8x4
.
T H E J O U R N A L O F I N D U S T R I A L A N D E N G I N E E R I N G C H E M I S T R Y Vol.
We find t h a t within the temperatures a t which tests were made t h e best carbon is obtained by heating t o the highest temperature, full red heat. It is possible a n d even probable t h a t still better carbons might be prepared by heating t o even higher temperatures, b u t this would hardly be of practical interest. One experiment was made in which a quantity of B was heated in a clay crucible in a Fletcher furnace, b u t observation showed t h a t the temperature was not a n y higher t h a n we could obtain with the iron cylinder in t h e muffle furnace. The resulting carbon, after washing with acid and water, produced a color of 0 . 3 6 , which is very close t o the 0 . 3 2 shown in the above table for the muffle heated carbon. Another experiment was carried out in order t o see whether a good carbon could not be made in one operation. The iron cylinder described above was filled with dried kelp, and one of the caps was only screwed on loosely, so t h a t the fumes might escape, without giving t h e air free access t o t h e char. After heating t o full red heat the carbon was boiled o u t with acid, and washed with water. I t produced a color of 0 . 7 5 , a n d was therefore much less effective than the carbon produced in two operations. We have also found t h a t i t is not necessary t o extract t h e carbon directly with hydrochloric acid. T h e water-soluble salts can first be removed with this solvent, and the greater p a r t of t h e remaining ash is then dissolved with hydrochloric acid, after which the acid is again washed out with water. Summarizing briefly, our tests have shown t h a t a carbon which has a much greater decolorizing power than Norit can be prepared in the laboratory by quickly carbonizing dried Pacific Coast kelp in such a way t h a t the fumes can freely escape. After they cease t o come off, the char is transferred t o a closed iron receptacle and heated for 2 hrs. or so to red heat. Instead of charring dried kelp, “incinerated” kelp may be used directly. The carbon is then boiled out either with dilute hydrochloric acid, or first with water a n d then acid. This is again washed out with water, and the carbon dried. I t remains to be seen whether t h e process can be worked successfully a n d economically on a large scale, a n d whether the price t o be gotten for the finished product will warrant its manufacture. The most logical place to work out t h e first problem is the United States Experimental Kelp Potash Plant in California, and we hope t h a t t h e Bureau of Soils may be willing and able t o take u p this project. The great decolorizing power of the kelp carbon is probably due t o two factors. We had found before t h a t active decolorizing carbons can be prepared from cellulose materials by first impregnating them with either infusible substances like lime, alumina, silica, or else with such substances as chlorides, etc., which are volatile a t the temperature a t which the carbon is made. I n all these cases t h e carbon must be heated t o red heat t o get good results, and the impregnating substances must then be removed with proper solvents. I n the particular case of potassium chloride as impregnating substance t h e carbon obtained was
IO,
No.
