ANALYTICAL CHEMISTRY
1616 (3) Cheng, K. L., and Bray, R. H., AN.~L.CHEN.,25, 655 (1953). (4) Fortune, W. B., and Mellon, iM. G., IND.ENG.CHEW,ANAL. ED.,10,60 (1938). (5) Gillis, J., Bull. centre belye &/de et document. eaux (Lidge), No. 22,233 (1953). (6) Gillis, J., Hoste, J., and Fernandex-Caldas, E., A d e s edafol. fisiol. vegetal (‘Madrid), 9, 585 (1950). (7) Hill, Rolland, Ed., “Fibres from Synthetic Polymers,” p. 61, Elsevier Publishing Co.. Houston, 1953. ( 8 ) Ibid., p. 63. (9) Hoste, J., Ar~al.Chirn. Acta, 4, 23 (1950). (10) Hoste, J., Research, 1,713 (1948). (11) Hoste, J., Eeckhout, E., and Gillis, J., A d . Chim. Acta, 9, 263 (1953). (13) Hoste, J., Heiremans, A , , and Gillis, J., Mikrochernie uer. mikrochim. Acta, 36/37,349 (1951).
. 24, 991 (13) Rlarteiis, R. I., and Githeiib, R. E., A N ~ L CHEM., (1952). (14) Maute, R. L., and Owenh, .\I I... Jr., Ibid., 26, 1723 (1954). (15) Sandell, E. B., “Colorinieti ic Determination of Traces of Metals.” 2nd ed.. u. 87. Inter>cience. New York. 1950. (16) Ibid., p. 304. (17) Smith. G. F.. and Richter, F. P., “Phenanthroline and Substituted Phenanthroline Indicators,” p. 67, G. F. Smith Chemical Co., Columbus, Ohio, 1944. (18) Smith, G. F., and Wilkins, D. H., ASAL. CHEM.,25, 510 (1953). (19) Stone, I., Ettinger, R., and Sanz, C., Ibid., 25, 893 (1953). (20) Timberlake, C. F., Chemistry & I n d u s t r y , 47, 1442 (1954). I
.
RECEIVED for review April 23, 1955. Accepted July 13, 1955. Division of Analytical Chemistry,127th Meeting ACS, Cincinnati, Ohio, 1955.
Colorimetric Submicromethod for Determination of Ammonia P. G. SCHEURER and F. S M I T H Division o f Biochemistry, University of Minnesota, St. Paul, Minn. The blue color, formed when sodium phenate is added to a solution of ammonia that has been treated with hypochlorous acid, forms the basis of a method for the determination of submicro amounts of ammonia. The method has been used for the determination of the molecular weight of compounds containing nitrogen which is easily transformed into ammonia. Coupled with the cyanohydrin reaction it can be utilized for the determination of the average molecular weight of aldehydes, such as sugars and certain polysaccharides possessing a free reducing group. The procedure might lead to the simplification of protein determination by the Kjeldahl method.
