Impurities in White Sugar IV. Determination of Nitrogen J. A. AMBLERAND S. BYALL,Bureau of Chemistry and Soils, Washington, D. C. s u s p e n s i o n through a 150-mesh TOTAL nitrogen in white direct-consumption screen.) The whole distillation the effects of non-sugars sugars has been determined by a Kjeldahl digesmust be carried out in an atmoson the properties of sugars phere free from ammonia and its tion of the sugar followed by nesslerization of the and sugarhouse p r o d u c t s , atvolatile compounds, or in a closed distillate. The nitrogen so found includes that system. The ammonia-free water tention has long been focused so obtained is used for the prepapresent as nitrates in the sugar. o n t h e v a r i o u s f o r m s of niration of the standard ammonium trogen which may be present (5, Protein nitrogen has been determined by prechloride comparison solutions and 10, 13, 15, 18, 20, 26, 27). Infor aliquotidg the distillate when cipitating the protein as tannate and determining necessary before nesslerization. organic and organic compounds the nitrogen in the precipitate by the same method 11. What will hereafter be reof this element are p r e s e n t in ferred to as “magnesia water” is as used for total nitrogen. Alpha amino acids the raw materials entering into prepared by concentrating to twoand related compounds have been determined by thirds or one-half its original volt h e m a n u f a c t u r e of w h i t e ume a s u s p e n s i o n of the finely ninhydrin. Nitrates have been defermined by sugars, and while the clarificapowdered magnesium oxide in distion (defecation) processes are reduction with Deuarda’s alloy to ammonia tilled water. It is allowed to cool designed to remove the nitrogeand settle in an ammonia-free atfollowed by distillation and nesslerization of the mosphere. The clear or slightly nous o r g a n i c s u b s t a n c e s as distillate. Nitrites have been determined by turbid supernatant magnesia water completely as possible, no comis suitable for use as ammonia-free Ilosuay’s modification of Peter Griess’ colorim e r c i a l l y feasible method of water in making solutions and in metric test. completely e l i m i n a t i n g them diluting solutions before distillation. It cannot be used for dilutfrom the iuices (93) has been found. E;en in the refinery they are not completely removed ing a solution to which Nessler’s reagent is to be added. OXIDE. Powdered copper oxide is ignited to a bright from the liquors (1). Neither are the inorganic nitrogen com- redCOPPER heat to burn off all nitrogenous matter. It is cooled and pounds completely removed by bone-char refining (14). stoppered closely t o exclude ammonia and the condensation of Methods of estimating the various types of nitrogen com- ammonium salts from the air. SULFURICACID. Pure concentrated sulfuric acid of specific pounds in juices, sirups, and molasses have been extensively gravity 1.84 is used. studied by many investigators, and i t is now possible, in such POTASSIUM HYDROXIDE SOLUTION.A solution containing 200 lower purity sugarhouse products, t o determine total nitro- grams of potassium hydroxide (stick form) per liter is prepared gen (16, go), ammonia (4, %), amido nitrogen (4, 27), with magnesia water. SODIUMHYDROXIDE SOLUTION.A saturated solution of amino acid nitrogen (10, 27), protein nitrogen (10, 24, 26)) sodium hydroxide (stick form) in magnesia water is prepared. betain nitrogen (19, 20, II), nitrates (17, %), and nitrites (9W). NESSLER’S REAGENT (9,25). Such a thorough partition of the nitrogen in white STANDARD AMMONIUM CHLORIDESOLUTION.Exactly 1.9093 sugars, however, is not possible at present on account of the grams of ure ammonium chloride are dissolved in 1 liter of extremely small quantity of the total nitrogen in the sugars. ammonia-gee water. Of this solution, 20 cc. are diluted to 1 liter with ammonia-free water. The resulting solution contains It is possible, by using the methods described in the following 0.01 mg. of nitrogen as ammonia in each cubic centimeter. paragraphs, t o determine total nitrogen, protein nitrogen, APPARATUS amino acid nitrogen, ammonia, nitrates, and nitrites in white The apparatus used was a regular Kjeldahl digestion and sugars. The samples of sugar used for the analyses here reported comprised various commercial grades of refined cane, distillation outfit and ordinary 100-cc. Nessler tubes. Before determination, the stills must be freed from nitrogen comof plantation granulated cane, and of granulated beet sugars. each pounds by distilling a suspension of the finely powdered magnesium oxide in distilled or magnesia water until no color deTOTAL NITROGEN velops when 2 cc. of Nessler’s reagent are added t o 100 cc. of the An excellent method for the determination of nitrogen in distillate. This may take some time when the apparatus is low-purity products by micro-Kjeldahl technic