Determination of Starch in Plants W. Z. HASSID, R. M. MCCREADY, AND R. S. ROSENFELS Division of Plant Nutrition, University of California, Berkeley, Calif.
T
HE methods of Denny (I),Sullivan (6),and Pucher and Vickery (6) for determination of starch based on preliminary extraction with calcium chloride, although highly selective for true starch, are laborious. The procedure described b y Hanes (3) is relatively simple, strictly specific for starch, and satisfactory for such plant tissues as apple fruit. However, since in this method the aqueous plant extract used for the starch determination is not clarified, the procedure was found unsatisfactory for many plant materials, especially leaves, because of the presence of considerable amounts of noncarbohydrate reducing substances. The reducing values for blank determinations (samples of aqueous extracts containing starch treated with inactivated amylase) for such materials were therefore high, and in some cases several times greater than the reducing values due to the hydrolyzed starch. I n the present method the hydrolysis of starch v a s carried out v i t h salivary amylase. The noncarbohydrate reducing substances were removed by clarifying the hydrolyzed solution with neutral lead acetate and the clarified solution was deleaded with disodium phosphate. The hydrolysis with salivary amylase had the advantage over the barley &malt amylase used by Hanes of considerably shortening the time of the procedure. The degradation of starch proceeded JTithin 2 hours at 37" t o 40" C. t o a definite "hydrolysis limit" of 0.890 mg. of maltose per 1 mg. of anhydrous starch used. Hanes's method, using P-malt amylase, requires 42 hours at room temperature to reach a hydrolysis limit of 0.600 mg. of maltose per 1 mg. of starch. The limiting hydrolysis value of 0.890 was remarkably constant when starches from different sources (canna, potato, wheat, corn, rice, and whitetop root) and of different concentrations mere hydrolyzed with salivary amylase under controlled conditions. The reducing product of the action of this amylase was found to consist almost entirely of maltose. This was shown b y the pure maltose phenylosazone prepared from the hydrolyzed solution after it had been treated with alcohol t o precipitate the small amount of dextrins. The specific rotation of this solution also corresponded to pure maltose. The reducing value due to the precipitated dextrins was less than 1 per cent of the maltose. The procedure used by Hanes of "solubilizing" the starch by boiling with 95 per cent ethyl alcohol containing 1/100 volume of concentrated hydrochloric acid was adopted in the present method. This step is of distinct advantage because the treatment with dilute alcoholic hydrochloric acid converts starch into a highly soluble form, without producing any appreciable reducing products and also facilitates the rapid action of amylase on starch. The treatment with the dilute alcoholic hydrochloric acid was also found to remove all the soluble sugars from the plant tissue. Preliminary extraction with 80 per cent alcohol can therefore be avoided Tl-hen the determination of sugars is not required. The grinding of the samples in a ball mill to a very fine powder for starch analysis, as emphasized by workers using other methods, is not necessary in this procedure. Grinding to 50- or 60-mesh was found to be sufficient when the subsequent solubilizing treatment with dilute alcoholic hydrochloric acid was applied. The starch could then be removed quantitatively by extraction with hot water. The residue left after extraction gave a negative test for starch with iodine. The maltose produced from the hydrolyzed starch was determined
by oxidation wit'li alkaline ferricyanide and titrated with ceric sulfate in acid solution (4). The reducing value of pure maltose was determined by this method. The value of Giragossint'z, Davidson, and Kirk ( 2 ) for maltose equivalent to that of 80 per cent of glucose was confirmed in this laboratory. Whereas 3 cc. of 0.01 M ceric sulfate solution are equivalent to 1 mg. of glucose, 2.4 cc. of this standard solution are required for the titration of 1 mg. of maltose.
Reagents Ethyl alcohol, SO per cent: 840 cc. of 95 per cent alcohol diluted to 1 liter. Buffer, pH 5.6: a mixture of 905 cc. 0.2 JY sodium acetate and 95 cc. of 0.2 V . acetic acid. Saliva. Saliva is obtained by chewing paraffin; it is diluted with an equal amount of water and filtered through a small Buchner funnel containing a layer of talc on a filter paper. The filter is prepared by shaking talc powder with water until a uniform suspension results. The suspension is poured through the Buchner funnel supplied with a wet filter paper using slight suction. The uniformly thin layer of talc is sucked free of liquid, washed with water, and sucked free of liquid again. The diluted saliva is filtered through the dry filter, applying suction. When kept under toluene in the ice box this saliva retains its activity for at least 2 weeks. Alkaline potassium ferricyanide, 0.01 111 ceric sulfate, and Setopaline C indicator are prepared as described by Hassid (4). Sulfuric acid, 5 N : 139 cc. of concentrated sulfuric acid diluted to 1 liter.
