INDUSTRIAL AND ENGINEERING CHEMISTRY
July 15, 1931
341
111-Determination of Labile Organic Sulfur'sz J. A. Ambler BUREAUOF CHEMISTRY AND SOILS, WASHINGTON, D. C.
HE presence of proteids
The quantity of the impurities in sugar which conT-tube, F , through which the in the sugar cane and tain labile sulfur may be determined by spectrophotostem of a dropping funnel, G, t h e sugar beet and metric measurement of the amount of methylene blue extended into the condenser. their juices has long been a produced under definite conditions from the hydrogen The T-tube was attached to m a t t e r of common knowlsulfide formed during digestion of sugar solutions with the stem of the dropping funedge, and since cystine is a alkaline lead acetate. The results obtained indicate nel by rubber tubing. The common cleavage product of that small amounts of compounds containing labile other opening of the T-tube protein, it is r e a s o n a b l e to sulfur are Present in all types of white direct-conwas attached by rubber tubs u p p o s e that derivatives of sumption suk!ars. Sucrose, by virtue of its degradation ing t o a glass tubeleading to a this s u l f u r c o m p o u n d are with alkalies, acts as a catalyst for the desulfuring of wash bottle, H , whichwas&cystine by alkaline lead solution. present in the mill and diffurectly connected to the absion juices and p o s s i b l y in sorption bottle, J , and this to small amounts in the white sugars crystallized from them. the trap wash bottle, K . The gas inlet tube of the digestion That such is indeed the case was shown qualitatively flask, D , was connected through a series of three wash bottles, by boiling with alkaline plumbite solution, thereby taking A , B, and C , with a tank of compressed nitrogen gas. Bottle advantage of the reaction characteristic of cystine and its A , connected directly with the nitrogen tank, contained a derivatives in which lead sulfide is formed by degradation. solution of potassium permanganate to remove oxidizable Fifty to 100 grams of a white sugar were dissolved in water, impurities in the nitrogen, bottle B contained alkaline pyroand the sucrose was removed a t room temperature as barium gallol solution t o remove any oxygen, and bottle C contained sucrate. The excess of barium ions in the filtrate was w a n - sodium plumbite to remove any trace of hydrogen sulfide. titatively precipitated with sulfuric acid. To the neutral The wash bottle, HI between the condenser and the abfiltrate were added a few cubic centimeters of lead acetate sorption bottle contained a solution of potassium dihydrogen solution and sufficient sodium hydroxide solution t o redissolve phosphate to absorb any volatile acids which may be carried the lead hydroxide a t first formed. This solution was boiled over (7). The absorption bottle, J , contained St. Lorant's for 1 hour and allowed to cool and stand overnight. In the zinc acetate solution (7). The trap wash bottle, K , contained morning there was a small amount of a finely divided, heavy water and served merely to prevent air from backing into the black precipitate on the bottom of the beaker. Attempts to absorption bottle, est>imatethe quantity of labile sulfur by this simple method Solutions failed to yield reliable results. Because of the extremely small quantity of the organic CYSTINE STANDARD-TWO-tenthS gram of cystine was dissolved sulfur compound present and because of the interference of the in a little 1:4hydrochloric acid and made up to 100 cc. with dislarge quantity of sucrose, it was impossible to detect the tilled water. Five cubic centimeters of this solution were diluted to 500 cc., giving a solution which contained 0.02 mg. of cystine presence of organic sulfur by the nitroprusside test ($1 or by per cubic centimeter. Sullivan's method ( I O ) , even after desugaring the solutions LEAD SOLUTION--Asaturated neutral lead acetate solution with barium hydroxide. A modification of the plumbite was used as the lead solution throughout the whole procedure. ALuLI-For the digestion, a 5 per cent suspension of powdered digestion method combined with the methylene blue colorimagnesium oGde. For making sodium Plumbite, a 30 Per cent metric method for hydrogen sulfide (1, 4, 7 , 8,Q) and with solution of sodium hydroxide. spectrophotometric measurement of the methylene blue WASHLIQUIDSFOR NITROGEN-(a) Two per cent potassium formed as described in the following paragraphs, proved permanganate solution. ( b ) For alkaline pyrogallol solution, 10 grams of pyrogallol successful and disclosed the fact that sucrose acts as an acdissolved in 30 cc. of water, and a solution of 175 grams of for the formation of lead sulfide in the degradation of were sodium hydroxide in 60 cc. of water were added. This solution cystine. must be renewed frequently, before it becomes completely ex-
T
Apparatus Since the method consists in part of an alkaline lead acetate
digestion. it is essential that no rubber or cork be used t o make unions a t those points where they will come in contact with hot vapor or liquid. Any all-glass apparatus for reflux digestion of the type of the Zeissel methoxy apparatus, which has provision for introducing a stream of gas and which has a digestion flask of a t least 503 cc. capacity, will be satisfactory. An apparatus, cheaper than a Zeissel outfit of the size needed, although more unwieldly because of its rigidity, was made by welding a 500-cc. Pyrex Claissen flask D, from which the small side outlet tube had been removed, to a Pyrex condenser, E , and sealing in a gas inlet tube as shown in Figure 1. The top of the condenser was closed by a rubber3 stopper bearing a 1 Received 2 1
April 20, 1931. Contribution 110, Carbohydrate Division, Bureau of Chemistry and
Soils. a All rubber stoppers and tubing used were boiled with alkali and then with dilute acid and finally with water before being used.