has been de- first set up. Any rubber stoppers or connections should be out thoroughly with dilute sodium hydroxide solution scribed by Stanek and Vondrak (go), but i t is not convenient boiled before they are used. If the apparatus must be in a room where nor economical to submit to Kjeldahl digestion sufficient other chemical work is being carried on, it is necessary to perform white sugar to yield enough ammonia for even a microtitra- all distillations in a closed system and, when not in use, to protion. Hence resort was had to a Kjeldahl digestion on smaller tect the stills and condensers from nitrogenous fumes. amounts of sugar, followed by estimation of the ammonia in METHOD the distillate by means of Nessler’s reagent. But here i t Kjeldahl flasks of 800 cc. capacity were cleared of ammonia was found necessary to modify the Kjeldahl technic because by concentrating to one-half its volume a t least 600 cc. of magit was impossible t o procure potassium sulfate sufficiently free nesium oxide suspension. The residual suspension in the flasks was discarded, and the unrinsed flasks were immediately used for from nitrogen. the digestion, Ten cubic centimeters of a magnesia water REAGENTS solution, containing 10 grams of sugar per 100 cc., were pipetted AMMONIA-FREE WATER. I. What will hereafter be called into the flask. (In analyzing sugars which are low in total “ammonia-free” water is prepared by boiling distilled water nitrogen, lar er quantities of the sugar should be taken. Two with very finely powdered magnesium oxide until the distillate and five-tentis grams of the dry sugar were used for the analyses gives no test with Nessler’s reagent, and the water, which subse- of this type of sugar in this investigation.) To this were added quently distils, is collected in ammonia-free containers. (Mag- about 1 gram of copper oxide powder from the tip of a spatula, nesium oxide was used here in preference to the more usual sodium 25 cc. of concentrated sulfuric acid, and, very cautiously, 25 cc. hydroxide or carbonate because its suspensions boil more evenly of the potassium hydroxide solution. The mixture was cauif the oxide is very finely’divided. For this purpose the finest tiously heated with a small flame until frothing ceased and then powdered oxide obtainable is necessary, that which will pass in with a strong flame until the carbon was completely oxihzed and
I
N INVESTIGATIONS of
34
January 15,1932
INDUSTRIAL AND ENGINEERING CHEMISTRY
the solution was of a clear green color. The digestion was completed in about 2 hours. After the mixture had cooled, it was diluted with 400 CG. of magnesia water and made alkaline with 50 CC. of the saturated sodium hydroxide solution. A teaspoonful of the finely powdered magnesium oxide was added at the same time. The flask was immediately connected with the still and 200 cc. of distillate were collected. This operation must also be carried out in an ammonia-free atmosphere or in a closed system. To each 100 cc. of distillate 4 cc. of Nessler reagent were added as in ordinary water analysis, and after 15 minutes the colors which had developed were compared with those which had developed simultaneously from known quantities of the standard ammonium chloride solution diluted to 100 cc. with ammonia-free water. An alternative method of measuring the ammonia obtained, which is not only convenient but economical if a large number of analyses are to be made, is to compare the color of the nesslerized solutions in a spectrophotometer against distilled water. The transmittancy (T) a t 510 mp of each of a series of nesslerized solutions containing known and differing quantities of ammonium chloride was determined within 15 to 45 minutes after the addition of the reagent. If the transmittancy is expressed as a decimal and the logarithm of its reciprocal (log 1/T, or - log 5") is plotted as ordinate against the quantity of nitrogen as ammonia in each solution as abscissa, a straight line is obtained which has a slope corresponding to an increase of 0.0530 in the - log T value for each increment of 0.01 mg. of nitrogen. The line as plotted does not pass through the origin, but a little above it, depending on the depth of color of the Nessler reagent itself. Hence the position of the line on the graph must be determined with each new batch of Nessler reagent used. From the line so obtained, the quantity of nitrogen in an unknown is readily and easily determined by finding the - log T value of the nesslerized distillate, without the necessity of making up new standard solutions for each set of determinations. The results obtained by either method of measurement, after deducting the quantity of nitrogen as ammonia which was present in the reagents and which was determined by a blank determination on the reagents without sugar, were calculated to parts per million of sugar. The nitrogen content