IO
rather poor, and the potassium chloride content of t h e kelp alone would not explain the decolorizing power of the kelp carbon. There is also too little infusible ash t o account for. it. However, a distinguishing feature of kelp is its high nitrogen coptent, a n d i t seems reasonable t o suppose t h a t this is largely responsible for t h e great effect of kelp carbon. T h e great decolorizing power of carbons made from highly nitrogenous materials, like blood charcoal, or the carbon made from the residues of the manufacture of ferrocyanide a n d from similar materials has long been well known. We noticed t h a t in every case where we obtained a good carbon from kelp, Prussian blue was formed when the carbon coming from t h e muffle was extracted with hydrochloric acid. It imparted t o t h e wash waters a deep blue color, being dissolved in colloidal form. The r6le played b y t h e nitrogen is not known definitely, b u t the effect of its presence is quite plain. SUMMARY
It is shown in this paper t h a t under proper conditions a decolorizing carbon much more effective t h a n Norit can be prepared from Pacific Coast kelp. T h e f?ctors affecting the decolorizing power of the carbon are discussed, and a method for making the most effective carbon is described. LOUIRIANA SUGAREXPERIMENT STATION N E W ORLEANS.LOUISIANA
THE ROLE OF OXIDASES AND OF IRON I N THE COLOR CHANGES OF SUGAR CANE JUICE‘ By F. W. ZERBAN
If the methods now being used in t h e manufacture of white sugar directly from t h e cane are t o be placed on a strictly scientific basis, it will be necessary t o gain a more accurate knowledge of the coloring matters which have t o be removed or the formation of which has t o be avoided. A great deal of work has already been done in this direction, but much more still remains t o be done. Any such investigation must first take into consideration t h e coloring matter found in the cane itself and in the raw juice obtained from it by applying pressure or diffusion. Previous investigators have found t h a t t h e cane contains chlorophyll, saccharetin, and, in. the case of dark-colored canes, also anthocyanin. Neither t h e chlorophyll nor t h e saccharetin dissolve in t h e juice upon milling, but pass into i t mechanically with t h e finely divided bagasse. They therefore do not affect t h e color of the juice itself, except in t h e form of solid suspended particles, and do not make their presence felt until the juice is treated with lime, a n excess of which causes t h e saccharetin t o t u r n yellow. Anthocyanin, however, is quite soluble in t h e raw juice, a n d this is the reason why dark-colored canes give a darker juice than light-colored ones. But all these facts do not explain the dark color of raw juice from light-colored canes. C. A. Browne2 1 Presented before the Division of Agricultural and Food Chemistry at the 56th Meeting of the American Chemical Society, Cleveland, S e g tember 10 to 13, 1918. 2 Louisiana Bulletin, 7 5 , 249; 91, 17.
Oct., 1918
T H E J O U R N A L OF I N D U S T R I A L A N D E N G I N E E R I N G C H E R I S T R Y
states t h a t t h e darkening of raw cane juice is due t o t h e effect of a n oxidizing enzyme, closely related t o oenoxydase, upon certain polyphenols present in t h e juice. This enzyme was found t o act upon hydroquinone, b u t not on tyrosin. Browne concluded from this t h a t tyrosinase was absent in cane juice. However, he found a peroxidase in some cases. Previously, Raciborskil had discovered t w o oxidizing enzymes in cane, b u t did not investigate their connection with t h e darkening of cane juices. H e found in t h e growing parts of t h e cane a n oxidase (a laccase) which turned tincture of guaiac blue in t h e absence of hydrogen peroxide. It was very unstable,. soon losing its oxidizing action upon standing, and being destroyed rapidly by heating above 60’. But besides this laccase he detected in all parts of the cane, young or old, a n enzyme giving a dark blue coloration with tincture of guaiac t o which hydrogen peroxide had been added. This enzyme was therefore what we now term a peroxidase. It was found t o be very stable, and resistant t o temperatures of g o o t o 9j O. H e called this substance leptorrtin. Further tests showed it t o be present in all t h e higher plants investigated, and Raciborski drew from this t h e conclusion t h a t his leptomin performed an important function in t h e life of all higher plants, being comparable t o t h e hemoglobin of the animal kingdom. Prinsen-Geerligs found in 1 9 0 5 ~t h a t the gray color of certain sugars is associated with t h e presence in them of small quantities of iron, and he suggested t h a t t h e iron was in t h e form of a saccharate. I n 1913 Shilstone2 again called attention t o t h e fact t h a t the iron, of which practically all modern sugar machinery is made, was responsible for t h e dark color of certain sugars, and I. F. Morse3 made a similar suggestion. Shilstone also recognized t h e fact t h a t t h e iron must be in t h e form of extremely dark-colored compounds, because otherwise t h e very great effect of mere traces of iron could not be explained. H e suggested t h a t t h e iron was combined with “organic acids.’’ It remained for Schneller4 t o show what particular class of organic iron compounds could cause t h e grayish tint of sugars and t h e abnormally dark color of other sugar products. Schneller based his explanation on t h e discovery by Szymanski5 and b y Brownee of “tannins” in cane, i. e., of aromatic compounds giving the well-known color reaction with ferric salts, and which need not necessarily be true tannins in t h e chemical sense of t h e word, b u t may be a n y of t h e numerous polyphenols or phenolcarbonic acids. As a further support of his explanation, Schneller called attentior, t o t h e researches of Gonnermann’ and of Grafes on the darkening of beet juices. Both of these authors had concluded from their investigations t h a t t h e dark color of beet juices is caused by t h e action Archief u. d . Java-Suzkerind., No. 8 , 1906. Loursiana Planter, 49, 402. 8 Modern Sugar Planter, No. 6 , 4s. 1 Louisiana Bulletin, 157. 6 B e y . der Vers.-Stat. f u r Zuckerrohr i n West Java, 2 , 13. 6 Louisiana Bulletin, 91, 9. 7 Z . V e r . D . Zuckerind., 67, 1068. 8 Oesleur.-Ung. Z . Zuckerind., 37, 5 5 . 1
2
81 5
of tyrosinase on pyrocatechin in t h e presence of ferrous salts. According t o Gonnermann, the pyrocatechin which both he and Grafe found in beet juices is not present as such in the beet itself, but is formed, as soon as t h e juice is extracted, by the action of tyrosinase on t h e tyrosin of the beet. I n view of these facts Schneller’s explanation seemed so plausible t h a t it was decided t o test it further, and t o see whether the dark color of cane juices could be experimentally explained on the basis of Schl;leller’s hypothesis. It was decided t o make the necessary tests with material as free as possible from the natural coloring matters of the cane which we have enumerated above. For this reason we used young cane shoots from which the leaves were entirely removed, and which therefore contained practically no chlorophyll. Neither could they contain a n y anthocyarin, and the presence of any saccharetin could not make itself felt t o any extent, because it does not dissolve in the natural juice. The young shoots have the further advantage t h a t they are rich in “tannin,” and t h a t t h e color reactions would therefore he more pronounced. We first undertook the identification of the oxidizing enzymes of the cane, and a study of the effects of iron salts. T h e nature of the “tannins” will form t h e subject of a later paper. A young cane shoot was ground t o pulp with a little water in a porcelain mortar, and we were surprised t o find t h a t t h e color of the solution obtained was entirely unlike t h a t of a mill juice. It was dark brown, instead of the characteristic muddy green color of mill juice. This a t once suggested t h a t t h e iron of t h e mill had something t o do with t h e color of the mill juice. In a second test we addeYl a crystal of ferrous sulfate, the size of a pin-head, t o t h e water in which t h e cane was macerated, and we a t once obtained a dark green juice, exactly of t h e same tint as mill juice, only more pronounced. It might be objected here t h a t t h e cane itself contains iron. This is perfectly true, b u t i t is well known t h a t t h e iron is in organic combination and not in ionized form. We would conclude from these tests t h a t t h e brown color obtained in the first instance is due t o t h e action of oxidases on t h e polyphenols, while in t h e second case t h e ferrous salt reacts with both t h e oxidase and t h e polyphenols yet unattacked. The result will be t h e green color due t o t h e reaction of ferric salts with certain polyphenols which will entirely mask t h e brown color of the oxidation products of polyphenols. I n order t o study this question further, the following experiments were m?de: Six flasks were prepared, with equal quantities of water in each Test I-A cane shoot was cut up into fine chips, and they were dropped directly into the water. The mixture was then allowed to stand, with frequent stirring. The juice gradually turned dark brown. Test a-Same as Test I , but water was first heated to boiling and was kept boiling while the cane slices were dropped in. This juice remained colorless for days. Test 3-Same as Test I , except that 15 mg. of iron in the form of ferrous sulfate were dissolved in the water, before the cane was added. The juice rapidly turned a muddy green to a greenish black.
T H E J O U R N A L O F I N D U S T R I A L A N D E N G I N E E R I N G C H E M I S T R Y Vol.
