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papers and remaining fertilizer are t h e n ready t o be dissolved. T h e contents of the funnel, including both filter papers, are p u t back into t h e Erlenmeyer flask, I O cc. concentrated sulfuric and j o cc. of dilute nitric acid ( I : I ) are now added and t h e flask placed on t h e bare hot plate. T h e digestion is allowed t o proceed undisturbed until all of t h e nitric acid has been boiled off which is shown b y t h e appearance of t h e white sulfuric acid fumes. About I or z cc. of concentrated nitric acid i s now added t o t h e boiling sulfuric acid and t h e digestion continued until v-hite fumes appear again. Concentrated nitric acid is then added again and t h e digestion continued. This is repeated till the solution is water-white, then i t is allowed t o cool, made up t o volume and a n aliquot taken. This is neutralized rritEl ammonium hydroxide, ammonium nitrate added and t h e phosphorus precipitated in the usual way. By using this method of digestion a clear solution is easily obtained in a n hour or less. T h e second addition of t h e nitric acld will almost always clear it up. H o w e l w . if the sample is heated after t h e sulfuric acid is added a n d t h e n the dilute nitric acid added considerable difficulty with foaming will be experienced. OK1 A I I O V 4
AAGRICULTURAL EXPERIMENT S T A T I O h
__
Yol. 8 , SO.1 2
in France as “gelose,” i t is synonymous with Bengal or vegetable isinglass. T h e Ceylon and Chinese agars are derived mainly from Gracillaria coTzjzSerooides and give with water a clear, transparent jelly. The product obtained from J a v a and Malaysia conies principally from Eucherna spiiiosztm and is of inferior quality. The Japanese agar, obtained from G d i d i u m corncum, is ordinarily accounted t o be of superior quality t o other agars. hlr. Y. S. Djang, of Tientsin, Southern China, has informed t h e author t h a t t h e Japanese product is also considered best in China from a dietary standpoint. “Carragheen” or Irish moss should not be confused with agar. I t is derived from Clzrondus crispus mainly, an alga which is very abundant in t h e North Sea. I t gives a gel resembling agar Agar is prepared for market in two ways: One method consists merely in Trying and bleaching t h e thallus of t h e algae i n , t h e sun, previous t o shipping. Such a product contains many impurities like diatomaceous refuse and other mineral or vegetable matter foreign t o t h e plant T h e other method consists in making a jelly of t h e seaweeds, allowing t h e water t o freeze out, and finally cutting t h e residue into thin strips and drying thoroughly. T h e commercial product usually consists of a number of different species of algae and hence its composition i s fairly constant.
~
THE ANALYSIS, PURIFICATION AND SOME CHEMICAL PROPERTIES OF AGAR AGAR By CARL R. FELLERS Received M a p 8, 1916 S O U R C E S O F AGAR
Agar is t h e commercial name applied t o t h e dried a n d more or less purified stems of certain kinds of marine algae. Of these t h e group Florideae or red algae is b y far the most common, They are char-
A N A L Y S I S O F AGAR C O X P I L E D F R O M V A R I O U S S O U R C E S
T h e folloming analyses are thirty t o forty years old except t h e one by Forbes, Beagle and Mensching‘ which is more recent and which gives a fairly complete analysis of a sample of dried agar. These results show how widely the composition of agar varies from different sources or even from t h e same source. The high - ash content shown by Analysis 2 , points t o gross mineral impurities, while the low
TABLEI-ANALYSES OF ALGAEPRODUCING AGAR Euchema & Unknown Euchema Gelidium Gelidiuin corneum Dried Dried Dried SPECIES 4 5 2 Analysis h-0.. . . . . . . . . . . . . . . . . . . . 1 3 18.5%; 49.80 17.33 Water . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22.8% 19.56 2.88 3.62 9.8 h’itrorenous M a t t e r . . . . . . . . . . . . . . . . 1 1 . 7 1 2.53 0.24 0.20 .... F a t . .T. . . . . . . . . . ..... 12.02 3.15 18,96 2.89 Ash . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.44 Dried -. ........Plant 5.00 3.20 0.47 Woody Fiber.. . . . . . . . . . . . . . . . . . . . . . . . . ..... Carbohydrates.. . . . . . . . . . . . . , . , . . . . . 65.05 .... ..... .......... 52.00 N-free E x t r a c t . . . . . . . . . . . . . . . . . . . ..... . . . . . . . . . Fat DRIED PLANT 12.02 .......... Nitrogenous M a t t e r . . . . . . . . . . . . . . . . . 14.06 3.15 !. 9 2 .......... 2.35 0.50 Nitrogen.. . . . . . . . . . . . . . . . . . . . . . . . . . .(.‘ N-free Extract $. Crude Fiber.. . . . . . . . . . . 73.6 .... .......... Fat Crude Fiber -t. N-free Extract.. 62. 05 .... 19.16 45.00 ..... N-free E x t r a c t . . ........................ i88p About 1885 Date of Analysis.. . . . . . . . . . . . . . . . . . . 1884 1883 ......................... Kellner(o) A‘agai & Iionig(a) Sack and Van Eck ANALYST.. Murai(a) and Greshoff ( a ) J. Konig, Chemie der Menschlichen n’ahru ngs und Ge nztssmiltel, Band I, S. 721.