Procedure The green plant material was rapidly dried in a vacuum oven at 90" C. A ventilated oven, provided with a fan to circulate the air over the tissue at 70" to 80" C., can also be used for this purpose. The dried material was ground to pass a 50-mesh sieve. One-half t o 3 grams of the dry, ground material were weighed into a 300-cc. Erlenmeyer flask, 200 cc. of 95 per cent ethyl alcohol were added, and the suspension was boiled on a hot plate under a reflux condenser for 20 minutes. (In case the determination of soluble sugars is required on the same samples, the ground material should be extracted with 80 per cent alcohol in Soxhlet extractors for at least 6 hours, and the double solubilization and subsequent procedure then applied.) The starch was solubilized by adding 2 cc. of concentrated hydrochloric acid to the boiling mixture through the top of the condenser and continuing the boiling for 15 minutes. The hot mixture was filtered with suction through a Buchner funnel, using a 7-cm. Whatman KO.1 filter paper, and washed with 100 cc. of 95 per cent alcohol. The residue, containing the solubilized starch, was transferred to a 400-cc. beaker, boiled for 5 minutes with 100 cc. of water, and then kept on a water bath a t 100" C. for at least half an hour. The hot mixture was filtered on a Buchner through KO. 1 filter paper until the residue was sucked dry, and the residue was then washed with 30 cc. of hot water. The residue was transferred to a 300-cc. Erlenmeyer flask, care being taken to break up the lumps, and solubilized again by boiling with 100 cc. of 95 per cent alcohol and 1 cc. of hydrochloric acid under a reflux condenser for 15 minutes. The mixture was filtered on a Buchner funnel and XTashed with 50 cc. of 95 per cent alcohol. The residue was transferred to a 400cc. beaker, boiled for 5 minutes with 80 cc. of water, and kept for half an hour on the water bath a t 100" C. The mixture was filtered on a Ruchner funnel and washed with about 20 cc. of hot water. The filtrates from the two extractions were then transferred to a 250-cc. volumetric flask and made t o volume. This solution was used for the subsequent hydrolysis of starch and also for the blank determination. The hydrolysis of starch was carried out as follows: A 25-cc. aliquot was pipetted into a 50-cc. volumetric flask, 2 cc. of the acetate buffer of pH 5.6, 2 cc. of 0.25 N sodium chloride, and 2 cc. of the diluted saliva were added, and the mixture was kept in a water bath at 37" to 40" C. for 2 hours. [Phosphate buffer was avoided because of the subsequent clarification of the digest with lead acetate. When acetate buffer is used the optimum pH for 142
h 3 1 L Y T I C A L EDITIOS
MARCH 15, 1940
TABLEI. h! ALTosE P R O D r j C T I O S BY SALIVARY . h . i L A s E STARCHES OF DIFFEREST SOURCES Source Canna Potato KheaL Corn
Starch Digested in 2 5 - C c . Volume
Starch per 5 Cc. of Digest
Mg.
Mg.
5
1
10 12.5 5 10 12.:
2
2.5 1 2 2.5 1
J
10 12.5
2
2 3 1
D
10
2
12.3
Rice
1
10 12.5
2.5 1 2 2.5
FROM
0.01 M Ceric Sulfate Used Maltose Production (Including Per 5 cc. From 1 mg. Blank) of digest of starch
cc .
Mg.
.vg.