816
Test 4-Same as Test 1, except that 15 mg. of iron in the form of ferric chloride were dissolved in the water before adding the cane. Similar result as in Test z.
-
Test 5-Same as Test 2, except that after short boiling the flask was rapidly cooled in running water. Then 15 mg. of iron as ferrous sulfate were added. The juice remained colorless for some time, but gradually became green, as the ferrous iron was oxidized. Test 6-Same as Test 5 , but substituting ferric chloride for the ferrous sulfate. Juice at once became dark green after addition of the ferric chloride solution. The. results clearly show t h a t t h e color of a raw juice depends on several factors, viz., t h e presence or absence of oxidizing enzymes, t h e presence or absence of iron salts, and the form in which any iron salts are present. The result of Test I is due t o t h e effect of t h e oxidizing enzymes on t h e polyphenols of the cane.
IO,
No.
IO
t h a t they rapidly turn greenish black, and all of t h e juice t h a t passes later assumes a green color. Having ascertained t h e exact r81e played b y t h e iron, the next step was t o identify t h e oxidizing en'ymes present in the young shoots. Previous work along this line has alreadv been mentioned. Browne's results showing the absence of tyrosinase seemed surprising in view of ,.he fact that t h e writer found tyrosin in cane.l However, for this very reason tyrosinase could not readily be identified b y t h e addition of tyrosin t o t h e juice. It was therefore necessary t o look for tyrosinase with t h e aid of other reactions. Besides, not mucbreliance can be placed on any identification of enzymes made on t h e basis of tests with cane juice, or even' with solutions obtained by dropI
--MATERIAL NOT EXTRACTED WITH ALCOHOL-----MATERIAL EXTRACTED WITH ALCOHOL-No addition Acid Alkaline No addition Acid Alkaline No. TEST RBAGBNT A B C D E F Dark, muddy brown Brownish yellow Dark,muddybrown Very slight dark- No change Very slight darkening ening 2 Pyrocatechin, 1 per Quickly turns golden Quickly lemon-yel- Quickly turns pink- Darkens very Same as D, but less Same as D but cent soln.. yellow, darkens t o low, darkens to ish yellow, darkquickly through quickly more quickiy medium brown brownish yellonens t o dark brown yellow 3 Resorcinol, No change No change No change No change No change
......... ........
4
Hydroquin
5
Pyrogallol, 1 per Golden yellow, dark- Golden yellow, Cent soln. . . . . . . . . . ens t o brownish lighter than A yellow Phloroglucinol, 1 per No change No change cent soln. . . . . . . . . . Guaiacol, 1 per Purplish pink Light pink cent s o h . . . . . . . . . Paracresol, 1 per Brownish yellow Reddish brown cent soln. ........ Paracresol, 0.1 per Reddish brown Brownish yellow cent soln. plus crystal of glycocoll, . Tincture of guaiac., Blue Very light yellow
6
7 8
9
10 12
Purple, darkens t o
Light purple, much lighter than A
Turns purple rapid- Purple, darkens to Same as D , but less Same as D , bat ly, darkens to pur- brown quickly more quickly plish brown Turns yellow rapid- Like A Like B Like C ly and soon darkens to brown No change No change No change N o change Purple Brownish yellow
Dark reddish brown Orange-red
.
Tyrosin, 1 per cent Dark, muddy brown soln..
............