+
+
acterized by a leaf-like growth and upright thallus and grow almost exclusively in tropical waters. T h e most important of these are Gelidizim c o r n e w n , G. c a r t i l a g i n e m z , F u c u s my lace us (also called Gracillarin coizjervoidesj, and E u c h e r n a s p i n o s z t m A g . , h u t agar may also be obtained from cert.ain species of Telzar a n d Gigartineae. Most of t h e agar of commerce comes from China, Japan, Malaysia, Ceylon and neighboring coasts. I n C h i m i t goer b y t h e name Rai-Thao or Ta-o,
DRIED AGAR
Fuczis Amylaceus
6
I
Water Albuminoids Solution in Cold Water Ash 0.89 Solution in Dilute N a O H 1.77 Gelose (Metarahin) Ca, 0.66 M g , 0.483 Solution in Alcohol 0.114 Wood gum h-a, K, 0.112 Cellulose C1, 0.034 P, 0 020 M a t t e r removed by KKO3 77.34 1882 1913 3reenish Forbes, Beagle & Mensching 15:29 ! .88 0.37 4.23
s,
+
Loss
15.07 7.48 2.7 10.24 6.52 36.71 0 10 4.17 10.17 3.40
ash and high carbohydrate percentages in Analysis j s h o v careful purification. Euchema is seen t o be inferior t o Gelidium as a source of agar. Analysis 7 gii-es some interesting information as regards t h e crganic composition of agar. Forbes’ results show a very high perce2tag.e of sulfur. Frankland2 reports t h a t he found one liter of agar jelly t o contain 0 . 3 0 ~ 6g. sulfur. Arsenic is reported t o be present by Leroide 1 2
Ohio Agr. Exp. Sta., Bd1. 265. “Microijrganisms in Water,“ pa 14.
Dec., 1916
T H E JOL’RNAL OF I N D U S T R I A L A N D ENGINEERING CHEMISTRY
a n d Tassidy.’ They found 0.02; and 0 . 0 2 mg. As, respectively, in two samples which they tested. This arsenic may owe its presence t o t h e fact t h a t t h e agar may have been bleached by means of SOz gas, as this process is often used in Japan. Loewit a n d Bayer2 show t h a t no reaction for protein can be obtained with agar, unless t h e latter has been first hydrolyzed by means of HzS04 when t h e ninhydrin reaction is positive showing agar does contain protein. Czapek reports pentosans in agar t o t h e extent of 1.66 per cent; Sebor3 also attests t o their presence, especially xylan. Bauer4 states t h e Carbohydrate principle of agar is galactin while Czapek6 says there is enough iodine present t o impart t o CS9 a red-violet color.
1129
have been mentioned as being very closely related t o agar since t h e products of hydrolysis of both these substances are very similar. Seyfert1 points out t h a t 2 2 per cent mucic acid is obtained by hydrolysis of agar with dilute nitric acid. Tollens2 states t h a t lactose and a mixture of glucoses have been crystallized from agar treated with dilute acids. Rouviers attests t o the presence of small quantities of amidon in agar. From these early investigations i t may be concluded with a fair amount of certainty t h a t the substance which is chemically t h e basal principle of agar and t o ’ which t h e latter owes its jellifying properties is dgalactan. Considerable work has been done on t h e jellifying &Galactan or gelose, t h e carbohydrate pectinlike basal priiiciple of agar, gives t h e latter its char- power of agar. It is a n ideal substance for the study acteristic swelling a n d jellifying properties. I t has of reversible colloids. Hardy4 used agar in his s t u d y on t h e theory of phases. He showed t h a t t h e liquid been repeatedly investigated. phase of t h e system agar-water is a solution of agar Payens was t h e first t o extract gelose from algae. in water while t h e solid phase is a solution of water He characterizes i t by its ternary composition, absence of nitrogen, complete solubility in boiling water and in agar. Further work on t h e mechanism of gelatinizaits remarkable power of forming on cooling a colorless, tion has been accomplished by Von Weirnarn,b VoigtWagner7 and Levites.8 Levites, for instance, transparent jelly, a n d coagulating in this form 500 found t h a t chlorides, bromides, iodides, salts of di-, times