2.45 4.60 5.65 2.45 4.60 5.65 2.45 4.55 5.55 2.45 4.55 5.70 2.45 4.35 5.5;
0.895 1.790 2.230 0.895 1,790 2.230 0.893 1.770 2.190 0 . 89.5 1.790 2.250 0.895 1.770 2.190
0.893 0 893 0.892 0.893 0.893 0.892 0.893 0.883
0.873 0 . 893 0.875 0.900 0.895 0.883 0.875
salivary amylase is 5.6 ( 7 ) . ] At theend of this period the n-atersoluble proteins and noncarbohydrate reducing substances vere removed by adding 1 cc. of saturated neutral lead acetate to the digest in the same flask. The flask \vas shaken and the mixture alloir-ed t o stand for about 5 minutes. The excess lead was removed by adding 4 cc. of saturated disodium phosphate. The solution was then diluted to the 50-cc. mark, mixed, and filtered through a dry, folded filter paper. [It is believed by some investigators that removal of the excess lead in the same medium in which the lead precipitate (precipitated impurities) exists is likely to decompose the lead complex, thus liberating reducing impurities back into the solution. The more approved procedure, therefore, is to dilute to volume after adding the lead acetate, mix, filter through a dry paper, precipitate the excess lead in the filtrate with dry, powdered disodium phosphate, and filter again through a dry filter paper. However, it is the experience of one of the authors (Hassid) that the removal of excess lead with phosphate carried out in the solution being clarified, as described in this method, is entirely satisfactory and no difference in the reducing value could be detected in the two procedures. The present method has the advantage of requiring only one filtration.] Another 25-cc. aliquot was taken for a blank determination and treated exactly as before, except that, instead of saliva containing the active amylase, saliva which had been inactivated by boiling for a few minutes in a test tube was used. Extreme care should be exercised not to contaminate the blank with traces of saliva when the sample and the reagents (buffer, inactive saliva, lead acetate, and disodium phosphate) are pipetted out. The upper parts of the pipets used for that purpose should be plugged with cotton wool, or, preferably, the reagents added from burets. The clear filtrates from the hydrolyzed sample and the blank were used for the determination of their reducing values according to a previously described method (4)as follows: Five cubic centimeters of the solution containing not more than 4 mg. of maltose were mixed with 5 cc. of the alkaline ferricyanide in a 150 X 25 mm. Pyrex glass test tube. The tube was heated in a boiling water bath or immersed in a steam bath and heated for exactly 15 minutes. The tube with the contents was then cooled to room temperature by immersing in running water for about 3 minutes. Five cubic centimeters of 5 N sulfuric acid were added and the contents mixed by shaking the tube. Seven to 10 drops of the Setopaline C indicator were added and the solution was titrated with the 0.01 M ceric sulfate from a 10-cc. buret until a golden brown color appeared. Estimations were conveniently carried out in batches of eight, The titration due to the blank is subtracted from that given by the hydrolyzed starch and the net result calculated in terms of maltose (2.4 cc. of 0.01 M ceric sulfate is equivalent to 1 mg. of maltose). The conversion into starch is obtained by dividing the amount of maltose by the value of the hydrolysis limit, 0.890. If, for example, a 5-cc. aliquot of the hydrolyzed starch, obtained from the diluted extract of 2.5 grams of plant material, requires 5.75 cc. of 0.01 M ceric sulfate, the blank being 0.5 cc., the calculation is made as follows: (5.75 2.4
- 0.5) 2500
X 100 o,890 X 100 = 9.83 per cent of starch
143
ExDerimental Results Starches from six different sources n-ere used for the determination of the hydrolysis limit. The samples were purified by bursting the granules and reprecipitating from alcohol as
follows : A 3 per cent suspension of the starch was heated on the steam bath 71-ith continuous stirring until a paste was formed. After heating for 30 more minutes the starch was precipitated by the addition of 95 per cent alcohol. The supernatant liquid was poured off and the precipitated starch lvas filtered on a Biichner funnel. I t n-as then ground in a mortar in the presence of alcohol, filtered again, washed with alcohol, and dried in a vacuum oven at 90" C. Weighed amounts of the dried starches n-ere suspended in cold water, boiled for a fem- minutes, held ai: 100" C. on the steam bath for 20 minutes, cooled, and diluted to volume. Aliquots of 25 cc. each, containing three different concentrations of the samples of purified starches, were hydrolyzed with saliva in a pH 5.6 acetate buffer for 2 hours at 37" to 40" C. The results are given in Table I. The t'itrations for the blank determinations, when the starch concentrations given in Table I and 2 I C C . of the inactirated saliva n-ere used, in all cases were 0.3 cc. of 0.01 M ceric sulfate. The slight reducing value is due t'o the saliva. The purified starch samples Jyere practically norireducing. The values for maltose produced from 1 mg. of starch were closely reproducible with starch specimens from different plant sources and also n-ith varying starch concent,ration. The average value of maltose produced by the salivary amylase under these conditions was 0.890 rng. of maltose per mg. of starch, the extremes being 0.900 and 0.875. This value mas not affected when samples of saliva from six different individuals were used for hydrolysis. The action of saliva was tried on a mixture of the following substances under the same conditions: cherry gum, a glucosan O F ST.4RCH FROM DRIEDI'LANT hIATERIAL TABLE 11. RECOVERY
(250 mg. added t o 2.5-g1am samples:
--
Starch Found-Recovered when 250 mg. o! pure I n sample starch were added
hlaterial
Jf g
.