Brownish yellow
Turns purple quickly Pinkish yellow
Lighter than D
Darker than D
No darkening, turns milky N o darkening
Yellowish red Red, next morning blue,with coppercolored reflex
Yellow
Blue No change
No change
No change
Greenish black
Muddy gray
No change
Muddy gray
I n Test 2 the enzymes are destroyed by boiling, and since there were no iron ions present, t h e juice did not change at all. Test 3 is a duplicate of t h e one already described and discussed above. I n Test 4 there is no need for t h e oxidase t o act upon t h e iron salt because it is already in the ferric form, The color of a juice extracted in the presence of iron salts will depend on t h e quantity of these salts. When this is infinitesimally small t h e brown color of t h e oxidation products of the polyphenols will overbalance t h e green color of t h e polyphenol-iron compounds. As t h e iron gradually increases we get mixtures of green and brown, and finally t h e green will become the dominating color. This is reached with only very small quantities of iron. I n Test 5 t h e enzymes are eliminated b y boiling. The juice therefore contains only unoxidized polyphenols, as in Test 2 . They give no reaction with ferrous salts, b u t as t h e oxygen of t h e air gradually oxidizes the iron t o t h e trivalent form, the color of t h e phenol-iron compound appears. I n Test 6 this happens at once after t h e ferric salt is added. Now we can readily understand why cane juice t h a t has not come in contact with iron turns brown (Test I), and why juice obtained by t h e mill in t h e sugar factory turns green (Test 3 ) . We have found, when passing young cane shoots through a clean laboratory mill, t h a t the first part of t h e juice is brown, b u t soon t h e particles of fiber in direct contact with t h e iron of the mill form enough organic ferrous salt so
ping cane slices directly into the test solutions, became the PolYPhenols Present in t h e cane are also acted upon by t h e enzymes, a n a t h e reactions are t h u s liable t o be obscured., A more reliable method consists in dropping thin slices of t h e material into strong alcohol contained in a mortar and macerating them with a Pestle. The alcohol Precipitates t h e enzymes, while t h e PolYPhenols go into solution. The alcohol is then filtered off rapidly, t h e residual Pulp dried quickly between filter Paper, and t h e remaining Pulp may now be used for carrying out t h e tests with t h e various PolYPhenol solutions. Several series of experiments were made, both b y dropping thin slices of cane shoots directly into t h e water solutions of the various reagents, and also b y using t h e alcohol-extracted pulp in t h e same way. I n every case parallel tests were made in solutions slightly acidified with acetic acid, and in others made slightly alkaline with sodium bicarbonate. I n every experiment a drop of toluene was added t o the test solution. The preceding table gives t h e results of these experirnents. The resuits show t h a t the tests in which the material was not extracted with alcohol in some cases gave characteristic reactions, while in others they were more affected b y t h e polyphenols naturally present in t h e cane t h a n b y t h e test reagents used. This is especially marked in t h e tests with paracresol, Nos. 8 1
Original Communication, 8th In#. Congr. A p g . Chem.. 8 , 103.
'
.
Oct., 1918
T H E J O U R N A L OF I N D U S T R I A L A N D E N G I N E E R I N G C H E M I S T R Y
and 9. Here the water solutions gave in every case colorations similar t o those obtained in t h e absence of the phenols used as reagents, a n d t h e tint was only slightly influenced by t h e latter. But t h e alcohol-extracted pulp gave, in these cases, very characteristic reactions which clearly show t h a t t h e cane shoots contain not only a laccase (8E, g E ) , but also tyrosinase (8D, S F , gD, and especially g F , which is such a characteristic reaction t h a t i t cannot be mistaken). The presence of a laccase is further shown by t h e tests with hydroquinone, pyrogallol, guaiacol, a n d tincture of guaiac. The effect on pyrocatechin may be due t o both laccase and tyrosinase, as pyrocatechin seems t o be acted upon by both of these enzymes. T h e reaction obtained with tyrosin itself was not very characteristic, for reasons already explained above, and also on atcount of the well-known interference with this reaction by other substances, particularly amido acids, which often have either a n inhibitory effect on tyrosinase, or may entirely