its weight of water. I t is free from water and resembles closely t h e pectins. He called this interesting tri- and tetrabasic acids, polyhydric alcohols and carbosubstance gelose, which recalls a t t h e same time its hydrates t o a lesser degree, all tend t o accelerate t h e origin, its applications and its most interesting gelatinization of agar. The action depends mainly on t h e anion, t h e part played by t h e cation being less property. He assigned t o it t h e formula C6H1006. striking. ReidemeisterQ determined t h e influence of Not until 1875 did Reichardt’ again take u p t h e a large number of acids and salts on the solidity of subject of agar. He identified t h e carbohydrate agar gel. Cooper and Nutall’“ show the application principle as pararabin (C12H22OI1),a substance which of agar t o photography. They show t h a t a n agar he had previously prepared from carrots and beet film need be but one-eighth as thick as a gelatin film. roots, Morins did much experimental work on gelose. Of course this is a decided advantage. Other adHe found i t gave mucic and oxalic acids on hydrolysis vantages which they s t a t e agar possesses over gelatin with dilute HNOJ. He determined t h e rotatory are cheapness and insolubility in water (except when powers of solutions of gelose a n d concludes t h a t its prop- t h e latter is very hot). Other investigators, however, erties are very similar t o those of the gums. Biot do not advocate its use in photography. and Persozg show i t is not a true gum, while PoramT h e colloid chemistry of agar plays a very important uburn10 assigns t o it t h e formula C ~ H I O O and ~ states r6le in t h e preparation of all agar media for bacterioi t is analogous t o lichenin a n d tunisin. The latter logical purposes. This is especially true as regards also determined t h e products of hydrolysis with various t h e concentrations of t h e agar and the other substances acids. I n 1882,Greenish” repeated most of t h e work which make up t h e medium. Very little is known which h a d been done on agar previous t o this time. concerning t h e changes which take place when agar is To gelose he assigns t h e formula ~ C ~ H ~ O O ~ItH Z O . sterilized a t high temperatures. No systematic does not reduce Fehling’s solution and is not ferwork has ever been done on t h e determination of t h e mentable by yeasts even after hydrolysis with dilute proper consistency or concentration of agar in t h e HzS04. He obtained seven carbohydrate-like comvarious media used so widely in bacteriological analysis. pounds by various treatments of gelose. Among Antagonism between salts and t h e effect of salts upon these were arabinose and glucose. A few years later t h e agar itself need further study. Possible reactions Bauer4 identified gelose with galactin, a product obmay occur during t h e process of making t h e media, tained b y Muntzl2 from lucerne seeds and various nonespecially when acids or alkalies are added t o adjust starchy plants. It has t h e formula CeHl006. Pectins t h e reaction (acidity). Agar which has been boiled 1 Bull. SOC. chim., 9, 65. a long time filters more readily t h a n when boiled only Cent?. allgem. path., 24, 745. Oeslerr. Chem.-Zlg., 3 (1900), 441. 4 J . Prakt. Chem., 8 0 , 367. 6 “Biochemie der Pflanzen,” p . 520. 8 ComQt. rend., 49 (1859). 7 Deut. chem. Ges. Bedin, 8 (1875). 807. I Comfit. rend., 90 (1880), 924. 9 A n n . Chim. et Phys., 62, 72. 10 Comfit. rend., 90 (1880). 1081 11 Arch. Pharm., 20, [3] 241 and 321 11 Bull. SOC. chim. de France, 17 (21, 709. 2
3
1
2 8
6
8 7 8 9 10
Deul. chem. Ges., 21, 298. Tollens et Bourgeoir, “Hydrates d e Carbone.” Compt. rend., 104, 128, 729 and 1366. Proc. Roy. Soc., 66, 95. J . Russ. Phys. Chem., 43, 653. J . Chem. Soc., Abs., 66 (1889), 817. Monatsh., 112. J . Russ. Phys.-Chem., 34, 110. and 85, 253. Z . Wiss.Mzkr., 26 (1908), 42. 7 t h Inleun. Congr o f d p p l i e d Chem., London, p. 62.
