290
INDUSTRIAL AND ENGINEERING CHEMISTRY
separatory funnel operations. When a n et'her solut.ion containing 2.5 micrograms of vitamin A was evaporated in the usual manner in colorimeter tubes in the presence of 0, 2, 4, 6, and 8 ml. of anhydrous ether which gave a positive peroxide test, losses of vitamin A were 0, G, 18, 27, and 29,$&,respectively. LIGHT.Vitamin A solutions are unstable in bright indoor light (6). During a 2-hour period, losses of vitamin h were approximately 38C0 greater when saponified samples were extracted with ether and the extract washed and dried near a north nindow than when the same operations were performed in subdued light'. Losses of vitamin 9 in ether solutions placed 180 em. (6 feet) from a north window, in subdued light, and in the dark over a period of 5 hours were 60, 1.2, and Oyo,respectively. Losses in petroleum ether solution under similar conditions were 41, 3, and Oc7,, respectively. SUMMARY AND CONCLUSIONS
The vitamin A content of liver can be determined by a singlc ext,raction of the saponified sample with petroleum ether, followed by treatment of the evaporated extract with antimony trichloride reagent in the usual manner, The procedure eliminates several manipulations and avoids undue exposure of vitamin A solutions to light. Large amounts of soap in alcohol-water
Vol. 18, No. 5
mixtures interfere with the extraction of vitamin A by petroleum ether. Approximately 95y0 of the vitamin can be recovered, however, when the quantity of soap in the alcoholwater phase does not exceed that equivalent to 0.05 gram of fat. LITERATURE CITED (1) Benham, G. H., Can. J . Research, 22, 21 (1944). (2) Berl, S., and Peterson, R. H., J . Sutrition, 26, 527 (1943).
(3) Boyer, P. D., Spiteer, R., Jensen, C., and Phillips, P. H., IND. ESG.CHEM., ; ~ N A L .ED.,16, 101 (1944). (4) Dann, IT. J., and Evelyn, K . A . , Biochem. J . , 32, 1008 (19381. (5) Embree, N . D., IXD.EKG.CHEJr., ~ X . A L ED., . 13, 144 (1941). (6) Kaser, SI.,and Stekol, J. d.,J . Lab. Clin. M e d . , 28, 904 (1943). (7) Kimble, M . S., Ihid., 24, 1055 (1939). (8) Koehn, C. J., and Sherman, W , C., J . Biol. Chem., 132, 527 (1940). (9) B. L.. Melnick. D.. and Pader.. M... IND.ESG. CHEM.. \ - , Oser. . ~ - . A L . ED:, 15, 724 (1943). (10) Oser, B. L., Melnick, D., Pader. >I., Roth, R., and Oser, M., Ibid., 17, 559 (1945). (11) Woodman, -4.G., "Food dnalysis", 3rd ed., p. 523, Kew Y o r k , SIcGraw-Hill Book Co., 1931
Determination of Uronic Acids R. M. MCCREADY, H. A. SWENSON, AND W. D. MACLAY, Western Regional Research Laboratory, Albany, Calif.
A modified method for determining uronic acids has been developed. Theoretical quantities of carbon dioxide were obtained for all uronic acids and their derivatives by heating the sample with 19% hydrochloric acid in an oil bath maintained at 1 4 5 " C. for 1.5 to 2 hours. The apparatus is simple, compact, easy to construct, and offers a substantial saving of space and time. Analytical results on alginic acid were higher and more reproducible than those obtained by accepted methods.
T
H1.: objective of t h r n-oi.k wporttd here
I Y R ~ to devisr iin rifective procedure for drtcxrmining the uronide content of' pectic and other polyuronide substances in a n appreciably shorter out this type of analysis by cui-time than rh:it required to c a rently recomrr.endeti modification;. (1-7, 9-16) of the IxfevwTollcns (8) mcthod. \Thereas a minimum time of 4 hours is consideretl necerisnry for romplete decarboxylation of certain pectir materials with boiling 12.5qc hydrochloric- acid and even a longer treatment dc.es not sufficr for alginic, arid, quantitative yields of rarbon dioxide were obtained from a variety of pectic substancer and dginic acid when they were heated in 195< hydrochloric acid, :it :I hath t(,mpernture of 145' C. for 1.5 and 2 hours, rwpt~ctively.
APPARATUS AND METHOD
The apparatus used in the investigation is shown in Figure 1. Two assemblies of this type were used simultaneously in the same heating bath. Air, the carrier gas, passes through an Ascarite or soda-lime column 4,which remows traces of carbon dioxide. A mercury valve, b;allows gas to pass in one direction through the apparatus. This valve is connected through a side tube, C, to a reaction flask, D, by means of a rubber connection. D is a 100-ml. round-bottomed, long-necked boiling flask, Lvith a 24/40 ground joint attached. The oil bath, E , is maintained a t 145" C. by means of a thermoregulator and immersion heater. From the reaction flask the carrier gas passes upward thr0ugh.a 20-em. reflux condenser, F , through a trap, G , containing 25 grams of 20-mcsh granulated zinc or tin, and finally into the absorption flask, H . This 250-ml. Erlenmeyer flask is equipped with a 24/40 ground joint and a side tube attached a little belox the ground joint, as shown. The gas passes up\\-ard through an absorption to\\er, I . The lower part of the toner consists of an 18-mm. tube, fitted with a medium fritted Pyres disk and sealed to the lon-er end of the inner part of a 24/40 ground joint. The bubbling disk should terminate 1 or 2 mm. above the bottom of the absorption flask when the joint is in place. A bulb of approximately 100-ml.