of sugars representative of many studied by this method is given in Table I. The nitrogen obtained by this method includes that present as nitrates. This was demonstrated by making determinations on a series of four solutions of nitrate-free sugar to which were added before the Kjeldahl digestions 0.0, 10, 20, and 40 micromilligrams of nitrogen as potassium nitrate. There were recovered 0.0, 10, 17, and 39 micromilligrams of nitrogen, respectively. PROTEIN NITROQEN The protein nitrogen in juices has been extensively investigated by Vondrak and others ( I O , 24, 26). The amount of protein in white sugars is generally so small that it is impossible to obtain a definite color test with protein reagents or to obtain more than a turbidity with such protein precipitants as copper sulfate, mercuric nitrate, phosphotungstic acid, and tannic acid. In two unusual sugars, sufficient protein was present to yield a biuret test when the precipitate obtained with copper sulfate was collected on a filtei paper and moistened with a little dilute sodium hydroxide solution. When analyzed for protein nitrogen by the method to be described, the one giving the stronger biuret test was found to contain 8.6 parts per million of protein nitrogen. REAGENTS Prepare the same reagents as were used for total nitrogen and also TASXICACID SOLUTION.Ten grams of tannic acid are dissolved in distilled water and made up to 100 cc. METHOD According to Vondrak (%), the tannic acid method of determining protein in the presence of sugar is the most accurate. T o a solution of 25 grams of the sugar in 100 cc. of water, 5 cc. of the tannic acid solution were added. After the mixture had been heated on the steam bath for 2 hours to facilitate coagulation of the precipitate, it was filtered on a quantitative filter paper. The paper and the precipitate were washed free from sugar with ammonia-free water, placed in an 800-cc. Kjeldahl flask, and analyzed for nitrogen by the method described above
35
for total nitrogen. Blanks were determined on a similar solution of protein-free sugar. The quantity of nitrogen found in these blanks was deducted from that found in the tannic acid precipitate, and the result was calculated to parts per million. The quantity of protein nitrogen in the representative sugars is shown in Table I. IN WHITEGRANULATED SUGARS TABLE I. NITROGEN
SAMPLE
1 2 3 4 5 6 7 8 9 10
11 12
13
14
15 16 17 18 19
20
21 22 23 24
26 26 27 28 29 30 31 32
TOTALPROTrmIN AMINOACID N A 8 N N N NHs P . p . m. 35 45
5
10
33 45 7
23
64 24 20
5 13 52 25 82
P.p,m. 0.3 3.9 0.0 1.0 0.8 1.6 0.0 4.4 0.4 0.3 2.8 0.5 0.4
1.3
P.p.m. 3.5 4.0 0.0 5.5
5.5 1.0 0.0
6:O
0.0 5.0 2.5 0.0 7.5
4.1
4.0
6.8 8.4
7.5
N A8 NOa-
P.p.m.P.p.m. 0 4 8 0 1 5 2 4 0 8 2 30 2 2 2 0 1 3 2 10 0 6 0 6 2 8
N
A8
NOIP . P . ~ . 0.004 0.004 0.0 0.014
0.024 0.0 0.020 0.0 0.002 0.003 0.001 0.0
4 0
14 10
0:002 0.004
2 2 2 4 0 12 2 6
14 12 8 12 10 12 2 10
0.009
36 17
26 55 72 86 30 22 17 186 7 33 30
4 16
18
2.4 0.9
4.6
2.1 0.0
2.4 4.8 0.0 1.4 0.0
6.0 5.0 5.0 4.0 10.0
0.0 7.5
8.0 0.0 3.5 2.5
6
1
0 6
8 2 8 8
0.049
0.001 0.001 0.001
0.001 0.007
0.001 0.039 0.0 0.014
0.001
AMINOACIDNITROGEN The amino acid nitrogen content of the sugars was determined by the ninhydrin method ( I , 9). This test, it must be remembered, is not specific for amino acids b u t is given by compounds having an amino group in the alpha position to a carboxyl group. The quantity of nitrogen present in this atomic grouping in the representative white sugars is shown in Table I. Some sugars when treated with ninhydrin give a color which is redder than that of the standard solutions containing aspartic acid. This is due t o the fact that different amino acids produce slightly different tones of purple, that given by aspartic acid representing the most commonly encountered color. The reddish nuance causes some difficulty in matching the colors of the solutions, but satisfactory approximations are possible. The fact that proteins themselves respond to this test does not mean that the results found by this method must be greater than those found in the tannic acid-protein method, which shows the total nitrogen in the protein. Only a small proportion of the total nitrogen in any protein is present in the grouping, R.CH(NH2).COOH, which reacts with ninhydrin. Nitrogen in other groupings is inactive Of the amino acids themselves, proline and oxyproline, containing no alpha amino groups, do not respond to the test. Tryptophane, which contains, besides the active grouping, nitrogen bound in a ring, reacts for only one-half its total nitrogen, and arginine and histidine similarly would show only one-third of their total nitrogen. These facts must be borne in mind in interpreting the values found by this method, which is specific for no individual compound but is characteristic for an atomic grouping occurring in varying proportions in many compounds.
