19 X 10.5 mm., were used. SUGAR SOLUTIONS
The procedure employed will give readings between 10 and 85% transmittance with the following concentrations of sugars (mg. per 10 ml.): lactose 3 to 30, fructose 0.3 to 2.7, glucose 1.5 to 19, invert sugar 0.4 to 4.7. The purity of each sugar used was assumed to be the purity found as the result of an analysis by the Quisumbing and Thomas method. Lactose concentrations are espreswd as anhydrous lactose. PROCEDURE
The sugar solution tu b e analyzed (10 ml.) is introduced into a dry 50-ml. Florence flask and placed in a water bath, maintained a t 25.0"C., for 10 minutes to allow the flask and solution to reach a constant temperature. A blank rolution using distilled water is also placed in the water bath. Ten milliliters of alkali are added to each flask and the time is recorded. The flasks are swirled to mix the solutions, returned quickly to the water bath, and held in the bath for 6 minutes to d l o w equilibrium temperature to be reached. At the end of 6 minutes, 2 ml. of the tetrazolium salt solution are added to each flask. The flasks containing the solutions are agitated and returned to the water bath for exactly 30 minutes after the addition of the tetrazolium salt. At the end of this period 10 ml. of acid-pyridine are added to each flask. The pyridine dissolves the formazan and a clear red qolution results, except in the case of the blank which is colorless. The per cent transmittance is read a t a wave length of 490 mp by means of a photoelectric colorimeter or a spectrophotometer. From this value the sugar content can be determined by means of a standard curve previouqly prepared.
the tetrazolium reagent by glucose followed by the addition of pyridine had a minimum transmittance at 480 to 500 mp. A similar study on a solution of recrystallized triphenylformazan dissolved in chloroform gave a value of 490 mfi as the wave length of minimum transmittance. Therefore 490 mfi was chosen as the wave length for transmittance measurements. STANDARD CURVES
Standard curves for lactose, invert sugar, fructose, and glucose were established. Upon plotting the optical density (2 - log G ) against sugar concentration, straight-line graphs passing through the origin were obtained. The slopes of these lines were evaluated by averaging the individual K values for each particular sugar concentration used. The K values were calculated by the equation : K = L/C (1) where L equals optical density (2 - log G ) , and C equals sugar concentration in milligrams per 10 ml. of sample solution. The K values for lactose, glucose, fructose, and invert sugar are presented in Table I. A comparison of the amount of lactose found by using the determined K value with the amount actually present in known lactose solutions is shown in Table 11.
Table I.
Relative Reducing Power of Some Reducing Sugars K
FACTORS IYFLUENCIWG THE DETER\ZIhATIO>
Alkalinity. The alkalinit7 of the reaction is one of the less critical factors. The I S sodium hydroxide used may vary by ~ 3 without ~ ; causing an appreciable error. Temperature of Reaction. Because triphenyltetrazolium chloride is readily reduced by reducing sugars a t room temperature in alkaline solutions, 25" C. Tvas chosen as the standard reaction temperature. The reaction is sensitive to rather small changes in temperature. '4 change of f1O C. from 25 O C., for instance, will result in an error of about lOy0 in a lactose determination. By controlling the water bath to *0.lo C. the error in per cent transmittance due to temperature variations is reduced to 1 % or less. Length of Reaction Time. The reaction time was chosen for convenience as 30 minutes. This reaction time did not allow too much color to develop and furthermore allowed a number of samples to be run simultaneously. In order to control the variation in transmittance to less than lye, it is necessary to limit differences in the length of reaction time to +30 seconds. The reartion is terminated a t a definite time by adding arid t o lower the alkalinity of the medium below that at which the reaction will occur a t room temperature. By incorporating the acid and the pyridine in one reagent, the reaction is stopped and the formazan is dissolved in one step. The color developed under these conditions is stahle for several hours if the solution is kept out of direct skylight. Wave Length. The pi eparation of a spectral-transmittance curve showed that the red qolution resulting from the redurtion of
Lactose 0.0268 Glucose 0.0512 Fructose 0.362 Invert sugar 0.212 a In range 100 to 150 mg.
