ANALYTICAL CHEMISTRY
472 Table I I. Added Solvent ,\.I1.
50 Xylene
40 RIethyl n-amyl ketone 100 Di-n-butyl ether
.+cetic Acid Determination Acetic Acid Known Found Gram Gram 0.993
0.905
0.564
0.564
0.497
0,485
Chlorinated Acid Content Grams (50 RIonocliloroacetic acid 11 Dichloroacetic acid 50 RIonochloroacetic acid 50 RIonochloroacetic acid
impossible to prepare a completely neutral sample of the ketone by distillation from calcium oxide. -4pparently the precision of the method can be improved to any estrnt desired by increasing the size of the sample and amount of additive, as the acetic acid is very easily separated. T h r method should be capable of application to any series of homologous compounds for which a series of inert additives can hrl found. It n-ould have to be established that relative volatilitics w r e not too much altcrrd. LITER-ITCRE CITED
that the acid was not completely extracted from the syl(,ne. The> procedure using the ket,one is t,he simplest in that the Icctorir~ i i soluble in a reasonable quantity of Tvater, and extraction i q thtsi~bi t \v:i,fore unnecessary. This is c q x ~ i a l l yintrresting k~c~~:iiisc~
(1) Pool, J. 1;. A , , Pharm. Teekblad'. 42, No. 8 , 16.5 (190.5) ( 2 ) U. S.. k m y Specifiratioris 4-503-15 (May 9, 1924).
Determination of Alcohol-Insoluble Solids and Sugar Contents of Vegetables J 4 3 I E S C. 3IOYEK -\YD KEA-SETH C . H O L G i T E .Yew Ebrk State .Igriculttcral Experiment Station, Genera, S. 1.. With the aid of a Waring Blendor and 8 5 7 ~alcohol, sugars are extracted froin frozen vegetables. The alcohol-insoluble solids content is determined hj filtering the macerate and drjing the residue. The alcohol in a small aliquot of the filtrate is evaporated and an aqueous solution of the residue is clarified by Somogyi's barium hydroxide-zinc sulfate procedure. For estimation of the total sugar content, an aliquot of the clarified extract is inverted with iniertase. The sugar content before and after in\ersion is determined bj Nelson's colorimetric method, using Somop! i's nelc copper reagent.
D
URISG a study of the losvc~sof soluble constituents in the
processing of vclgdahles, the need arose for a rapid mc~thod of determining the reducing arid total sugar contents. T h r d~ sirability of combining the sugar determinations with that oi alcohol-insoluble solids also became apparent : there w t w indications that the calculation of the constituent losses based o n the dry weight or total solids content would be erroneous becaust' of the altwed composition of the solid material after contact ~ i t h water. T o avoid this error, Lee ( 4 ) suggested that the alcoholinsoluble solids content of a vegetable may be used as a reliahl(~ basis for the computation of vitamin losses during blanching. This investigation was undertaken to devise a proccadurc. whereby the reducing and total sugar contents and alcohol-insoluble solids determinations might be combined and appreciably shortenrd. The formulation of such a procedure requires that consideixtion be given to the rapid and thorough extraction of the supai's from the vegetable, the removal of pigments and interfering cornpounds which vould normalll- react with the chromogen of a colorimetric procedure, the hydrolysis of sucrose for estimation as reducing sugars, and the formation of a reproducible and stahlca color which is proportional in intensity to the sugars present o w r a fairly \vide range. EXTRACTION
Sugars are usually extracted from plant tissues with ethyl alcohol, which, if present in sufficient concentration, precipitate .< polysaccharides and proteinaceous materials and inhibits enzymatic action. Thorough alcoholic estraction is greatly aided hy a maceration of the tissues in a TTaring Bleridor ( 2 , J ) . The following extraction procedure employs the ll-aring Blendor and a resulting alcohol concentration of a t least 75%, depending on the moisture rontcnt of the veyetal,le.