Sawdust 0 (starch-free) 0 Young barley tops 0 Tomato leaves5 0 Plant material 44.5 (containing 1.78% starchi 44.5 a Original starch content was 10.50 per cent. ing in dark for several days.
TABLE111. COMPARISON
Material Tomato leaves
Blank Bean leaves Blank Squash leaves Blank Whitetop roots A Blank Whitetop roots B Blank Whitetop roots C Blank
248 99.2 246 98 5 246 98.5 252 100.1 286.5 97.3 288.5 98.0 Leaves destarched b y keep-
GRINDKGIT BALL ~O-MESH
OF
Grinding t o 50-11esh 0.01 M ceric sulfate per 5 cc. of digest Starch Cc. 2.75 10.50 2.70 10.30 2.80 10.75 2.75 10.60 0.50 ... 2.40 8.88 2.20 8.85 2.40 8.88 ... 0.40 1.00 3.02 1.00 3.02 0.35 ... 3.90 16.40 3.95 16.60 3.90 16.40 0.40 ... 8.80 39.40 8.80 39.40 0.40 8.00 35.60 8.00 35.60 0.40
'ro
...
...
Recovery
%
.Mg.
?vIILL A S D T O
Grinding in Ball Mill 0.01 M ceric sulfate per 5 cc. of digest Starch
cc
.
2.70 2.70 2.75 0:45 2.20 2.20 2.20
0.40 0.95 0.95 0.30 3.90 3.90 3.90 0.40 8.80 8.70 0.40 7.90 8.00 0.40
% 10.50 10.50 10.75
... ...
8.42 8.42 8.42
...
3.02 3.02
...
16.40 16.40 16.40
...
39.40 38.80
...
35.20 35.60
...
INDUSTRIAL AND ENGINEERING CHEMISTRY
144
isolated from barley roots, two polysaccharides synthesized b y bacteria, and a- and P-methylglucosides. No hydrolysis occurred. This shows that saliva is specific for starch as far as the above-named starchlike substances are concerned. I n another experiment, three samples of plant material devoid of any starch and one plant sample containing 1.78 per cent of starch (found by previous analyses) were mixed with 250 mg. of potato starch, extracted with 80 per cent alcohol, and analyzed by the described procedure. The results of the recovery are shown in Table 11. Table I11 gives the results of starch analysis when the plant material mas ground to 50mesh and also when it was ground in a ball mill to a very fine powder. The results are practically the same. The laborious work of grinding the plant material in a ball mill can therefore be avoided.
Summary After plant material to 50- or it is treated with dilute alcoholic hydrochloric acid to convert the starch into a soluble form, and the solubilized starch is completely
VOL. 12, NO. 3
extracted with hot water. The extract containing the starch is buffered with acetate buffer, p H 5.6, hydrolyzed with salivary amylase, determined as maltose b y oxidation with ferricyanide, and t'itrated with ceric sulfate. The method does not require grinding the plant material in a ball mill. It is specific, rapid, applicable to small amounts, and gives results duplicable within 5 per cent.
Literature Cited (1) Denny, F. E., Contrih. Boyce Thompson I n s t . , 6, 129, 381 (1931).
(2)
Giragossintz, G., Davidson. C., and Kirk, P. L., Mikrochemie, 21, 21 (1936).