Relative Reducing Power Quisumbing Tetrazolium a n d Thomas method method 0.523 l:oo 1.00 0.02Q 7.07 4.14
__
~Table 11. Determination of Lactose Sample
Present, Mg.
Found, hfg.
1 2 3 4 5 6 7
24.7 23.7 22.3 20.7 19.8 18.5 17.8 14.8
24.9 23.4 22.3 21.2 19.8 18.7 18.0 14.9
8
Sample 9 10 11 12 13 14 15 16
Present, hZg. 13.8 12.4 10.4 9.9 7.4 6.9 6.2 5.9
Found. Mg.
14.1 12.4 10.3 9.7 7.4 6.7 6.1 5.8
ANALYSIS OF MILK
Milk samples were clarified by the addition of neutral lead acetate (2) followed by ammonium oxalate. The copper sulfate method of clarification could not be used, as i t interfered with the subsequent determination of lactose by the proposed method. The clarified filtrate was used for analysis by both the proposed method and the Quisumbing and Thomas method ( 1 ) . The 10-ml. aliquot used for the proposed method corresponded to 0.5 gram of milk. The per cent lactose was calculated by use of Equation 1, substituting the value 0.0268 for the constant K . il comparison of the results obtained with milk samples by using the proposed method and the Quisumbing and Thomas method is shown in Table 111.
ANALYTICAL CHEMISTRY
184
..
-
whom -*-.
Table 111. Lactose in Milk Lactose, ?& Quisumbing and Thomas Tetrazolium method I method I1
Sample
No.
Ratio 1/11
1
4.44 4.44
4.34
1.023 1.023
2
4.25 4.31
4.32
0.984 0.998
3
4.39 4.36
4.39
1,000 0.993
4
4.28 4.31
4.29
0.998 1.005
5
4.56 4.56
4.54
1.006 1.006
G
4.53 4.50
4.48
1.011 1.006
7
4.76 4.76
4.65
1.024 1.024
8
4.76 4.76
4.69
1.017 1.017
9
4.19 4.16
4.07
1.030 1.022
10
4.16 4.13
4.09
1.017 1,010
11
4.25 4.25
4.17
1.019 1,019
12
4.37 4.31
4.28
1.021 1.007
13
4.46 4.43
4.38
1.018 1.011
14
4.43 4.37
4.35
1.018 1.005
15
4.40 4.40
4.32
1.018 1.018
16
4.46 4.40
4.35
1.025 1.011 Total Mean
32.404 1.0126
S and S’ are equal to the apparent per cent total sugars ex-
pressed as glucose by the tetrazolium method and the Quisumbing and Thomas method, respectively. a and a’ are constants denoting the relative reducing power of fructose in a mixture of fructose and glucose by the respective methods. F equals per cent of fructose. G equals per cent of glucose. Subtracting Equation 2 from 3:
(S
-
8’) =
(a
- a’) F
(4)
( S - 8’) = AF
(5)
where A is a constant that can be evaluated by substituting experimentally determined values of S and S’ corresponding t o known sugar concentrations. The value of A so determined was found to be 6.48. After the value of constant A has been established, EqJation 5 can be used to calculate the percentage of fructose in unknown mixtures of glucose and fructose from experimental values of S and 8’. The per cent glucose may be determined by substituting the value 0.92 for constant a’ in Equation 3 and solving this equation for G after the per cent fructose has been determined. Experimental values obtained using known sugar mixtures itre shown in Table IV. ANALYSIS O F HONEY
The percentages of glucose and fructose in honey were determined by using the method previously outlined for determining glucose and fructose in a mixture of these sugars (Equations 3 and 5). The honey was clarified with dialyzed cream of alumina according t o the usual procedure ( 3 ) . A rapid method of inversion by heating after the addition of acid was used. Ten-milliliter samples equivalent to 3 mg. of honey were used for the proposed