As a means of preserving the samples until a convenient time for analysis, the vegetables are frozen and stored a t -23" C. This also affords an opportunity for obtaining a representative sample for analysis by g1,inding one or more pounds of the frozen material twice in a food chopper at -23" C. Twenty grams of the finely ground vegetable are weighed into a 100-ml. beaker in the cold room. A i troom temperature the ground sample is washed into a Blendor cup (500-nil. capacity, having a rubber gasket under the ,screw top) with 150 nil. of 8 5 5 rthl-1 alcohol (specific gravity 0.850). After 5 minutes of maceration, t,he contents of the Bleridor cup are washed into a 600-ml. beaker with 8jC< ethvl alcohol from a Ivash bottle. The solids are allowed to settle and the contrnts of the bealter are poured onto a weighed 5.5-cni. S o . 40 \I-hatman filter paper in a Ruqhner funnel inserted throukh H two-holed rubber stopper placed in the mouth of a 500-ml. Kohlrausch sugar flask. Khere heavy-walled or Pyres Kohlrausch flasks are not available, a 500-ml. Pyrex volumetric flask can be adapted bl- sealing onto the neck a piece of tubing, 37 mm. in outside diameter and 6 em. long. The use of a Kohlrausch flask and the danger of possible collapse of the flask by the vacuum can be avoided by collecting the filtrate in a 500-ml. volumetric flask under the high-form glass cover of a Fisher Filtrat or. Suction is applied to the interior of the Kohlrausch flask through a small piece of glass tubing bent a t right angles and inserted into the other hole of the rubber stopper. By decanting off most of t,he alcoholic solution before adding the solids to the funnel, the filtration is greatly accelerated. The beaker is then rinsed out with more S5yc ethyl alcohol n-hich is poured into the Biichner funnel. The residue is washed with alcohol three or four times. After each u-ashing the residue is allowed t o become partially dry but care must be taken that t,he precipitate is not too thoroughly dried or the filter paper n-ill pull away from the sides of the funnel and the nest addition of alcohol \vi11 wash the solids into the flask. K h e n the volume of the filtrate approaches the d00-nil. mark. the residue is allowed to dry and carefully removed to a weighing dish for complete drying at 9 5 " C. overnight. The weight of the dried residue represents the alcohol-insoluble solids content of the sample. The volume of the filtrate is then made up to the 500-nil. mark with the 85cr alcohol and the contents of the flask are thoroughly mixed.
473
V O L U M E 20, NO. 5, M A Y 1 9 4 8 The completeness of the extraction was tested by further extracting several residues with alcohol and no sugar vias found. The precision of this alcohol-insoluble solids method was determinrd on ten replicate samples of frozen lima beans and an average v a l w of 29.37% was obtaint,d n i t h a standard deviation of *0.16 for a single dt~termination. CL4KIFICATIOh
of the beaker are washed into a funnel 50 mm. in diameter and the filtrate is collected in a graduate test tube. The precipit,ate is washed with a fine stream of water until a filtrate of 35-ml volume is att,ained. For the det,ermination of the reducing sugar content, a 2-ml. aliquot of this filtrate is used in the colorimetric procedure. When det,ermining the total sugar content, a 5-ml. aliquot of the filtrate is used for invnsion. IYVERS[OY FOH TOT4L SU(:4H
CONTENT
Of the two nirLthodscornmoiily used t o hydrolyze sucrosjt, the authors have found enzymatic action better suited for use viith the colo1,imetric procedure. IYith acid hydrolysis the concentration of acid necessary for inversion required. very careful neutralization antl did not lend itself to routinc, bnalyses.
.\n dcoholic vstixet of vrgetablv tissues usually contailis noiisugar reducing substancrs and pignitlnts Lvhich would interfere in a coloritntitric procedurcl. These coloring materials can usually be removed by adsorbents. I n preliminary experiments with such decolorizing agents as S u c h a r SXS,Suchar KLk,Darco G60, Reagents. To prepare t h r sodium acetate buffer, 13.6 grams of Xorite A, activated charcoal, Hyflo-Superrel, and Celite in the sodium act:tate trihydratr are dissolved in distilled water, 8 ml. of form of filter beds on Hirsch funnels, it was hopcd that the p e e n glacial acetic. ailid arp a d d d , and t h r mixture is diluted to 500 ml. and y ~ l l o wpigments would be adsorbed and the alcohol in the For the invertase solution, 200 mg. of Kallerstein Laboratories filtrate w.ould evaporate rapidly in the vacuum. I'ridtxr these Blue Label invertase scales are dissolved in 100 ml. of distilled water and the solution is kept in the irc. box under a layer of conditions only Xorite ,4and S u c h a r NSS completely removed toluene. This solution contains an excess of invertase activity the pigments. :md some of the filtrate was lost hl- bumping of the for the amounts of sugar encountered in the analysis. Informaalcohol during evaporation. Subsequent r ~ o v e r yrJxpc.rinieuts tion supplied kiy the manufacturer indicates that the solution will indicatcd that the S o r i t r .4 and Suc-hai. SSS had t o washtad have a k valur in the neighborhood of 0.02, and can be tested by the procrdure ( 1 ) . with unduly larg;r~arnouiitsof Fvtitorif a l l thc, rtducing wgar was t o Procedure. .I 5-nil. aliquot of thr c~larifietfextract is pipetted be rcbrlairned. into a gratluattfd test tubex. Then 2 drops of the sodium acetate Because of the difficulties in the removal of pigments from an buffer and 5 tiriips of t,he invertase solution are added t o the test alcoholic .solution by idsorption and the realization that the nontube ht.fore incubation overnight a t 35'