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a short time, hence i t is probable t h a t changes do occur when acid is used t o neutralize the medium a n d t h e latter subsequently sterilized a t high temperatures in a n autoclave. The character of t h e precipitates formed during the sterilization, their effect on t h e nutritive value of t h e medium, and t h e changes Khich may take place when media are stored a I.ong time are mentioned merely as suggestions for furtber work on agar media. THE AiiALYSIS O F AGAR
As a starting point for t h e s t u d y of agar media in more detail, it was decided t h a t t h e composition of a product so widely used as agar, should be better understood. A large number of samples of commercial agar were collected. These were obtained from chemical a n d bacteriological supply houses in t h e United States. The foreign samples were procured for t h e author b y Mr. I:. S. Djang, of Tientsin, China. They represent both crude and purified material. These results may also be of value from a dietary and nutrition point of viev.; t o show t h e exact s t a t u s of agar as a food for man. DBSCKIPTION OR SAMPLES OI“ AGAR
Isinglass (Agar), Schieffeliii and Co., New York. Guaranteed under Pure Foods and Drugs Act. This sample was finely ground, grayish white in color with a somewhat decided, aromatic odor. No dirt was visible to the naked eye but the microscope revealrd a considerable amount of foreign matter. 2-Shred A , p 7 , E:. Leitz, New York. This sample consisted of long shreds of a dirty gray color, spotted here and there with a reddish stain. The microscope showed the presence of enormous numbers of diatom tests. The large amount of impurities was also noted on the filters through which media made from this sample were passed. S--Shued (1gnr, Arthur H. Thomas & Co., Phila., Pa. The general appearance ol this sample was good, no impurities being evident. 4-Agur Lt’hite. E. Leitz, S e w York. -4 firicly ground white, clean appearing sample j--An impure blackish colored sample obtained from China. This sample had not been purified in any way. 6---A reddish colored unpurified Chinese sample. This sample was derived from a different species of alga than was Sample V. 7, 3 and 9-Comparatively high-grade samples of white Chinese agar; this is the product used so widely in China as a food. IO, I I and rz--Exceedingly pure white samples of Chinese agar. This is the best product obtainable on the markets of China, and so far as the author was able to determine was practically free from foreign matter. 13-Slzred Agar, Eimer and Amend, New York. Guaranteed under Pure Foods and Drugs Law. The appearance of this sample was good. 14-Coarsely ground sample, Merck 8i Co., New York. Guaranteed under Pure Foods and Drugs Acts. rg---Shred Agar, Merck and Co., New York, This sample had a good outward appearance, 16--“Becto.” “IlC;fco,” Digestive Ferments Co., Detroit, Mich. An especially prepared purified agar for bacteriological purposes. The coarsely ground shreds or granules are of a grayish white color and are free from visible dirt. The product was packed in an air-tight container. The methods used in the analysis are those of t h e ,4. 0. *I.C.’ except in the determination of crude fiber, where t h e method given in Ohio Bull. 2 2 5 was followed. I-Jupuizese
’
1
U. S.Dept. Agr., Bur. of Chem., Bull. 107.
Vol. 8. S o .
12
In this method the use of fine sand on top of the asbestos film in a Gooch crucible greatly facilitates filtration. All determinations except those of crude fiber, ether extract and arsenic m r e run in duplicate, the figure recorded being t h e ayerage. T o determine t h e solubility in cold water, j - g . portions of t h e air-dried substance were placed in a flask containing 2 0 0 cc. of water and allowed to stand 18 hours, with occasional shaking. Arsenic m-as tested for by a microchemical test,’ and iodine b y t h e carbon disulfide method. Sulfur was determined b y t h e sodium peroxide fusion method and nitrogen b y t h e Kjeldahl digestion method with KzSO4. T o determine the acidity of agar, j-g. portions were placed in 2 5 0 cc. of water ( 2 . 0 per cent solution) heated until t h e agar was dissolved and finally titrated while hot (100 cc. portions). using phenolphthalein as indicator. T h B L E II-PROXIXATE
Moisture 17.60 z . . . . . 15.75 3 . . . . . 16.14 4 . . . . . 17.65 5 . . . . 16.74 $ . , . . . 16.72 i . . . . . 16.25 8 . . . . 15.89 9..... 16.18 10 ... , 16.52 11 . . . . . 17.05 1 2 . . . , . 16.79 1 3 . . . .. 16.85 14 . . . . . 14.57 15. . . . 17.84 16(a). , 5.72 Av . . . . 16.57 ( a ) Sample No. l,....
.
h N h 4 Y S E S OF .4G.UZ
NitrogenProtein free Ether Crude (NX6.25) Extract Extract Fiber 2.40 0.43 0.51 73.38 0.30 0.71 3.26 76.70 0.31 0.63 1.93 77.66 72,72 2.93 0.45 1.50 1.40 74.61 2.38 0.27 0.29 0.77 74.82 2.94 0 . 3 0 1.12 74.84 2 72 0.81 78.21 0.34 1.53 77.48 0.29 0.60 2.35 0.66 77.55 0.35 1.84 0.72 0.1, 77.06 1.80 0.39 17.62 1.82 0.18 0.46 77.25 1.78 0.28 0.90 i7.11 2.76 0.31 0.80 0.25 2.39 75.13 89.25 1.14 0.45 0.32 2.34 76.15 0.30 0.80 16 not included in the average.