AMMONIAAND NITRATES The determination of ammonia in low-purity products has been thoroughly worked out by Baerts and Delvaux (4) and
e
ANALYTICAL EDITION
36
by Vondrak (26), who have shown that the true value for ammonia may be obtained by distilling with magnesium oxide a t 40’ to 50” C. under reduced pressure. If the distillation is carried out a t atmospheric pressure, the amides present are hydrolyzed to ammonia even by magnesium oxide. I n white sugars, the ammonia present is so small that it was not deemed practical to use reduced pressure for the distillation. I n fact, the values given in Table I for ammonia are really the values of blanks for the nitrate determinations, representing the nitrogen from all compounds present which yield ammonia when treated with alkali under the conditions obtained during the determination of nitrates. Of the various methods of determining nitrates, the only feasible one in which the large excess of sucrose does not interfere is that in which the nitrates are reduced to ammonia. The reducing agent chosen was Devarda’s alloy (6, 8), and the method of reduction was essentially the same as that used by Chibnall
(7). REAGENTS MAGNESIAWATER. This is prepared as described under “Total Nitrogen.” SODIUM HYDROXIDESOLUTION.A solution containing 400 grams of stick sodium hydroxide per liter is prepared with magnesia water. DEVARDA’S ALLOY. The best grade of alloy is ground fine enough to pass through a 50-mesh screen. NESSLER’SREAGDNT(82). STANDARD AMMONIUMCHLORIDE SOLUTION. The standard solution is prepared as described under “Total Nitrogen,’’ METHOD Two and five-tenths grams of sugar were dissolved in 250 cc. of magnesia water in an 800-cc. Kjeldahl flask. One gram of powdered Devarda’s alloy, 50 cc. of the sodium hydroxide solution, and a teaspoonful of the finely powdered magnesium oxide were added, and the flask was immediately connected to the distillation a paratus. At the same time a similar solution was prepared wittout the alloy and connected t o the still. .After the had stood at ordinary temperature for at least 30 minutes a n i t h e vigorous evolution of hydrogen had subsided, the mixtures were slowly heated t o boiling and &stilled as in the determination of total nitrogen, until 200 cc. ?f distillatetehadbeen collected in Nessler tubes. The ammonia in the distillate was determined colorimetricall and corrected for .ammonia and nitrates in the reagents as &und in blank determinations carried out without sugar. The amount of nitrogen as ammonia in the distillate from the mixture t o which no alloy had been added represents the nitrogen as “ammonia” in the sugar. When this quantity is deducted from that found in the distillate from the mixture to which the alloy had been added, the difference is the nitrogen as nitraQ in the sugar. Although Devarda’s alloy reduces nitrites as well as nitrates, and therefore the nitrate value so found is really the.sum of the nitrate and the nitrite nitrogens, no further correction is generally necessary, since the nitrite nitrogen is such a small quantity (see Table I). When Nessler’s reagent is added to thege distillates, a greenish color is generally formed, due t o volatile organic substances formed by the action of the alkali on the sugar. (A similar color is formed when Nessler’s reagent is added t o a dilute solution of alcohol.) This coloration does not interfere with the development of the color due to ammonia, but its presence in the solutions precludes the measurement of the colors spectrophotometrically. If, however, both the standard ammonium chloride tube and the unknown tube are. viewed through a Wratten K-3 No. 9 yellow light filter, the interference is overcome for colorimetric comparison and perfect matching is possible. The quantities of ammonia and of nitrates in the representative sugars as determined by these methods are shown in Table I. NITRITES The presence of nitrites in sugar-beet products has been ascribed by Urban (25) to the introduction of oxides of nitrogen with the carbon dioxide used for carbonation. I n the burning of limestone in kilns for the production of lime and carbon dioxide, a small quantity of nitrogen is unavoidably fixed as oxides which are absorbed by the alkaline juice along
