Semimicro-Kjeldahl Procedure for Control Laboratories JOHN 0.COLE
AND
CARL R. PARKS
Research Laboratory, The Goodyear Tire & Rubber Co., Akron, O h i o
N
acid and about 1.5 grams of catalyst mixture are added. Digestion is started with a low flame, gradually increasing the heat until the mixture boils briskly. For most samples the solution becomes clear after about 10 minutes heating. Digestion is continued for 25 minutes after clearing; for difficult compounds such as pyridine derivatives the afterboil should be extended t o 1 hour. The flask and contents are cooled to room tempefature, 20 ml. of m t e r are added, and the solution is well mixed. A blank flask is fitted into place in the distillation unit and the apparatus is well steamed. The blank flask is replaced by the flask containing the sample and a moderate current of steam is passed over until the apparatus is completely filled with steam. A 125-1111. Erlenmeyer flask containing 10 ml. of 45!4 boric acid solution and 3 drops of mixed indicator is placed under the delivery tube, with the tip of the delivery tube below the surface of the acid. During the course of several minutes 12 ml. of 48% sodium hydroxide solution are slowly added. Dropwise addition of base is necessary until most of the acid is neutralized to prevent too violent a reaction. About 4 ml. of sodium thiosulfate solution are added immediately after the base and the solution is steam-distilled as rapidly as possible. After 25 to 30 ml. of distillate have been collected, the receiver is lowered and about 5 ml. of additional distillate are collected.
ITROGEX analyses must often be run either on a very limited amount of sample or in the shortest possible time. While the usual macroprocedure is not satisfactory under these conditions, micromethods often cannot be employed because of limitations on equipment and personnel. In recent years sufficient improvement and simplification have been made in the s e m i ~ c r o m e t h o dto permit its use in almost any laboratory. Among the modifications lyhich have been made are the distillation unit of Redemann (4),the adaption of the Rinkler boric acid method to the semimicro scale by Wagner ( 6 ) , the introduction of an improved indicator for use in presence of boric acid by Ma and Zuazaga ( I ) , and the discovery of highly efficient digestion catalysts of the mercury-selenium type by Osborn and Krasnitz ($7).
Table
1.
Determination of Nitrogen in Simple Compounds % Nitrogen
Compound Acetanilide
p-Bromoacetanilide Benzanilide
Found 10,44 10.34 6.47 6.43 7.14 7.07
Theory 10.36 6.54
Table 11.
7.10
Determination of Nitrogen in Ring Compounds
Compound Pyridine hydrochloride 2-Aminopyridine
This paper describes a procedure which incorporates these recent modifications and may be used with equipment available in almost any laboratory. The procedure has been checked by two different laboratories and has given excellent results for more than 3 years.
P-(Pyridyl-Z) ethyl phenyl amine 2-Chloroquinoline Triphenyltriazine 2-Benzoyl-5-phenylglyoxaline 2,5-Diphenyl-3-keto-3,4dihydropyrazine Z-Methyl-4-phenyl-5-ketodihydroglyoxaline
APPARATUS
The balance employed should be adjusted to give a precision of 0.1 mg. or better when weighings are conducted by the method of swings. All weights used should be checked to assure accuracy within the precision of the balance. Samples are introduced into the digestion flask by means of a long-stemmed weighing tube ( 2 ) to prevent errors resulting from particles of sample adhering to the neck of the flask and escaping decomposition. A digestion rack can be made by attaching six buret clamps to a n iron bar which is clamped to a ring stand. The 100-nil. Kjeldahl flasks are heated with a Bunsen or Fisher burner. The Redemann distillation unit can be made by a glassblower of ordinary skill or may be purchased from Scientific Glass Apparatus Co., Bloomfield, S . J., catalog S o . 3f-1285.
Found 11.99 11.93 29.54 29.55 14.06 13.90 8.54 8.53 13.46 13.52 11.27 11.23 11.33 11.24 15.93 16.10
yo Nitrogen
Theory 12.12 29.76 14.13 8.57 13.61 11.29 11.29 16.09
The boric acid solution changes from pink to a bluish green as soon as it comes in contact with ammonia. The solution is titrated with 0.015 N acid. Since the true end point is difficult to detect, the titration is continued until a faint pink color appears. The volume of acid required t o produce a pink color of this same intensity is determined by adding standard acid to a solution of the same volume, containing the same quantity of boric acid and indicator. The blank (usually about 0.20 ml. of 0.015 N acid) is subtracted from the volume of acid required to titrate the sample.