hausted. ( 6 ) Alkaline lead acetate solution. To 5 cc. of the saturated lead acetate solution sufficient 30 per cent sodium hydroxide solution was added to redissolve the initial precipitate of lead hydroxide. .The solution was then diluted as much as necessary to give a minimum depth of 2 inches (5.08 cm.) of liquid through which the nitrogen must bubble. ABSORBING SOLUTION FOR VOLATILE ACIDS-Fifty cubic centimeters of a 10 per cent solution of potassium dihydrogen phosphate ( 7 ) . SUCROSE SOLUTION FOR STANDARDS-Twenty grams Of cube sugar or of specially purified sucrose were dissolved in water and diluted to 100 cc. with distilled water. volume of concentrated hydroHYDROCHLORIC ACID-one chloric acid was diluted with four volumes of water. ST. LORANT'S ABSORPTION SOLUTION (7)-Fifty grams of zinc acetate, 10 grams of sodium acetate, and 0 5 gram of sodium chloride were dissolved in water and diluted to 1 liter. ST. LORANT'SDEVELOP~NG SOLUTIONS (7)-(a) One gram of dimethyl-+phenylenediamine sulfate was covered with 100 cc. of water, and immediately, with cooling, 400 cc. of 60" Be. sulfuric acid were added. With vigorous cooling, the solution was diluted to 2 liters. ( b ) Twenty-five grams of ferric ammunium sulfate (clear,
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342
ANALYTICAL EDITION
purple crystals) were moistened with 5 cc. of 60’ BC.sulfuricacid, and immediately dissolved in water and diluted to 200 cc. Procedure
The digestion apparatus was washed twice with tap water, cleaned with concentrated nitric acid, and rinsed thoroughly twice with tap waterand twice with distilled water. This thorough cleaning was done before each determination. The reagents were introduced through a clean tube inserted into the condenser and extending to a point below that at which the vapors will condense. One cubic centimeter of lead acetate solution was added first, next a measured volume of the standard cystine solution, and then 25 cc. of the magnesia suspension. The liquid adhering to the tube was washed into the flask by rinsing the inside and outside of the
Figure 1-Apparatus for Labile Sulfur in Sugar
tube and the inside of the condenser with a total volume of 125 cc. of distilled water. For the standards containing sucrose, 50 cc. of the 20 per cent (by volume) sucrose solution (10 grams of sucrose) were added through the tube instead of the first portion of the rinse water, and the washing down was completed as before with a total volume of 75 cc. of water. No lead compound must be allowed to remain in the condenser above the highest point washed by condensing vapors. Determination
ALKALINEDIGESTION-The flask was connected by the inlet tube and the T-tube with the properly charged wash bottles. After 5 cc. of water had been placed in the dropping funnel as a seal, nitrogen was passed through th6 apparatus until the air was completely displaced from it. While this was taking place, the absorption bottle, J, was charged with 20 cc. of St. Lorant’s absorption solution and 10 cc. of water and connected to the apparatus together with the trap, K. At the same time another absorption bottle was similarly charged and connected to the end of the train of bottles until all the air had been displaced from it, when it was disconnected and immediately closed to the air. Its contents served for the development of the blank standard solution for comparison in the spectrophotometric measurements later. The stream of nitrogen was slowed down to about one bubble a second, and the mixture in the digestion flask was boiled gently for 2 hours. At the end of this period the stream of nitrogen was speeded up so that there was a steady
Vol. 3, No. 3