Vol. 4, No. 1
with the carbon dioxide and give rise to nitrites. Urban determined them in the juices by reduction with Devarda’s alloy. This method is not feasible with white sugars because the quantity of nitrites present is so small. The same objection holds in regard to the gasometric method used by Pellet ( l a ) and by Andrlik and Stanek (6.However, when analyzing white sugar, conditions are ideal for the use of Ilosvay’s modification of Peter Griess’ extremely sensitive colorimetric test ( l l ) which, , of course, cannot be used with colored sugars and juices. Since this test is slightly less sensitive in the presence of sucrose the standard comparison solutions must contain the same concentration of sucrose as the unknowns. The reagents, sulfanilic acid and alpha-naphthylamine hydrochloride solutions, and the standard nitrite solution were prepared according to the directions recommended by the Association of Official Agricultural Chemists (3). The tests were made on solutions containing 20 grams of sugar in each 100 cc., after objectionable turbidity had been removed by filtration with Filter-Cel. The comparison solutions were made from a similar solution of nitrite-free granulated sugar, to which known volumes of the standard nitrite solution were added. After 2-cc. portions of each reagent had been added to 100 cc. of each known and unknown solution in Nessler tubes, the solutions were thoroughly mixed, allowed to stand a t room temperature for a t least 30 minutes, and then compared colorimetrically. If, as rarely happens, the color of the unknown is tinged with yellow or brown, the color of the standards may be given a similar hue by the addition of ti few drops of a very dilute caramel solution. As shown in Table I, the quantity of nitrites in white sugars is generally very small. ACKNOWLEDGMENT Thanks and appreciation are extended to J. B. Snider of the Carbohydrate Division, Bureau of Chemistry and Soils, who kindly performed most of the analyses whose results are recorded in Table I. LITERATURE CITED (1) (2) (3) (4)
(5) (6) (7) (8) (9) (10) (11) (12) (13) (14) (15)
Ambler, Intern. Sugar J.,29,382,437,498 (1927). Andrlik and Stanek, 2.Zuckerind. Bfihmen,26, 228 (1901-2). Assocn. Official Agr. Chem., Methods, p. 229 (1925). Baerts and Delvaux, Sucr. belge, 50, 52 (1930); Deut. Zuckerind., 55, 1331 (1930). Browne, ”Handbook of Sugar Analysis,” p. 270, Wiley, 1912. Cahen, Analyst, 35,307 (1910). Chibnall, Biochem. J., 16, 349 (1922). Devarda, 2. anal. Chern., 33, 113 (1894). Grrybowski, Gaz. Cukrownicza, 37, 291 (1930). Homuth, Centr. Zuckerind., 36, 388 (1928). Ilosvay de Nagy Ilosva, Bull. chirn., (3) 2, 347 (1889). .Pellet, Ann. chim. anal., 5, 361 (1900). Pucherna, 2. Zuckerind. cechoslovak. Rep., 55, 145 (1930-1). Rice and Murray, IND.ENQ.CHEM.,19, 214 (1927). Rumpler, “Die Niohtzuckerstoffe der Ruben,” pp. 36-40,
260-374, 401-450, 459-71, 499-505 (1898). (16) Riimpler, Ibid., p. 357. (17) Riimpler, Ibid., p. 358. (18) Spencer-Meade, “Handbook for Cane-Sugar Manufacturers and Their Chemists,” pp. 10 and 11, Wiley, 1929. (19) Stanek, 2. Zuckerznd. Bohmen, 29,410 (1904-5). (20) Stanek and Vondrak. Z Zuckerind. cechoslovak. Rep., 46, 227 (1921-2). (21) Stoltzenberg, 2. Ver. deut. Zuckerind., 49, 440 (1912). (22) Sutton, “A Systematic Handbook of Volumetric Analysis,“ p. 486, Blakiston, 1924. (23) Taegener, Centr. Zuckerind., 32, 852 (1924). (24) Tovarnizkii and Maksimovich, Zhur. Sakharnoi Prom., 4, 77 (1930). (25) Urban, 2. Zuckerind. cechoulovak. Rep., 44, 93 (12/31/1919). (26) Vondrak, Ibid., 46, 691 (1921-2). (27) Vondrak, Ibid., 51, 41, 261 (1926-7). R ~ C H ~ I VJuly E D 10, 1931. Contribution 112, Carbohydrate Division, Bureau of Chemistry and Soils.