change the color produced. Tests for oxidases with potassium iodide-starch solution also gave positive results with both extracted a n d unextracted pulp. This reaction is not due t o nitrites, because boiled juices do not give it, nor is it obtained in the presence of N / 2 sulfuric acid. The activity of both laccase and tyrosinase diminishes very rapidly, as was already pointed out b y Browne and b y Raciborski. But the peroxidase reaction with tincture of guaiac and hydrogen peroxide could be obtained in juices t h a t had been kept for weeks, preserved with toluene. I n t h e light of Bach a n d Chodat’s theory of oxidases this may be due t o the fact t h a t the organic peroxide p a r t of the oxidase is quickly used u p when acting upon the polyphenols also contained in the cane itself, b u t t h a t the peroxidase part of t h e oxidase is much more stable. Since the amount of tyrosin found in t h e cane by the writer was extremely small, i t would appear t h a t t h e dark brown color of cane juices in the absence of iron is due largely t o t h e effect of the laccase upon t h e polyphenols of the cane, and only in small part
817
t o t h a t of the tyrosinase upon tyrosin. The reaction is therefore greatly different from t h a t taking place in beet juices. The question as t o t h e nature of the polyphenols has so far not been taken up. Schneller suggested pyrocatechin from analogy with the beet and from t h e green iron reaction. During the past grinding season several pounds of eyes which are rich in polyphenols were cut off from sugar canes, and dropped directly into alcohol. Cane tops also were sliced and treated in the same way. But in spite of these precautions the materials, which were kept in the laboratory, after several mofiths’ standing, had darkened so much t h a t i t was found impossible t o isolate any polyphenols from them in a pure state. So far we have been unable to find a n y pyrocatechin in these solutions, although the positive result of Wolff’s test’ would point t o the possibility t h a t pyrocatechin or some related substance is present in t h e sugar cane. We intend t o approach this problem by a different method a n d hope positively t o identify any polyphenols or phenolcarbonic acids. SUMMARY
The presence in young cane shoots of a laccase, of tyrosinase, and of peroxidase has been established. I t is shown t h a t t h e dark brown color of cane juices obtained in the absence of iron is due to t h e action of the laccase upon the polyphenols present in the cane, and to a:small extent, t o t h a t of the tyrosinase upon the tyrosin of the cane. The dark green color of cane juices from the factory mill is due t o t h e interaction of the laccase, the polyphenols of the cane, a n d of the ferrous salts formed by the action of t h e organic acids of the cane upon the iron of the mill. The ferrousfsalts are rapidly oxidized by the oxidases of ,the cane t o t h e ferric state, and these give the characteris tic idark-colored compounds with the polyphenols of the cane. LOUISIANA SUGAR EXPERIMENT STATION New
ORLEANS,
LOUISIANA
LABORATORY AND PLANT METHODS OF ANALYSIS USED IN THE COAL-TAR INDUSTRY. 11-DISTILLED TARS AND P I T C m S By J. M. WEiSs Received August 20, 1918
DISTILLED TAR TESTS CZ-WATER.
TEST
C3-SPECIFIC
Identical with B2. (PYCNOMETER).
Iden-
GRAVITY (PLATINUM PLAN).
Iden-
GRAVITY
tical with B j . TEST C4-SPECIFIC
tical with B6. 1
THISJO~URNAL, 10 (1918), 732.
Identical with B 7. Identical with BIO.
IN B E N Z O L .
TEST C7-CONSISTENCY
I n Paper I of this series’ the author presented t h e testing methods of The Barrett Company for crude t a r together with introductory matter t o which we would refer t h e reader of this paper All references t o previous tests noted in this paper refer t o t h e preceding paper.’ TEST
T E S T CS-INSOLUBLE TEST C~-VISCOSITY.
(SCHUTTE)
APPARATus-Schutte penetrometer (see Fig. IV) .z Stop watch. METHOD-The collar shall be filled by placing i t upon a flat tin roofing disk which has been coated with a thin film of vaseline and pouring a n excess of material into t h e collar. After cooling and contraction the‘excess material shall be cut off level with t h e upper edge of t h e plug by means of a heated knife blade. The collar shall be then immersed in water of t h e required temperature and left a t t h a t temperature for 1 5 min. T h e collar with roofing disk attached shall be screwed into the tube while t h e tube 1
Ann, inst. Pasteur, 31, 92.
a Figures are numbered consecutively in this series
of articles.
~