REAGENTS
Catalyst Mixture. Grind together 150 grams of anhydrous potassium sulfate, 5 grams of mctallic selenium, and 10 grams of mercuric oxide. Rlixed Indicator. Prepare 0.1% bromocresol green and O.1y0 methvl red solutions in 95% alcohol separately. Mix 10 ml. of brombcresol green solution &th 2 ml. ofmethyl red solution. Boric Acid, 4yo. Dissolve 20 grams of boric acid in 500 ml. of boiling distilled water. Sodium Hydroxide, 48y0,. Dissolve 480 grams of sodium hydroxide in 520 ml. of distilled water and allow to stand until carbonate-free. Sodium Thiosulfate, 44yo. Dissolve 88 grams of sodium thiosulfate pentahydrate in 112 ml. of dist,illed water. Standard Hydrochloric Acid, 0.015 N . Prepare a 0.015 N solution and standardize against pure sodium carbonate, using methyl red as an indicator.
Table Ill.
Determination of Nitrogen in High Polymeric Materials
Polymer Butadiene-acrylonitrile 70130 copolymer Butadiene-acrylonitrile 60/40 copolymer Butadiene-2-vinylpyridine 75/26 copolymer Butadiene-2-vinylpyridine 60140 copolylner Butadiene-2-vinylpyridine 60/50 copols-mer Chloroprene-2-vinylpyridine copolymer
Found 7.69 7.64 9,l5 Y .06 3.26 3.25 5.16 6.19 6.19 6.32 1 ._34
Nitrogen Calculateda 7.65 9.14 3.27
5.00 6.27 1.30
1,32
Polymeric salt of sebacic acid and p xylylenediamine Polyhexamethylene adipamide (nylon)
PROCEDURE
8.02 8.13 12.03 ll,Y9
8.28 12.11
a Calculated values were obtained from analysis of polymer by an independent method such as Dumas nitrogen or determination of oarbonhydrogen ratio.
A 15- to 50-mg. sample (30 to 50 mg. for balance with precision of 0.1 mg., 15 to 25 mg. with precision of 0.03 mg.) is weighed into a 100-ml. Kjeldahl flask, and 4 ml. of concentrated sulfuric 61
62
INDUSTRIAL AND ENGINEERING CHEMISTRY RIULTS
Table I indicates that the procedure gives excellent results. With the exception of compounds which contain a nitrogen to nitrogen or a nitrogen to oxygen linkage, practically all organic nitrogen compounds can be analyzed by the Kjeldahl method. So far the authors have encountered no exceptions to this rule. In Table I1 analytical results are listed for a number of nitrogen ring compounds including several pyridine derivatives. For pyridine compounds the digestion time had to be extended to about one hour. Shirley and Becker (6) observed that the use of a copper sulfate catalyst gave low results with pyridine-type compounds while correct results were obtained with a mercury or mercury-selenium oxychloride catalyst. This observation has been confirmed by the authors. The literature contains little information as to the reliability of
Vol. 18, No. 1
the Kjeldahl method for high polymeric materials. All high polymeric materials investigated by the authors to date have given the correct results by this method. However, the same limitations as to structure should apply for high polymers as for the usual compounds. Table I11 gives analytical results for a number of high polymeric substances. LITERATURE CITED
(1) Ma, T. S., and Zuaaaga, G., IND.ENG.CHEM.,ANAL. ED.. 14, 280-2 (1942). (2) Niederl, J. B., and Niederl, V., “Organic Quantitative -Micro Analysis”, p. 44, New York, John Wiley & Sons, 1942. (3) Osborn, R. A., and Krasnitz, A., J . Assoc. Oficial Agr. Chem., 17, 339-42 (1934). (4) Redemann,C. E., IND. ENG.CHEM.,ANAL.ED.,11, 635-6 $1939). ( 5 ) Shirley, R. L., and Becker, W. W., Ibid., 17, 437-8 (1945). (6) Wagner, E. C., Ibid., 12, 771-2 (1940).