DETERMINATION OF GLUCOSE AND FRUCTOSE IN MIXTURES
Table IV. Mixtures of Glucose and Fructose The relative reducing power of fructose to glucose (Table I) (Basis, 100% total reducing sugars) varies greatly between the proposed method and the Quisumbing Actual and Thomas method. Using the proposed method, fructose has Glucose, Experimental Values, % ’ % G1u co 8 e Fructose Total sugars about seven times the reducing power of glucose, whereas when 20 20.8 78.9 99.7 using the Quisumbing and Thomas method fructose is only 92% 40 39.2 61.0 100.2 100.2 50 50.9 49.3 as active as glucose in the range of 60 59.5 40.4 99.9 100 to 150 mg. of sugar. The relative activity of glucose and fructose in a Table V. Determination of Glucose and Fructose in Honey mixture of these sugars has been found Apparent Total Sugars as to differ somewhat from the relative re% Glucose Total Sugars ducing power of the single sugars. In Quisumbing Quisumbing Tetrazolium a n d Thomas Combined a n d Thomas mixtures with glucose-fructose ratios of Sample method, method, Fructose, Glucose, methods, method, % 20-80 to 60-40 the relative reducing NO. Sbp) S’(4P) % ?& % (invert sugar)
power of fructose was found to be 7.40 times that of glucose. Using values obtained from sugar determinations by both the Quisumbing and Thomas and the tetrazolium methods it is possible to calculate the percentages of glucose and fructose in a mixture of these sugars. The development of the equations necessary for the calculation of glucose and fructose in a mixture is shown below.
S=aF+G
S‘
= a’F
+G
(2)
(3)
1
320 318
68.8
38.7 38.4
33.2 33.5
71.9 71.9
72.3
2
3 14 307
66.8
38.1 37.1
31.8 32.7
69.9 69.8
70.2
3
297 297
63.9
30.8 30.8
66.8 66.8
67.2
4
311 314 316 314
67.0
36.0 36.0 37.7 38.1 38.4 38.1
32.4 32.0 31.7 32.0
70.1 70.1 70.1 70.1
70.4
5
311 314 311 309
66.5
37.7 38.1 37.7 37.4
31.9 31.5 31.9 32.1
69.5 69.6 69.5 69.5
69.9
6
322 320 324 318
69.2
39.1 38.8 39.4 38.4
33.2 33.5 33.0 33.9
72.3 72.3 72.4 72.3
72.7
7
320 318 318 320
69.7
38.6 38.3 38.3 38.6
34.2 34.5 34.5 34.2
72.8 72.8 72.8 72.8
73.1
V O L U M E 22, NO. 1, J A N U A R Y 1 9 5 0
185
method, and a sample containing 150 mg. of honey was used for the Quisumbing and Thomas method. The results are found in Table ir. LITERATURE CITED
(2) Ibid.. p. 565. (3) Ibid., p. 582. (4) Kuhn, Richard, and
Jerchel, Dietrich, Ber., 74B,949-52 (1941). (5) Mattson, A. M., Jensen, C. O., and Dutcher, R. A., Science, 106, 294 (1947).
Chemists, “Official and Tentative hlethods of Analysis,” 6th ed., p. 133, 1945.
( 1 ) Assoc. Offic. Agr.
RECEIVEDJune 20, 1949.
Determination of Certain NitrogeRContaining Functional Groups in Organic Compounds WALTER W. BECKER Hercules Experiment Station, Hercules Powder Company, Wilmington 99, Del. Nitrogen exists in a considerable number of combinations with oxygen, hydrogen, and carbon in organic compounds. It is often necessary to determine the specific nitrogen-containing functional group present. This paper presents a review of available methods for determining certain of these groups. Included are literature references and an outline of the procedures for the determination of the nitrate, nitro, diazo, azo, amino, amide, alkimide, and nitrile groups in organic compounds.