I
N THESE investigations into polysaccharides the authors
sought t o devise a method for determining the reducing group and, hence, the average molecular weight of these substances by a chemical method. T h e reaction between an aldehyde and the elements of hydrocyanic acid, known to proceed to completion under certain conditions according t o the law of mass action with simple aldoses, has formed the basis of these studies (4, 6). In the formation of polysaccharide cyanohydrins of high molecular weight the amount of combined cyanide is relatively small, and its determination requires an extremely sensitive method unless unlimited amounts of polysaccharide are available. T h e difficulty has been overcome by the use of ryanide l a h l e d with radioactive carbon-14 (5). T h e blue color produced by the action of phenol and hypochlorous acid upon ammonia ( 2 , 6-9) can be used t o determine fairly accurately extremely small quantities of ammonia. T h e density of color developed by dilute solutions of ammonium chloride (measured by an Evelyn colorimeter and filter 620) was found to be a linear function of the concentration of ammonia from 0 t o 8 x 10-8 mole of ammonia per ml. T h e slope of this linear function, moles of ammonia per milliliter per abaorbance, hov*ever, was best determined by saponifying an aliquot of a standard solution of purified acetamide containing about 0.0002 gram of acetamide with ammonia-free base. The solution was steam distilled with steam generated from a dilute solution of sulfuric acid and the ammonia-containing distillate collected in about 15 ml. of ammonia-free distilled water. About 75 t o 100 ml. of distillate was collected and the exact volume determined by weighing. This standard ammonia solution was then used t o determine the slope of the linear relationship between concentration of ammonia and color intensity. Bcetamide
as a primary standard was determined by saponification, steam distillation into standard acid, and back-titration with standard base using methyl red indicator. T h e slope has been found to lie independent of the concentration of either the phenol or the chlorine water reagent. The slope obtained, however, depends upon the success with which ammonia has been removed from the saponifying base. T h e problem of its removal has not yet been solved and consequently the slope relationship muat he I edeterniinrd every time fresh base is prepared. In contrast t o the phenol-hypochlorite reaction ( d , 6, 9) no heating is necessary, as the reaction takes place quickly a t room temperature. In addition, more color is produced with the phenol-chlorine water than with the phenol-hypochlorite reagent. The color disappears only very slowly, 1% approximately in 24 hours. I t is possible t o detect 5 X 10-lo mole of ammonia per nil. of solution. By comparison, Nessler’s reagent has only one tenth of this sensitivity and, moreover, the color must be read shortly after its development. The values of slope obtained by four acetamide determinations differed from their average value by about 2%. The accuracy of this method is therefore within &2%. PREPARATION OF REAGENTS
Ammonia-Free Distilled Water. This reagent is prepared by distilling distilled water from a dilute solution of sulfuric acid in a n all glass apparatus. Hypochlorous Acid Reagent. Chlorine is bubbled into icecold distilled water until solid chlorine hydrate forms. The a p proximate chlorine content determined by the iodide-thiosulfate method should thereby exceed the required minimum value, about 0.08M chlorine. The molarity should, however, be determined approvimately before use by adding 10 ml. of 5y0 potassium iodide solution and titrating the liberated iodine with 0.2M sodium thiosulfate. The required minimum chlorine content is determined by plotting the maximum color development obtained for a given ammonia solution against the strength of the hypochlorous acid reagent (Figure 1). A suitable ammonia solution which gives a maximum color development of about Soy0transmittance is prepared by adding one drop of 0.4M ammonium chloride solution to 500 ml. of ammonia-free distilled water. Potassium iodide, 5y0 aqueous solution. Sodium thiosulfate solution, 0.2M, 5 grams of sodium thiosulfate (hypo) per 100 ml. of solution. Sodium Phenate Reagent. A cool solution of sodium hydroxide, 7.2 grams (0.18 mole), in 300 ml. of ammonia-free water is added t o commercial phenol, 16.7 grams (0.0178 mole). and shaken until the latter is dissolved. Manganous chloride solution, 0.003-W.
V O L U M E 2 7 , NO. 10, O C T O B E R 1 9 5 5
1617
Ammonia-Free Sodium Hydroxide Solution. Sodium hydroxide pellets are dissolved in ammonia-free water to give a 20% solution. The amount of ammonia in commercial sodium hydroxide varies from sample to sample. This ammonia cannot be removed effectively by dissolving the hydroxide in distilled water and passing ammonia-free steam through the solution. Several experiments with different samples of sodium hydroxide revealed that a steady diminishing rate of ammonia removal is obtained (Figure 2).