Ash 5.68 3.28 3.33 4.75 4.60 4.16 4.77 3.22 3.10 3.08 3.20 3.10 3.38 4.35 3.59 3.12
3.85
Si02 1.11 0.55 0.90 1 .os 0.85 0.77 1.11 0.46 0.35 0.40 0.36 0.31 0.55 0.83 0.55 0.29 0.68
The analyses in Table I1 show t h a t a striking similarity exists among all of the samples. This is probably accounted for b y the fact t h a t the original sources of t h e samples do not differ greatly, all originating no doubt from the coastal waters of Japan, China and Malaysia. Another possible reason for this similarity is t h a t the samples tested consist of several species of algae, which fact would tend t o minimize a n y differences which may exist in single species. The percentage of moisture and f a t t y substance is very constant. ,lgar is hygroscopic and if left exposed t o the air will absorb from 1 5 t o 18 per cent moisture. The relative amounts of protein, ash and fiber, are, however, more variable. These constituents appear t o have some relation t o t h e purity of t h e sample, t h e more impure the sample, the greater t h e percentage of these constituents. On microscopic examination the impure samples showed much foreign material, including many species of diatoms, sand and foreign plant tissue. On the other hand, those samples which had been carefully purified were almost constant in composition, showing t h a t impurities were not present. The siliceous diatom tests and sand in intimate contact with the agar shreds are the principal causes of the high percentage of ash in some of the samples. The very appreciable amount of protein present demands attention, as i t is generally accepted b y bacteriologists t h a t agar adds no nitrogen t o t h e cultural media. The f a c t is t h a t there is present in agar a n amount of nitrogenous matter equal in quantity 1
Chamot. “Elem. Chem. Microscopy,” p . 349.
Dec., 1916
T H E JOURNAL OF I N D U S T R I A L A N D ENGINEERING CHEMISTRY
t o t h a t present in our less rich foods. T h e high sulfur content of agar has already been shown by Forbes and by Frankland. The foregoing analyses check up these investigators. Since agar is sometimes bleached with SO2 gas, this may account for t h e high sulfur percentage in t h e samples. A point in favor of this view lies in t h e fact t h a t less t h a n one-halftheamount of sulfurfound in Samples I and z was obtained in the ash of Sample 16, a carefully purified product. Possibly t h e acidity of a water solution of agar may be ascribed t o this water-soluble sulfur.
1131
t o dryness. This procedure removes t h e part soluble in cold water and which does not set. Among the investigators recommending the use of dilute acids t o assist in the purification, are Mace,’ Schottelius,Z Marpmann3 and BessonS4 These methods appear t o effect a sort of purification if t h e acid is not too concentrated, in which case t h e jellifying properties of t h e agar are injured. Dominikiciew9 published a n article on t h e question of uniformity of composition and methods of preparation of agar. His method of purification is t o precipitate a lukewarm solution TABLE111-ANALYSIS OF AGAR (PERCENTAGES) b y a large volume of alcohol which has been weakly Sample No. 1 2 16(a) AVERAGE acidified with acetic acid. This agar precipitate is 1.02% 0.82 .... 0.92 0.595 0.540 ,.., 0.568 then filtered, washed and dried. ........................ 0.235 0.264 .... 0.25 0.062 0,072 .... 0.067 A method used by t h e author in t h e preparation of 0.57 0.052 .... 0.55 “purified” agar has given uniformly good results. 1 ..................... 1.11 0.55 0.29 0.83 ............. 2.65 0.264 l.ll(b) 2.645 A brief account of t h e procedure follows. ............. 0.056 0.048 .... 0.052 3 ........................ .... Clean agar shreds are cut into pieces about in. 0.26 0.17 + 0.22 +Q .... + in length (a large paper cutter serves very well for sans .................... 2.996 3.236 .... 3.12 tan. . . . . . . . . . . . . 24.34 21.40 . . . . 22.87 this), transferred t o a glass funnel containing a filter, on in HzO a t 20O 19.1 18.9 3.08 19.00 on in Hg0 a t 100 96.5 95.9 98.77 96.20 and washed with ether. The ether is saved and may Protein in alcohol precipitate. . , 0.94 1.30 .... 1.12 be used over a n d over. Then a little alcohol is poured Cc. excess N HC1 per gram agar 0.034 0.024 .... 0.029 (a) Not included in the average. (4-1 Detected but not estimated. over t h e shreds t o dissolve out t h e ether. The agar ( b ) SO3 in the ash. (-) Negative results in the tests. is transferred t o a beaker containing a sufficient No arsenic was detected b y t h e microscopic methods. quantity of a solution of 3 t o 4 per cent acetic acid t o used, hence t h e author was unable t o check up Leroide cover t h e swelled shreds. I n a n hour or thereabouts and Tassidy’s work already commented upon. A t h e water is changed in t h e beaker, no more acid being reddish violet color was imparted t o t h e CSn used in added. I n a few hours, during which period t h e water t h e test for iodine. I t was very faint, however, show- has been changed several times, t h e shreds are poured ing t h a t t h e amount of .iodine in dried agar is very small. on a piece d cheese-cloth and as much liquid is squeezed The inorganic elements Ca, Mg, Na, K, a n d P are out as possible. Washing a few times with water is present in varying amounts. These, together with sometimes advantageous a t this stage, especially t h e carbohydrate-like pentosans and galactan, form if t h e agar is still very acid. The swelled shreds all t h e elements necessary for microbial development. are weighed and calculating back from t h e amount of It is possible t h a t some of these substances may become original sample taken, enough distilled water is added available for bacterial food through t h e action of t h e t o give approximately a 5 per cent solution of agar. acid present in t h e media or else b y t h e high tempera- This is flasked, melted in a n autoclave and while ture used in media sterilization. still hot is poured through a cotton filter t o remove Agar is in itself acid t o phenolphthalein as t h e d a t a foreign matter. T h e filtered solution is now poured show. This also is contrary t o t h e .popular belief. very slowly into a large beaker containing 4 or 5 A reaction of about 0.3 cc. of N / I O HC1 per gram of times its own volume of 95 per cent alcohol or acetone. agar is considerable and for this reason agar media The latter should be constantly agitated and t h e agar should not be neutralized before t h e agar itself is added. solution poured in a very thin stream for precipitaAgar on being washed in water and precipitated b y tion t o take place properly. At Grst t h e alcohol bealcohol loses about 60 per cent of its original nitrogen comes opalescent, b u t t h e snow-white precipitate soon content. This method of procedure may, therefore, appears. After standing for a short time, t h e alcohol be used t o purify t h e crude agar, for use in refined is decanted from t h e precipitate which is thoroughly bacteriological investigations. washed with water and dried a t 100”C. This product, P U R I F I C A T I O K O F AGAR if kept in’ bottles with paraffined stoppers, will absorb The question oE purification has received some a minimum amount of moisture and may be kept inattention in t h e literature of t h e subject. At least definitely. T o test t h e efficiency of this method, t h e two patents have been taken out in Germany, one by percentage reduction in nitrogen due t o the puriSteinitzerl and another by Merck.2 The former is fication process was determined in four samples, .of little value as the jellifying power of the agar is namely, 2 , 4, 3 a n d ~ j .The reduction was 60, 82, injured by treatment with acids. Merck’s method 93 and 76 per cent, respectively, for these samples. consists essentially in dissolving low-grade agar in These results show t h a t a large part of t h e hot water, filtering and congealing by freezing. After nitrogenous matter originally present in agar is eradiit has become liquid again, i t is separated from the cated b y this method. Experiments were also made aqueous solution and washed with cold water until 1 See Mace’s “Traites de Bacteriologie.” 2 Centr. Bakt., 2 (1887), 1042. the washings show little or no residue on evaporation
+
3 1
German Patent pio. 269,088 (1912). German Patent No. 272,143.
4 6
Ibid., 10, 209. See Besson, “Bacteriologie,” 42 (translation). Centr. Bakt., Abt. 1, Bd. 47, S. 666.
THE JOCRNAL O F INDUSTRIAL A N D EXGISEERIA7G CHEMISTRY
1132
t o test out this pcriiied product b y comparison with ordinary agar, t o see if bacterial development had been inhibited on plates where both kinds were used in media. These results are reported b y t h e author in another place.’ Here i t is shown t h a t as many bacterial colonies develop on “purified” agar media as on ordinary agar, made up in the same way and using a soil infusion as t h e inoculating material. T h e “purified” agar gives a very clear medium, and since it contains a minimum amount of impurities and is cons’tant in composition, its use is strongly t o be recommended. The fact t h a t it contains little or no moisture is a n added point in its favor. T h e jellifying power of t h e agar was not injured in any way as a result of t h e purification process. T h e question arises, can microorganisms make use of the protein compounds and other nutrients present in agar? From d a t a presented elsewhere2 it is shown t h a t part of t h e nitrogen a t ieast is available under certain conditions. This was determined b y a series of ammonification experiments in which t h e agar itself was used as a basis of nitrogenous matter. However, in solid culture media, t h e amount used b y microorganisms was shown t o be very small, even though appreciable growths of a number of organisms including bacteria, yeasts and fungi, were obtained on plain agar. T h e failure of W a r i n g t ~ n t,h~e Franklands* and Winogradski5 t o isolate pure cultures of nitrifying bacteria on agar media, may probably be attributed t o t h e food in t h e agar itself. ACTION O F ACIDS O N AGAR
Three per cent HC1 caused agar t o lose its power of forming a gel with water. Alcohol or acetone cann o t reprecipitate it from this acid solution. T h e agar on dissolving in t h e acid leaves a brown insoluble residue which on drying becomes a humus-like substance. T h e solution after neutralization with Na2C03 and evaporating t o dryness, very slowly on a water-bath, gave a gray syrup; which tasted sweet and which on heating further gave t h e characteristic odor of caramel. All attempts t o cause this sugar t o crystallize failed. Solutions of this substance, with or without t h e nutrients, in concentrations of 0 . 5 , I and 1.5 per cent gave no gas in fermentation tubes when inoculated with either Fleischmann’s yeast or l?. coli. Three grains oE each of Samples I and z gave on hydrolysis with H N 0 3 (sp. gr” 1.15) 0.6216 a n d 0.j358 g. of mucic acid, respectively. This is a n average for the two samples of 28.93 per cent. Seyferts obtained z z per cent of mucic acid from Fucus c v i s p u s . hforin7 states t h a t oxalic acid is also formed b u t none was detected in this experiment. Two per cent K2S04 solution, allowed t o act on “purified” agar, dissolved it. T h e solution, after neutralization with I3aCO3, filtering and evaporating, yielded a n amorphous grayish brown powder. I t s See Soil Science, No. 3. 2 (1916), 260. Ibid., p. 257. a J . Chem. Sos. London, 59 (18911, 484. 4 Proc. Roy. Soc. London, 47 (18401, 296. 6 A n n . inst. Pasteur, 4 (1890), 213 and 257. Bar., ai3 (1888). 298. 1 Comfit. rend., 90 (18801,924. 1
2
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Vol. 8 . SO.1 2
aqueous solution is precipitable b y acetone or alcohol. One per cent solutions in fermentation tubes with or without added nutrients gave no evolution of gas in j days, on being inoculated with either Fleischmann’s yeast or B . coli. S o a t t e m p t was made t o determine t h e identity of this substance. It had a slight sweetish taste and could not be made t o crystallize. It probably is not a true sugar. An experiment mas performed for t h e purpose of determining t h e maximum concentration of KOH and IlCl which will cause a z per cent solution of agar t o lose its jellifying properties. The effect of heat and t h e addition of peptone and KC1 on these points was also determined. The sterilization of t h e agar was carried o u t in a n autoclave a t I a t m . for 15 min. TABLE IV-ERFECT OF ACID A N D ALKALI ON THE JELLIFYING POWEX OF AGAR,BEFORE AND AFTER STERILIZATIOX PLAIN2 7 , AGAR 2 % AGAR T 2 % PEPTONE Not After Xot After +0.6% KC1 REACTIOX Ster. Ster. Ster. Ster. (After Ster.) 5.0% HCl Solid-Liquid Liquid Solid Liquid Liquid Solid Liquid Liquid Solid 1.iqujd ,... Liquid Solid Liquid Liquid Solid Liquid Liqujd 2.5% HC1 Solid Liquid Solid Liquid-Solid Liquid Solid-Liquid 2.OyO HC1 Solid Solid-Liquid Solid Solid 1.5% HCI Solid Solid Sol!d Solid Solid 1.0% HCl Solid Solid Sol!d Sol!d Neutral Solid Solid Solld Solid Solid Solid Solid Solid Soljd Solid .... Solid S o l ~ d Solid .... Solid Solid Solid .... solid Solid Solid .... solid Solid Solid .... 4.5% NaOH Sol/d Solid Solid Solid ,... 5.0% NaOH Solid Solid-Liquid Solid Solid-Liquid . ..
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Table IV shows t h a t practically all t h e concentrations of acid and alkali U D t o i Der . cent caused no degelatinization of t h e agar; except when 5 per cent HC1 was used, t h e gel was rather soft and crumbly. I n these experiments t h e acid was added t o t h e h o t agar solution, thoroughly stirred and quickly cooled. Sterilization at I a t m . in t h e autoclave for r j min. caused all t h e agar containing more t h a n 2 per cent HCi t o become liquid. T h e j per cent alkali solution was also partly liquefied. Peptone seems t o aid slightly in the gelatinization process, as 2 per cent agar with peptone was much firmer t h a n t h e plain agar treated with a like amount of acid. A small amount of KC1 may cause a slight decrease in lellifying power, b u t t h e d a t a are too scanty t o draw- conclusions. Where over 2 per cent K a O H was present t h e agar became brownish black in color. I n connection with this experiment t h e least amount of clean pure agar which is able t o form a firm gel with water was found t o be 0.3-0.4 per cent. d j per cent agar gel is not harmed in any way b y heating a t a pressure of one atmosphere for 1 5 minutes. ACTIOIi O F O T H E R C H E M I C A L S O N AGAR
Ether extracts a n aromatic-scented, light, ambercolored. f a t t y substance of about t h e consistency of stearin. On account of t h e small quantity obtained no tests were made on t h e fat. IYith hot aqueous solutions of agar ether gives a dirty, gray-brown colloidal gel. This is rather porous in character and on driving off the ether by gentle heat, ordinary agar is obtained. Chloroform added t o a boiling agar solution, and vigorously stirred, gives a two-liquid layer system
Dec., I 9 16
T H E J O U R N A L OF I N D U S T R I A L A N D ENGINEERING CHEMISTRY
with water-agar a t the t o p and CHC18-agar beneath. T h e bottom layer consists of a snow-white porous, spongy mass, occluding large quantities of chloroform. On heating, the mass becomes less bulky, loses its chloroform, a n d apparently reverts t o ordinary agar. The precipitating action of alcohol and acetone has already been described. S U M RIA R Y