series of bubbles in the absorption bottle, one bubble leaving the bottom of the inlet tube just as the preceding one was about to break on the surface of the liquid. Without admitting air to the apparatus, 60 cc. of the 1:4 hydrochloric acid were added through the dropping funnel, which was again with about cc* Of water* Cracked ice was placed around the absorption bottle and the closed bottle containing the blank. As soon as the magnesia was all washed down from the sides of the flask, heating was stopped and the stream of nitrogen continued for a total of 1hour from the time of acidification. DEVELOPMENT OF METHYLENE BLUE-The absorption flask was disconnected, instantly closed to the air, and removed from the ice bath. With as little opening to the air as possible, 7.5 cc. of St. Lorant’s solution of dimethyl-pphenylenediamine and 2 cc. of his ferric sulfate solution were quickly pipetted into the absorption bottle and into the blank bottle. The bottles were closed and shaken. By applying alternately gentle suction and pressure on the inlet tubes with the outlet tubes closed, the insides of the inlet tubes were wet with the mixtures in the bottles. The closed bottles were allowed to stand for 1 hour, during which time they assume room temperature and the maximum depth of color of the methylene blue is developed. The colored solution was then transferred to a graduated cylinder containing 1 cc. of 95 per cent alcohol and diluted with water to 50 cc, After the solution had been thoroughly mixed by pouring it back and forth into the bottle twice, it was filtered through a dry paper. The first few cubic centimeters running through were discarded, and the rest of the clear filtrate was used to fill a 1-dm. color-analyzing tube. The blank solution was treated in exactly the same way and used as the standard in the spectrophotometric determination of the transmittancy, T , of the colored solution a t 610 mp.4 This method of using as the standard a blank solution containing all the reagents in the same concentrations and under the same conditions, practically corrects the value of T for the transrnittancy of the solution due to the reagents. From the value of T, that of - log T (or log 1/T) was obtained and plotted on coordinate paper against the quantity of cystine taken. By this means regular curves, as shown in Figure 2, were obtained. In determining the quantity of labile sulfur in a sample of sugar, the sugar was dissolved in distilled water in the proportion of 500 grams of sugar to 375 grams of water, and the solution was passed through a 150-mesh copper screen to remove suspended fibers, hairs, and particles of other foreign matter. After the lead acetate and the magnesia suspension had been added to the digestion flask, 350 grams of this sugar solution (equivalent to 200 grams of sucrose) were introduced to wash the lead-magnesia mixture from the tube and condenser. The latter were finally rinsed down with a little water from a wash bottle. The subsequent procedure was the same as before, If the color developed was too intense to be read accurately in the 1-dm. tube, either the colored solution was diluted to 200 cc. (or more if necessary) instead of to 50 cc., or shorter tubes were used. I n either case, the - log T value so found was multiplied by the appropriate multiple of 50 cc. used, or by the reciprocal of the length of tube used when this length was expressed as fractions of a decimeter. If the color was so intense that the - log T value was too great to fit on the standard curve, the determination was repeated with less sugar. The quantity of labile organic sulfur expressed as milligrams of cystine was found by means of the curve 4 Methylene blue has two absorption bands located at 609.3 and 667.8 mp (Schultz Farbstofftabellen). The former (or in round numbers 610 mu) was found more convenient than the latter, since it is in a portion of the spectrum where more accurate measurement is possible and may be used for a greater range of concentrations without resorting to dilutions or to shorter tubes.