The solution was acidified with 3 ml. of 0.4A7acetic acid, and the hydrogen cyanide was removed by blowing air through the solution for 1 hour. (The air used for this and other experiments was blown through phosphoric acid to remove possible ammonia contamination.) The cyanohydrin was saponified with ammonia free sodium hydroxide (10 ml., 20%) and the distillate (114.57 grams) investigated for its ammonia content; the absorbance was 0.367. Because the cyanide decomposes sloa ly to give ammoilin, a reaction blank was run for these determination. Mol. wt. glucose =
0.000386 1.60 X 10-7(0.367 X 114.57 - 29.0)
=
155
(where 29.0 is average blank of 6 determinations) Calcd. for CeH1206: mol. wt. = 180
MOLAR,-Y
OF
c n Lc-iv
E
Figure 1. Color development of standard ammonia solution us. strength of hypochlorous acid reagent Reaction time 5 minutes
However, if sodium sulfide is added t o the base, all the ammonia is removed with the first 50 ml. of distillate. This suggests that the above retention of ammonia in commercial sodium hydroxide is due t o the presence of heavy metal ammonia complexes. Unfortunately a small amount of hydrogen sulfide is obtained in the distillate which can react with the hypochlorous acid reagent and interfere with the color development. Heavy metals may also be removed by sodium silicate. Since sodium silicate forms slomly by the etching of the glass, this accounts for t,he observed gradual removal of the ammonia on steam distillation of hydroxide solutions. If a simple method can be discovered for the preparation of ammonia-free bitse, an absolute method of determination of submicro quantities of ammonia may become available. The slope relationship moles of ammonia per milliliter per absorbance, will be independent of the preparation of the reagents. Color Development. The solutions are added to the colorimetric tubes in the following order: 10 ml. of the unknown ammonia solution are mixed with 1 ml. of hypochlorous acid reagent. The initial reaction between the chlorine and ammonia is complete in 1 minute or less with 0.10M chlorine. At lower concentrations more time is required. After 5 minutes, 1 ml. of phenate reagent and one drop of manganous chloride reagent are added. After shaking the tubes, the color develops rapidly and reaches a maximum intensity in about 3 minutes. The intensity of color was measured in an Evelyn colorimeter using a KO.620 filter. APPLICATION OF T H E METHOD
Apparent Average Molecular Weight of Laminarin. Duplicate runs were made as follows: -4 mixture of laminarin, 0.00673 gram in 1 ml. of solution, potassium cyanide, 0.1 gram, in 1 ml. of solution, and 2 ml. of 0.4,V acetic acid was heated in a n-ater bath a t 40" C . for 20 hours, The solution was acidified, freed from hydrogen cyanide, saponified, and steam distilled as before. The reaction is incomplete after 3 hours. The result after 42 hours is approximately the same as that obtained after 20 hours. The cyanide blank, however, becomes so great after 42 hours that the results are no more than an indication of the chain length. As the absorbances obtained from the distillates in all cases were too great to be read with accuracy, aliquots were taken and diluted. Calculations show:
Laminarin
fi..
Blank z. 1%.
Distillate
Weight Aliquot
Diluent
Absorbance
104.24 108.83
20.33 19.85
70.02 68.87
0.297 0.276
114.10 115.50
19.33 l Y .90
68.35 62.53
0,240 0.258
Laminarin I.
ii.
A = 0.297 X 70.02 X 104.24/20.33 = 106.5 A = 0.276 X 68.87 X 108.83/19.85 = 104.2
Av. = 105.3 Blank
0.240 X 68.36 X 114.10/1!3.33 = 96.8 0.288 X 62.53 X 115.50/19.90 = 93.5 A v . = 95.1 0.00673 Mol. wt. laminarin = 1.60 X 10-7(105.3-95.1) 4130 4130 Chain length (av.) = - = 25 anhydroglucose units (approx.). 162 2.
ii.
= =
This result is in reasonably good agreement with a repeating unit of 20 as found by methylation studies (1, 3).
x
Molecular Weight of Gluconamide. This experiment is representative of many carried out with pure D-gluconamide (melting point 144') [a]'; +31° in water (c, 2.01, after recrystallization from ethyl alcohol. D-Gluconamide, 0.01 gram, was dissolved in 49.75 grams of ammonia-free water and 9.92 grams of solution withdrawn for saponificatioii with ammonia-free sodium hydroxide (10 ml.). The weight of distillate collected was 110.30 grams. Since a n absorbance of 0.491 was developed from 10 ml. of distillate, the molecular weight was calculated from the relationship Mol. wt. gluconamide = (where 1.95 X
0 .01 X 9.92 49.75 1.95 X lo-? X 0.491 X 110.30
ZOO
= 188.8
is the slope)
Calcd. for CeHiaOaX: mol. R t .