N
ITROGEN exists in organic compounds in probably more combinations with oxygen, hydrogen, carbon, and itself than any other element. Its several valences enable it to unite with these other elements to form a variety of nitrogen-containing functional groups. In early days only the total nitrogen could be determined by the Kjeldahl or Dumas methods; as time went on, methods for determining the specific groups began to be developed. This article presents a review of the available methods for the specific determination of certain of these groups, which have been arbitrarily divided into a number of classes for the purpose of presentation. The principle of the procedure, a few typical compounds which have been analyzed, and literature references are given. NITRATE NITROGEN (-ONOz)
The methods for determining nitrate nitrogen fall into three general classifications. By Nitrometer. The apparatus in general use today is the Du Pont compensating-type nitrometer (20), which is a modification of Lunge’s gasvolumeter (21). With the du Pont nitrometer, the nitric oxide generated may be readily brought to the volume it would occupy a t 20” C. and 760 mm.; this eliminates the need for temperature and barometric corrections. Elving and hfcElroy (IO)developed a semimicronitrometer of the compensating type, with a motor-driven shaker. The nitrometer is usually standardized with potassium nitrate, which reacts with sulfuric acid and mercury according to the following equation: 2ECX03
+ iHzSO4 + 3Hg +KzS04 + 3HgS04 + 4H20
+ 250
In assembling the nitrometer, care must be taken to dry all glassware and to use clean mercury. As a safety precaution, a cellulose acetate face mask must be worn during the generation of the nitric oxide, in case of an explosion. Khen carefully trained, the average analyst is able to operate the nitrometer safely. Complete directions for assembling and standardizing the Du Pont nitrometer, as well as the procedure for analyzing samples, are given by Scott (34) and by the American Society for Testing Materials ( 3 ) . The method can be applied to the analysis of nitroglycerin, nitrocellulose, pentaerythritol tetranitrate, and similar esters, or to compounds that liberate nitric oxide quantitatively. Ob-
viously, the method is inapplicable to nitrate nitrogen-containing compounds that liberate carbon dioxide, sulfur dioxide, or gases other than nitric oxide. Nitrogen in finished explosives usually cannot be determined by means of the nitrometer because they invariably contain stabilizers such as diphenylamine or diethyldiphenylurea. These stabilizers undergo partial nitration in the decomposing bulb, and cause low results. However, certain compounds undergo quantitative nitration under these conditions. For example, if weighed amounts of potassium nitrate and material containing mononitrotoluene are shaken with sulfuric acid in the decomposing bulb, quantitative nitration to dinitrotoluene takes place. The decrease in volume of the nitric oxide obtained is a measure of the mononitrotoluene present. Saponification Methods. The reduction of inorganic nitrates to ammonia by the use of Devarda’s alloy is well known (2). It would seem, then, that organic esters such as nitroglycerin and nitrocellulose could be saponified with sodium hydroxide, and then Devarda’s method applied. Unfortunately, when such esters are treated with sodium hydroxide, various reduction products including ammonia are formed. Muraour (24) avoided this difficulty by conducting the saponification in an oxidizing medium. He dissolved the sample in acetone, added sodium hydroxide, sodium perborate, and hydrogen peroxide, then let stand overnight for the saponification to sodium nitrate. He then added Devarda’s alloy, heated, and distilled the resulting ammonia into standard acid ( 2 ) . Considerable foaming occurs a t the start of the distillation, due to the presence of a floating layer of isophorone which is formed by reaction of acetone and sodium hydroxide. -4good distilling head must, therefore, be used to prevent entrainment of alkali during distillation. hfuraour’s procedure has been used in this laboratory for various products, with good results. It is particularly applicable to samples of lacquer containing nitrocellulose; in this case the nitrogen content of the nitrocellulose must be known or assumed in order to calculate the amount of the latter present. Volumetric Reduction Methods. Knecht (16) first determined nitric acid in aqueous solution by reduction to nitric oxide with ferrous iron in hydrochloric acid solution, then titrating the resulting ferric iron with a standard solution of titanous chloride, using ammonium thiocyanate indicator. In this laboratory, it was found that Knecht’s method could be used for the determination of the nitrate nitrogen in nitroglycerin and nitroglycol, after