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July 15, 1931
INDUSTRIAL A N D ENGl‘NEERING CHEMISTRY
developed from known quantities of cystine when the determination was made in the presence of sucrose, and,calculated to parts per million. Discussion
Although neither the production of lead sulfide from cystine (3, 6, 9) nor the formation of methylene blue from hydrogen sulfide (7, 8) is quantitative, it is possible to use these reactions in the estimation of labile sulfur in white sugars, if all conditions of the determination are maintained as strictly uniform as possible, as is shown by the results represented by Figure 2. The deviation of these curves from a straight line is due in part to the fact that the formation of lead sulfide is not quantitative under the conditions of the method, and is
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yields with potassium hydroxide a product (which he asserts is methylglyoxal), which yields iodoform on the addition of iodine solution. Methylglyoxal (CH,CO.CHO) is the aldehyde of pyruvic acid (CH3C0.COOH), and since Clarke and Inouye (3) have shown that the catalytic effect of pyruvic acid is to be attributed to the ketonic group, it is to be expected that methylglyoxal, which contains the same ketonic grouping and in addition an aldehydic group, would also act as a catalyst for the splitting off of hydrogen sulfide from cystine. The formation of iodoform from the cleavage products of sucrose when warmed with magnesia suspension was observed during this investigation by adding a solution of potassium iodide and iodine to mixtures of sucrose and magnesia suspension without the addition of sodium hydroxide. This would indicate that the action of magnesia suspension on sucrose is qualitatively similar to that of sodium hydroxide. Tn the experiments magnesia was used in the alkaline digestion instead of sodium hydroxide, because the solid magnesia in the suspension facilitates quiet boiling without bumping and maintains the pH of the solution more nearly constant than would a solution of a soluble alkali, and also because magnesia suspension is less destructive to the sucrose and causes less disooloration during the 2-hour digestion period. The method is accurate to within 0.05 mg. of cystine, within the range from 0.1 to 0.6 mg. Because of the steepness of the -log T curve, a comparatively large difference of -log T values on duplicate determinations makes very little difference in the results. Below 0.1 mg. of cystine the method is not accurate because of the extremely small amount of methylene blue formed and the resulting paleness and small -log T value of the solution. Determinations which fall on this portion of the curve are best reported as “traces” or “less than 0.1 mg.” The results obtained with representative white sugars are given in Table I, and indicate that all types of white sugars contain some labile organic sulfur compounds. The figures given are the averages of two or more determinations, except as noted. Table I-Labile
Organic Sulfur (as Cystine) i n White Sugars AMOUNT FOUND P.9 m
SAMPLE
DIRECT-CONSUMPTION
BEET SUGARS
a
0 8
b
15 1 1 0 8 0.8 16 2 9
G
d
i
h
MG. OF CYSTINE Figure 2-Curves
Trace
DIRECT-CONSUMPTION
for D e t e r m i n a t i o n of Cystine
i
3.4 0.84 0.4“
j
apparently a function of the original concentration of cystine. Another factor tending to cause deviation from a straight line is to be found in the different quantities of ferric ions in the standard and the colored solutions used for spectrophotometric measurement, since some ferric ions are reduced to colorless ferrous ions in the production of methylene blue. However, while this introduces an error its magnitude is relatively small a t 610 mp and may be wholly ignored, inasmuch as the method involves only relative values based on cystine and obtained by means of a specified arbitrary procedure. Curve I of Figure 2 shows the results obtained with solutions of cystine and demonstrates the feasibility of the method. Curve I1 shows the results obtained with solutions of cystine in the presence of sucrose, which increasgs the sensitivity of the method by accelerating the degradation of the cystine. This observation is in agreement with those reported by Clarke and Inouye (3), who found that salicylic aldehyde, benzaldehyde, and pyruvic acid catalyze the desulfurization of cystine. I n the case of sucrose, the decomposition products formed by heating the sugar with alkali act in a similar way. Fischler (5) has noted that sucrose
SUGARS
CANE
k
REFINED CANE SUGARS
a
1
14
m n
0.8
Trace
Single determinations.
Literature Cited (1) Almy, J . Am. Chem. SOC.,47, 1381 (1925). (2) Blankenstein, Bioch6m. Z , 218, 321-30 (1930). (3) Clarke and Inouye, J . Biol. Chenz , 89, 399 (1930). (4) Fischer, Ber., 16, 2234 (1883). (5) Fischler, 2. angew. Chem., 42, 682 (1929). (6) Morner, 2. physiol. Chem., 34, 210-2 (1902). (7) St. Lorant, Ibid., 186, 245-66 (1929). ( 8 ) St. Lorant, Ibid., 193, 56-8 (1930). (9) Sheppard and Hudson, IND.ENG.CHEM.,Anal Ed., 2, 73-5 (1930). (10) Sullivan, U.S. Public Health Refits. 41, 1030-56 (1926).
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