(3) Hanes. C. S., Biochem. J., 30, 168 (1936). (4) Hassid, TT. Z., ISD. ESG. CHEM.,Anal. Ed., 9, 225 (1937). (5) Pucher, G. TV., and Vickery, H. B., Ihid., 8 , 9 2 (1936). (6) Sullivan, J. T., J . Assoc. Oficial Agr. Chem., 18, 621 ( 1 9 3 5 ) . (7) Tauber, H., "Enzyme Chemistry", p. 7 , Kew York, John TTiley &- Sons, 1937. C O L L A B O R ~ Tof I O Dr. N Rosenfels was made possible by t h e cooperative project on control of noxious weeds conducted by t h e California Agricultural Experiment Station and t h e Division of Cereal Crops and Diseases, Bureau of Plant Industry, United States Department of Agriculture.
Determination of Tungsten A Volumetric Method &I. L. HOLT
AND
ALLEN G. GRAY, University of Wisconsin, Madison, Wis.
This proposed volumetric method for the determination of tungsten is based on the reduction of W"' to W"' by liquid lead amalgam in a specially designed reductor. The reduced tungsten is reoxidized with ferric iron and the resulting ferrous iron titrated with standard dichromate solution, using diphenylamine sulfonic acid indicator. The method is applied to the determination of tungsten in tungstate solutions, in ferrotungsten, and in electrodeposited tungsten-nickel alloys. ~~
T
HE importance of a rapid volumetric method for the
quantitative determination of tungsten has long been recognized, but u p to the present time no entirely satisfactory method has been suggested. Most of the reported volumetric determinations are based on the reduction of Wv' in acid solution, followed b y titration of the reduced product with standard oxidant. Pfordten ( 4 ) reported that WOs in hot concentrated hydrochloric acid is reduced to WO2 by zinc, while Dotreppe ( 1 ) found that WOa is reduced to W40,by the same reducing agent. Holt ( 2 ) reported that methods for the volumetric determination of tungsten based on the reduction of WvI by zinc are unsatisfactory because of difficulties in obtaining identical reductions of the tungsten. Someya ( 6 ) ,who was apparently the first worker to study the reduction of tungstates by various amalgams, reported that zinc, cadmium, and lead amalgams reduce WvI to W I ' in acid media, and concluded that lead amalgam used a t a temperature of about 60" C. was the most satisfactory. He also found that bismuth amalgam reduces Wvl to Wv. Tananaev and Davitashvili ( 7 ) reported that tin amalgam used in acid solution a t a
temperature of 50' to 60' C. effects a quantitative reduction of several metals, tungsten being one of those listed. Vlashk (9) in his analytical study of tungstates, using amalgams of bismuth, zinc, cadmium, and lead, concluded that lead amalgam was the most satisfactory reducer. The purpose of this paper is to describe a volumetric method for the determination of tungsten which is based on the reduction of TVv' to W"' b y liquid lead amalgam. The reduction is carried out in a specially designed reductor which is an essential part of the method. The W"', which is rather unstable, is not titrated directly but is reoxidized at once b y ferric iron. The resulting ferrous iron is titrated with standard dichromate solution using diphenylamine sulfonic acid indicator. T h e amount of tungsten (W"') present in the original solution is then readily calculated from the volume of dichromate solution used.
Reagents and Solutions IXDICATOR. Diphenylamine sulfonic acid indicator (barium salt in water) was prepared and used as directed by Sarver and Kolthoff ( 5 ) . This indicator is particularly satisfactory in the dichromate titration of ferrous iron in the presence of tungstates. REDUCING AMALGAM. Forty grams of granulated lead were covered with concentrated hydrochloric acid, and the mixture was stirred for perha s 10 minutes, after which time the acid was poured off and the read washed several times with water. It xms then carefully dried between pieces of filter paper and finally heated at 100" C. for 10 minutes. The metal was immediately added to 600 grams of pure mercury and heated in a casserole on a water bath for an hour with constant stirring (hood). The amalgam was allowed to cool and was filtered twice through a rather fine cloth filter to separate the solid portion from the liquid metal solution. The resulting liquid amalgam was then washed thoroughly with water and dried with filter paper. One batch of amalgam thus prepared was used for a large number of determinations. STANDARD POTASSICM DICHROMATE SOLUTION.The solution was prepared by direct weighing of the analytical quality reagent, and this value was checked by standardization against iron wire using diphenylamine sulfonic acid indicator.