=
195
Molecular Weight of Glucose. The following is representative of many experiments carried out with o-glucose. Pure anhydrous or-D-glucose (melting point 146') [ a ]22 +52.5" equilibrium value in water (c, 2.0), 0.000386 gram in 1 ml. of solution (obtained by diluting a weighed sample of glucose by weight not volume for greater accuracy), potassium cyanide, 0.1 gram in 1 ml. of solution, and 2 ml. of 0.4N acetic acid (ammonia-free reagents) were heated in a water bath a t 40" C. for 3 hours (4).
VOLUNE
300
400
I
CISTILLATE h l l
Figure 2. Evolution of ammonia during steam distillation of different samples of hydroxide solutions Samples 1 to 4
Apparent Average Molecular Weight of Corn Amylose. Corn amvlose cyanohydrin prepared as described for laminarin (reaction time 70 hours) q - a ~isolated by acidification with acetic acid followed by precipitation with ethyl alcohol (3 volumes). The amylose cyanohydrin s-as dialyzed for 7 days against distilled water, precipitated with ethyl alcohol, and dried, and the nitrogen
ANALYTICAL CHEMISTRY
1618 content determined as above. The apparent average mo1ec:ulur weight of the corn amylose in duplicate experiments was found t o be 32,000 and 39,000. SUMMARY AND CONCLUSION
Although the use of the hypochlorite-sodium phenat,e reiictioii is difficult to control for the quantitative determination of ammonia ( 6 ) , a method using hypochlorous acid gives reliable results for the det,erminatiori of minute amounts of ammonia. The procedure gives good results with simple nitrogen-containiiig compounds such as acetamide and n-gluconamide, which libemte ammonia directly upon treatment with alkalies. Thtx method could be employed in conjunction with micro or si111micro Kjeldahl determinations. I n conjunction with the Kiliani cyanohydrin reitctiori thct method has been used for the determination of the appiirc'nt average molecular weight of the polyglucosan and laminarin, which was found t o correspond t,o about 25 anhydroglucoac~ residues. As the Kiliani condensation requires considerable time to reach completion, hydrolysis of the cyanide ion must be taken into account. The effect is too large to be corrected for wcurately by a blank determination; and it seems necessary to remove ammonium ions produced by hydrolysis of cyanide ions I>y some chemical method, or by a physical method w c h ar , However, the polyqaccharide cyanohydrin (-:in be
purified by precipitation with alcohol as in the case of amylose , cyanohydrin. Water-insoluble polysaccharides such as celluloac cayanohydrin can be filtered off and washed t o remove ('ont:tniiiiating ammonium ions. ACKYOWLE:DG\I EYT
Thc authors wish t o thank t h r l 1'. I. du Pont de Kernour5 & C h . for a grant, which defi:t\ed the expenses of this work and provided a fellowship for P. G. S c h t w r r . LITER i T U R E CITED
(I) Barry, V. C.. AIcCormick, Joan E., and Mitchell, P. W . I).. .I. Chem. SOC.,1954, p . 3692. (2) Borsook, H., J . B i d . Chsm., 110, 48 (1935). ( 3 ) Connell, J. J., Hirst, E. L.. and Percival, E. G. V., J . ( ' h e m . SOC.,1950, p. 3494. (4) Frainpton, V. L., Foley. Lucia P., Smith, L. L., and Jlalone. J. G., ANAL.CHEM.,23, 1244 (1951). ( 5 ) Isbell, H. S.,Science, 113, 532 (1981). (6) Russell. J. A.. ,J. B i d . Chein.. 156. 457 (1944). (7) Thomas, P., bull. SOC. chii7z.. '11, 796 (1i12). ' (8) [bid., 13, 398 (1913). (9) Van Slyke, D. D.. and Hiller, .ilnia. .J. Bioi. Chem., 102, 499 (1933).