capacity is blown above thv ground portion of the joint to serve as a trap to prevent the possible loss of alkali by foaming. The outer portion of a 24/40 ground joint is sealed on above this bulb. The absorption tower, from the bottom of the disk to the top of the ground joint, is approximately 30 em. in length. The top of the tower is fitted n.ith a hollow, ground stopper, with a short, side tube attached. The carrier gas passes from the tower assembly to a soda-lime tower, J . 9 water pump, attached to a capillary-tube regulator, K , serves to sweep 1700 to 2000 ml. of carbon dioxide-free air per hour through the apparatus during the heating period. The sample to be analyzed is placed in the dry reaction flask. (The optimum size of sample used depends upon its uronic acid content-for example, about 250 mg. are sufficient for analyzing pectin.) Thirty milliliters of lSc; hydrochloric acid and a ,small boiling tube are added. The ground joint is lubricated with sirupy phosphoric acid and attached to the reflux condenser and mercury valve. -4stream of carbon dioxide^-free air is drawn through the reaction flask arid reflu\ condenser to remove traces of carbon dioxide before the ahsorption tower is at'tached. The ground joints of the absorption tower are lubricated with stoprock grease and inserted into t,he absorption flask, H . The
r
Air
A
To
acuum -P
Figure 1. Apparatus for Determination of Carbon Dioxide Evolved from Uronic Acids b y Heating with Hydrochloric A c i d
ANALYTICAL EDITION
May, 1946
flaqk and tower are w e p t free of carbon dioxide, :ind 25 ml. of 0.25 S sodium hydroxide and 5 280 drops of butanol-1 are added t,o the absorption K tower. Thc. side tube of the absorption flask is att,ached to the zinc trap :.nd the top of the tower to the soda-lime toner, .I, by means of rubber conncctions. The oil bat,h, previously brought t o 145' C., is a plawd in position, the lrvel of the oil being 1 or 2 mm. below that of tht' liquid Icvc.1 \Tithin the reacY K tion flask. After the initial rapid evolution of gas a W through the :tbs:~iption tower has ceased, the capilP lary tube. R,is .:ttarhrd to J and to thcl source of P vacuum. Heating of thv oil bnth at 145' C y , is continued while the apparatu> is swept for 1.5 hours. The bath is then rtrmcived, thtl ahsorption flask and tower are diacomiectrd from the apparatus, and the alkali is washed down from the tower into the absorption flask. Three or four nashings are needed to remove the last traces of alkali from the tower. Gentlr pressure with carhon dioxidefree air from the top of the tower can be used to hasten the washing. Ten milliliters of 10' h:irium chloride dihydratr solution and two drops of phenolphthalein indiTIME OF HEATING I N MINUTES cator are added to t h t ~ah.wrption flask and the Figure 2 . Rate of Evolution of Carbon Dioxide from Citrus Pectin, excess alkali present is titrated with 0.100 S hyGalacturonic, Alginic, and Pectic Acids with 19% Hydrochloric drochloric acid. Sormal precautions are .used A c i d in an Oil Bath Maintain ed at 145 O C. to exclude carbon dioxide during the storage, addition, and s~tbsequr~nt titration of the absorption alkali. A control standnrdization should be run without the Decartioxylation of citrus pertin (purified), pectic acid, :ind :ilgini(' sample and used in subsequent c&ulations of the ea.rbon :icid with 12.57, hydrochloric acid at a n oil bath trmperaturc ( i f rvolvrd from uronic. acids. 145" C. for 1.5 hours was approximately 70, 80, and 6 0 5 , r e s p w tively, complete. Equal quantities of carbon dioxide were obtaintvl EXPERIMENTAL RESULTS AND DISCUSSION from the pectic substances with 12.5Yc acid for 5 hours antl b>'I'litx results : u t ' pre,cnted on a moisture- and ash-fret. hisis. the proposed method. However, the yields of carbon dioxidv 1Ioistur.w rvrre dctc11,rninedby drying in vacuo for 24 hour.: at from alginic acid n-ere l o ~ :IS r compared with those obtainrd with 70" (',, a n d dry s:irnpl(~.~ wvrre heated 3 hour a t 600" C. t o drst Galacturonic acid (inonoh3 drute) Citrus pectin (purified)
... 16.0 I 6 8
20.7 20.2
20 I 20.8 21.1
Citrus pectin (unpurifird, Apple pectiii
16.5 ... 16.3 ... 21 4 21 2 . P e c t i c aciil 14 5 20 0 14.4 20.0 (enzymarir. .Ilginic aci