I-The sources, preparation and composition of agar have been discussed. 11-The analysis of sixteen samples of agar, obtained from widely different sources, show a remarkable uniformity in composition. High ash or silica content is indicative of a n inferior product. Considerable amounts of nitrogenous substances were found in all of t h e samples. P a r t of this nitrogen, a n d possibly some of t h e other nutrients may serve as a food for microorganisms, if grown on a medium containing agar. Some aqueous solutions of agar are acid t o phenolphthalein. 111-A method of preparing a “purified” agar is described. It consists essentially in washing t h e agar shreds in a solution of dilute acetic acid, washing out t h e acid and precipitating, while hot, a 5 per cent
1.133
solution of t h e agar, by means of a large volume of alcohol or acetone. It is shown t h a t much of t h e nitrogenous matter of t h e agar is removed by this method of purification. The method is recommended t o be Eollowed for t h e preparation of agar substrata t o be used in refined bacteriological work, especially where a jellifying medium containing a minimum of nutrients is desired. IV-Solutions of agar will solidify at all concentrations of HC1 and NaOH between 4 . j per cent HC1 a n d 5 per cent NaOH. Heating a t one atmosphere pressure for 1 5 minutes in a n autoclave narrows t h e range of solidification t o from z per cent acid t o 4.5 per cent alkali. Peptone increases t h e jellifying power of agar. KC1 appears t o decrease i t slightly. The author wishes t o take this opportunity t o t h a n k Dr. J. G. Lipman, of Rutgers College, for his valuable suggestions and kindly interest in t h e work, and Dr. E. M. Chamot, oE Cornel1 University, under whose guidance t h e work was begun. Thanks are also due t o t h e various companies, and t o Mr. Y . S. Djang, of China, who so kindly furnished samples of agar. AGRICULTURAL EXPERIMENT STATION NEW BRUKSWICK, NEW JERSEY
LABORATORY AND PLANT FORMULAS FOR THE FLOW OF GASES By
W. K. LEWIS
Received August 5, 1916
I n t h e formulas ordinarily employed for t h e flow of gases through orifices, pipes, and conduits, use is seldom made of t h e simplifications which t h e gas laws make possible, with t h e result t h a t in our engineering handbooks a n d textbooks we find formulas for t h e flow of steam, other formulas for t h e flow of air, and occasionally formulas for use with illuminating gas. On t h e other hand, all these formulas can be expressed in terms of fixed constants (the same for all gases) a n d t h e molecular weight of t h e particular gas with which t h e engineer is dealing. While such modification of t h e formulas involves nothing new in t h e dynamics of gases, none t h e less such simplification is of great value, most especially t o t h e chemical engineer, who frequently deals with gases other t h a n those familiar t o mechanical engineering practice. This is t r u e t o such a degree t h a t i t seems worth while t o develop and present such formulas in general form so as t o make them available t o t h e profession. The mechanical engineer always uses t h e gas law in t h e form pV = R T , applying this formula t o one pound of t h e gas in question. On the other hand, t h e chemist has learned t o use t h e gas law in t h e form pV = n R T , where a is t h e number of mols of gas in question a n d R is t h e gas constant, the same for all gases. The chemist, it is true, has hitherto used this formula exclusively in t h e metric system, but its advantages over t h e older form are no less real in t h e English system t h a n in the metric, and throughout t h e following we shall therefore express t h e gas law
in this way, pV = n R T , using throughout English engineering units, so t h a t p = t h e pressure on t h e gas in lbs./sq. ft., V = the volume of gas in question expressed in cu. f t . , n = the amount of gas in pound mols, one pound mol being a weight of t h e gas in question in pounds equal t o its molecular weight, R = t h e gas constant in English units, 1545, a n d T t h e temperature in O F. absolute. By t h e use of this formula and this nomenclature all t h e ordinary formulas for t h e flow of gases may be written in a common form applicable t o all gases, merely inserting into t h e formula t h e molecular weight or average molecular weight of t h e gas or mixture of gases in question. The molecular weight of a gas may be defined as the weight in pounds of 359 cu. ft. of t h e gas reduced t o or measured under standard conditions, this definition being identical with t h e ordinarily accepted definition familiar t o t h e chemist. The measurement of gas by volume when this is not absolutely necessary is intolerably bad practice, especially on t h e part of a chemical engineer. T o state t h e volume of gas tells nothing of its amount unless its temperature and pressure also be given. It is true t h a t gas quantities may be expressed as weight, but weight relationships in gases are always complicated in comparison with molal relationships, and t h e rational way t o express gas quantities is in mols. Inasmuch as most engineering work has t o be done in t h e English system, t h e most satisfactory unit is t h e lb. mol, i. e . , a number of pounds of t h e gas equal t o its molecular weight. The formulas for t h e number of pound mols of gas are therefore given below, and in general it will be found t h a t these are the easiest and best t o use.