(LO) Zalta. J. P., Bd1. SOC. chini bid.. 36, 444 (1954). RECEIVEDfor review January 18, 1953. -\rcepted June 10, 1955.
Paper No. 3310 Scientific Journal Series, I l i n r i e w t a .igricultural Exppriinent Station.
Absorptiometric Microdetermination of Total Sulfur in Rubber Products K. E. KRESS Firestone Tire and Rubber Co., Akron 17,
Ohio
The 1.0 to 2.5'30 of sulfur normallj present in ruhber products is oxidized with concentrated nitric acidbromine reagent, followed bj perchloric acid in the presence of excess lead nitrate. Sulfur as lead sulfate is precipitated and washed with acetone. The lead sulfate is dissolved in 50% hjdrochloric acid and absorbance of the lead chloride complex is recorded at 27'0 mp. Sulfur is calculated on the basis of the nieasured lead content of the precipitate. The high sensitivity puts the method in the micro range. 411 experienced analyst can analyze 40 to 50 samples a cia!. Precision and accuracy are comparable to those of the conventional barium sulfate gravimetric method at the low sulfur concetitrations normally found in ruhher products.
T
HE literature contains numerous references to deterniinatiori of total sulfur in rubber products as barium sulfate by gravimetric, volumetric, turbidimetric, or nephelometric methods. The latter methods were developed in the course of a search for a more rapid procedure than that afforded by standard gravimetric methods. Recently an amperometric titration has been applied for the indirect seminiicrodetermination of sulfur in organic conipounds aft,er precipitation as lead sulfate (8). However, digestion of the sample in a sealed Carius tube has serious disadvantages for routine cont.ro1 of sulfur in rubber products, and the range of sulfur concentration (about 1 to 6 mg. of sulfur) recommended rcquires rubber samples of above 50 mg. h new combustion apparatus for automatic determination of sulfur in steel has been applied successfully to rubber ( 2 ) ) but the cost of this single-purpose instrument precludes its use in most rubber laboratories.
I'ei~~liloric acid has been used to speed up oxidation of ruI>ljer 1)roducts during determination of sulfur (,9). Published methods have all been for work on a niitcro scale, where the possible explosion hazard has retarded generid acceptance of this strong osidant in rout,ine analysis of rubber. Even with the more rapid osid:ition provided by perchloric acid, the conventional gravimetric barium sulfate procedure ip used to precipitate the sulfur. This limits the speed of analysk. .1 very sensitive absorptiometric method for determining lead (/i) depends on the strong selwtive ultraviolet light absorbance near 270 mp of the lead chloride mrnplex formed in strong hydrochloric acid solutions. A rapid osidation ivith nitric arid perchloric acids on a safe micro scale has been developed and is reported here. Sulfur is insoluhilized and precipitated a s lead sulfate with acetone. Absorptiometric measurenient of lead as the lead chloride coinples in 50% hydrochloric acid provides data for calculating sulfur concentration. EQUIPNIENT, REAGEYTS, AYD PROCEDURE
Equipment. Sample test tubes, such as 15 X 100 mm. lipless w l t u r e tubes, of 12- to 15-ml. volume. Capillary-tipped pipet connected to a water pump vacwum source. It may be made by drawing out the lower end of a standard pipet over a hot flame until i t is of capillary size, with thin walls and an internal bore of 1 t o 2 mm. Standard high speed semimicrocentrifuge capable of holding sample tubes. Finger stall of pure gum rubber, or a finger from a rubber glove, extracted with a solution of 5% hydrochloric acid in acetone. Beckman DU quartz ultraviolet spectrophotometer with ultraviolet accessories, or a similar instrument. Roller-Smith microbalance, torsion type, of 25-mg. capacity with V-shaped pan, or similar balance weighing to 0.01 mg. Reagents. CONCESTR.4TED NITRIC -4CID-BROMINE. Add reagent grade bromine to concentrated reagent grade nitric acid. in