Sensitive and Selective Test for Gallotannin (Tannic Acid) and Other Tannins’ FRITZ FEIGL AND HANS E. FEIGL Laboratorio da Produggo Mineral, Minirterio da Agricultura, Rio d e Janeiro, Brazil
A
VIOLET flocculent precipitate is slowly produced when an ammoniacal solution of tannic acid is warned with a soluThe tion of ferrous a,a’-dipyridyl sulfate, Fe(~~,a’-dip)~SOd. precipitate, which filters well, forms quickly if the ammoniacal solution or suspension is treated with acetic acid and warmed. Precipitation is complete, as shown by the negative response of the filtrate to the most common tannic reagent-i.e., ferric chloride plus sodium acetate (2). A solution of ferrous a,a’-phenanthroline sulfate, Fe(a,a’-phen)3S04, shows a similar reaction toward tannic acid. Attempts to isolate precipitates of constant composition have not been successful. Nonetheless, the chemical basis of this new reaction of tannin deserves consideration. Some of the factors involved are: the phenolic nature of the tannin, the colloidal character of aqueous tannin solutions, the autoxidation of these solutions a t pH greater than 7, and the ability of Fe(a,a’-dip)a++ ions to combine with voluminous and complex anions to form red, slightly soluble salts ( 1 ) . Of the possible types of reaction, the writers believe the most probable to be the formation of an adsorption complex by combination of the oxidation products of the tannin with Fe (a,a’dip)8(OH)n, or Fe (a,~ ~ ’ - p h e(OH)z. n)~ Adsorption compounds of tannin with hydrous metal oxides have been reported and uscd for analytical purposes (3,4). The assumption that it is not tannin itself, but rather an oxidation product (of unknown composition) which takes part in this reaction, is supported by the observation that no precipitate is formed if air is excluded, or if much alkali sulfite is present. This is also in agreement with the fact that the deposition of the violet precipitate occurs gradually, and the precipitate always forms first a t the upper surface of the liquid. This effect is seen distinctly if the solution of tannic acid is not extremely diluta. The probability that autoxidation of tannic acid is responsible for the reaction with Fe(a,a’-dip)a+‘ ions made it likely that other autoxidizable phenolic compounds would exhibit an analogous behavior. Trials showed that ammoniacal solutions of pyrogallol give a very strong reaction. Gallic acid, in cold saturated (lyo)solution, gives a visible response, though much less decided 1 Translated from the German manuscript by Ralph E. Oesper, University of Cincinnati, Cincinnati, Ohio.
than taiinic acid and pyrogallol a t this dilution. Hydroquinone reacts weakly. Phloroglucinol, resorcinol, and pyrocatechol give no response. These findings led to the expectation that vegetable tannins would behave toward Fe(a,a’-dip)JSO, as tannic acid does, since they are all phenolic in nature, and their alkaline solutions are said (2) to absorb oxygen. This prediction has been realized with all the natural tanning agents that have been tested thus far, though the number of varieties available has not been large. Consequently, this reaction applies to gallotannin and to other tannins. As will be seen, the nature of the response to the tannin reaction with Fe(a,a’-dip)3SO, is not determined by whether the test material is a pyrogallol- or catechutannin. Synthetic tans, which mostly are condensation products of formaldehyde and sulfonated phenols, give no response. DETECTION OF TANNIC ACID (GALLOTANNIN)
REAGENT.Dissolve 0.25 gram of a,a’-dipyridyl andl.0.146 gram of ferrous sulfate heptahydrate in 50 ml. of water. The solution keeps well in closed containers. Before the test, it is well to render a portion of the reagent ammoniacal and boil. Remove the resulting slight precipitate of hydrous ferric oxide by filtering or centrifuging. This purification is essential when testing for small quantities of gallotannin or other tannins. PROCEDURE. Test Tube Reaction. Treat 1 ml. of the test solution with an equal volume of ammoniacal reagent solution and bring the mixture to boiling. Add acetic acid until the odor of ammonia vanishes and again heat the solution to boiling. A flocculent violet precipitate forms. If only minute quantities of the tannin are present, the precipitate has a brownish tinge. Turbidities produced by minute amounts of precipitate can be easily discerned in the red solution, if the test tube is held toward an intense source of light and a sheet of frosted glass interposed. Identification limit, 8 micrograms of tannic acid; limiting concentration, 1 to 128,000. Drop Reaction. Place one drop of the test solution in a small (0.05-ml.) centrifuge tube, add 2 drops of reagent solution and suspend the tube in boiling water for several minutes. After acidifying with acetic acid, again warm the solution and then centrifuge. Any precipitate collects in the constricted end of the tube. Very tiny precipitates are readily seen if, after centrifuging, the tube is held against white paper. A blank test is recommended when small amounts of tannic acid are suspected. Identification limit: 1 microgram of tannic acid; limiting concentration: 1 to 50,000. The test for tannic acid can alternatively be carried out as follows:
