.
Rapid Determination of Starch An Index to Maturity in Starchy Vegetables JOHN P. NIELSEN
Western Regional Research Laboratory, Bureau o f Agricultural Chemistry a n d Engineering, U. S. Department of Agriculture, Albany, Calif.
I
of starch, which can then be estimated by the starch-iodine colorimetric method as suggested by Pucher and Vickery (6).
and general food quality of t h e vegetables. Since the increase of starch is one of the major changes that occur during the maturing of these vegetables, it seemed logical that the estimation of starch should serve as an index of maturity and quality. This idea was supported by the fact that alcohol-insoluble solids, which give a rough measure of starch, correlate well with quality in green peas ( d , 4 ) .
Photoelectric colorimeter. For rapid work the colorimeter should have a test tube adaptor and at least a dozen matched test tubes. A red filter is necessary for maximum sensitivity. Disintegrator, a Waring Blendor or any similar instrument. Perchloric acid, 72 er cent rea ent rade solution; acetic acid, 2 N solution; sogum hydroxiie, 6 solution; potassium iodide, 10 per cent solution; and potassium iodate, 0.01 IN solution.
Common methods of starch analysis ( I , S, 6) usually involve drying the sam le and grinding in a ball mill or other device for disintegrating t i e tissues. The material is then extracted with alcohol and suspended in water, the starch is brought into solution with acid, after which it is hydrolyzed with an enzyme or acid, the resulting product is clarified, and the sugar is determined by some conventional method. Some methods include an additional step for separating dextrins, either by different solubility in various concentrations of alcohol or by a precipitation of the starch with iodine, after which the starch is determined by hydrolysis or colorimetrically with iodine (6). Hassid and coworkers (3) cpmbine the alcohol extraction and acid solubilization by adding a small amount of acid to the alcohol and refluxing.
A 100-gram sample of the fresh, frozen, or canned vegetable is placed in the disintegrator cup with an equal weight of water, and the instrument is allowed to run a t the high-speed position for 3 to 4 minutes. Two grams of the ground sample are weighed directly into a 30- or 50-ml. beaker on a torsion balance sensitive to 0.01 gram, 2 ml. of water are added, and then exactly 2.7 ml. of 72 per cent perchloric acid are slowly added with thorough stirring, so that there will not be momentary high concentrations of the acid in any portion of the sample. The mixture is allowed to stand with occasional stirring for about 10 minutes. After standing, the mixture is made up to 25 or 50 ml. with distilled water, depending on the starch content, and then poured into a suitable test tube to settle. A 1-ml. aliquot of the supernatant liquid is pipetted into a 100-ml. beaker and 6 ml. of water are added. A drop of phenolphthalein is added and the solution is brought to a pink color with a few drops of 6 N sodium hydroxide. Now 2 N acetic acid is added until the pink color disappears, 2.5 ml. are then added in excess, 0.5 ml. of 10 per cent potassium iodide and 5 ml. of 0.01 N potassium iodate are accurately added, and the solution is allowed to stand at least 5 minutes. If the solution is relatively pale bluish-green, it should now be made up to 25 ml., and if it is dark bluish-green, it should be made up to 50 ml. The color is estimated in a photoelectric colorimeter using a red filter having a transmission range from 640 to 700 millimicrons. The colorimeter should be set at zero absorption or the readings corrected, with
T IS well known that in certain vegetables such as peas, corn, and lima beans, the starch content of the seed tends to increase as the plant matures. This increase in starch is usually associated with a decrease in total sugar, tenderness,
Reagents and Equipment
5
Procedure
The above procedures require a good deal of time-consuming manipulation and they increase considerably the chances of loss of starch. Most methods do not attempt t o remove dextrins and other polysaccharides, which are determined along with starch upon hydrolysis. Pucher and Vickery (6) by a long procedure do remove most of the interfering substances. A determination b y any of the above methods will require at least a d a y and sometimes several days. If a large number of samples are run together a considerable amount of equipment is required. The method presented in this paper was developed mainly for speed without appreciable sacrifice in accuracy. Since manipulation is greatly reduced, errors from that source are minimized. The starch is estimated colorimetrically with iodine, using a red filter, which decreases considerably the error due to dextrins. Interfering glucosides and alkaloids are shown to be absent from the products examined. However, a rapid procedure for their removal is given, in case they are present in the sample t o be analyzed. A single sample can be carried through the whole procedure, including preparation, in 20 to 30 minutes. Forty to 50 samples can be analyzed in a day. Perchloric acid is unique, in that practically all its inorganic compounds are soluble in water. In order to ascertain whether this “solubilizing” effect might occur in organic combinations, various concentrations of perchloric acid were mixed with raw potato starch. It was found that after a certain normality was reached, which was well below the concentration of hydrochloric acid used for the same purpose, the starch granules lost their form and passed into solution in 3 to 4 minutes. This acid “solubilization” has now been applied to vegetable material disintegrated in a Waring Blendor. As a result, a rapid procedure has been devised for the extraction
FIGUREI 1. AESORPTION OF LIGHTBY STARCHIODINE SOLUTIONS, REDFILTER 176
ANALYTICAL EDITION
March 15, 1943
TABLE I. EFFECTOF PERCHLORIC ACID CONCENTRATION ON SOLUTION AND HYDROLYSIS OF POTATO STARCH Treatment Min.
Weight of Starch
Molality of Acid
MD.
Starch Recovery
%
a blank containing all of the reagents. I f the filtrate used in developing the starch-iodine color is turbid, an extra blank correction should be made if precise results are desired. This can be done by discharging the blue color with a few drops of 0.1 N sodium thiosulfate and comparing the turbid solution against water.
Standardization The percentage of starch is calculated from a curve prepared from the colorimeter readings of a known range of starch concentration. For solution depths of about 1.25 cm. (0.5 inch), the best range of starch concentrations is from 0 to 3 mg. per 50 ml. Figure 1 shows a typical standardization curve with the Klett-Summerson photoelectric colorimeter. Soluble starch cannot be used for standardization. If absolute results are desired, starch prepared from raw, unblanched material similar to that which is to be analyzed should be used. Equal weights of starch from different products, such as potatoes and peas, do not give the same amount of color with iodine; therefore one type of starch cannot be used as a standard for all products. Perhaps if factors were established between the various starches, a single standard such as potato starch would suffice. If a series of analyses is to be carried out on a given product and only relative results are desired, potato starch, which is easily prepared, could be used to prepare a standard curve. To obtain starch for standardization, disintegrate ram, unblanched material in a Waring Blendor type of disintegrator with an equal weight of water. Separate the fibrous material by washing the ground pulp through a 100- to 200-mesh screen, place the material that passed through the screen in a large beaker or pan, and stir with a large volume of water. Allow the starch to settle and decant off the water. Repeat this process until the starch is free of extraneous material. Xow wash the starch with alcohol and ether and dry in an oven at 70' to 80' C. for 30 minutes. This should give a starch that is reasonably pure. Analyses can be made to establish its purity if such accuracy is desired.
Effect of Perchloric Acid Concentration on Solution and Hydrolysis of Starch
It was stated above that a certain concentration of perchloric acid had to be reached before the starch granules went into solution. To study this further and to ascertain the hydrolytic effect of the acid, samples of potato starch were treated with known concentrations of acid for two different periods. The data in Table I indicate that a concentration near 4 molal is necessary to bring the starch into solution and that this range can extend to a t least 4.8 molal without hydrolysis of the starch when held a t room temperature for 15 minutes. A similar experiment carried out on corn as shown in Table I1 placed the lower limit slightly higher, probably because of the buffering effect of the sample. I n a third experiment on lima beans (Table 111), little hydrolytic effect was shown even after 30 minutes.
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Effect of Filtration on Solubilized Starch A Whatman No. 1 filter paper can be used to filter the sample after it has been solubilized and made up to volume. Curiously enough, a Whatman No. 2 paper, which is a good deal heavier, removes about 20 per cent of the starch from the solution. For this reason the method was devised so as to involve no filtration and thus eliminate any chance of absorption of starch by filter paper. Turbid solutions which had settled for only a few minutes were sampled and compared with some of the same solution which had been centrifuged. Starch results by the two procedures were identical.
Stability of Starch-Iodine Color If the sample is immediately made up to volume after the preparation of the starch-iodine color, a slow upward drift in colorimeter reading will be observed. However, if the sample is allowed to stand in the concentrated state for 5 minutes, this drift occurs to only a very limited extent. Twelve samples prepared by the latter procedure had an average upward drift of 1 division, equivalent to 0.008 mg. of starch in 40 minutes.
TABLE11. INFLUENCE OF PERCHLORIC ACID CONCENTRATION ON EXTRACTION OF STARCH FROM CORN Sample No.
Molality of Acid
Starch Found in Sample
73
4.8 2.4
74
4.8 4.0 3.4
5 20 2.52 4.03 3.41 3.30 4.25 4 a3 2.83 3.46 2.58 3.20 3.13 3.83
%
30
6.0
4.8
31
6.0 4.8
37
6.0 4.8
45
6.0 4.8
TABLE111. INFLUENCE OF TIMEIN 4.8 MOLALPERCHLORIC ACID ON PERCENTOF STARCH FOUND IN LIMABEANS Sample No. 66 66 66
66
Time in 4.8 M HClOi Min. 5 10 15 30
Starch Found in Sample
% 7.34 7.25 7.17 7.15
Effect of Alcohol Extraction Most of the procedures that have been proposed for starch involve drying the starch-containing material, grinding in a ball mill, and then extracting for several hours with alcohol. This serves to remove sugars which would interfere in a hydrolysis procedure for starch. According to Pucher and Vickery (6),it also removes certain compounds which give a blue color similar to that produced by starch with iodine. Since the method developed did not involve the determination of sugar produced by the hydrolysis of starch, the removal of sugars was of no concern. However, it is necessary to remove any compounds other than starch which would give a blue color with iodine. To check this point, 20-gram samples of the disintegrated material from several different varieties of frozen corn, lima beans, and peas were each mixed with 200 ml. of alcohol and reground in the disintegrator for several minutes. A 25ml. aliquot of this mixture was then filtered through a Whatman No, 1 paper in a Gooch crucible and washed several times
INDUSTRIAL AND ENGINEERING CHEMISTRY
178
Vol. 15, No. 3
OF PRELIMINARY ALCOHOLEXTRACTION TABLEIV. INFLUENCE ON THE DETERMINATION OF STARCH
Sample No. 86 87 36 30 31 37 45 51 882 877 874 3 57 10 3; 59 65
27
Starch after Alcoh?l Extractiuri
Product
Starch
Corn Corn Corn Corn Corn Corn Corn Lima beans Lima beans Lima beans Lima beans Soybeans Peas Peas Peas Peas Peas Peas Peas
2.43 1, 4 5 3.86 4.83 3.46 3.20 3 83
2.45 1.46 3.91 4.86 3.52 3.28 3.91
9.35 12.12 12.42 11.91 2.43
9 30 11 85 12 52 12.00 2.50
7.90 9.30 7.92
9.10 7.94
7.80 10.90 9.35 6.65
3
7.62
7 , s
10.50 9.13 6.50
with alcohol. That the extraction was complete \vas indicated by the pure white residue left on the filter paper. This residue was then suspended in 4 ml. of water and starch was determined by the procedure given above. Table IV shows that there were no interfering compounds in the products studied.
Influence of Dextrin
0
B
IO
0
I2 PER CENT
16
14 STARCH
FIGURE3. BRINEFLOTATION OF
The method herein described does not attempt to separate dextrins from starch. It is not necessary in most cases and to do so would greatly lengthen the procedure. There was no indication of the presence of dextrins in the peas, soybeans, or lima beans used for the experiments. HonTever, in corn an occasional sample had a slightly brownish color when iodine was added. To determine the influence of the red color of dextrins in the colorimeter reading using a red filter, a sample containing 125 mg. of bacteriological dextrin was analyzed. This sample gave an apparent starch content of 22 mg. or between one fifth and one sixth as much absorption of light
B A B Y LIMA BEASS US.
PERCENTSTARCH
Curve drawn b y inspection
as would the same amount of starch. This means that the error due to dextrins is much less than in a hydrolysis procedure for starch where the dextrins are not removed.
Recovery Experiments A known quantity of potato starch was added to samples of lima beans and soybeans whose starch contents had been previously determined. Table V indicates that the starch added can be determined within less than 3 per cent, which is a good recovery for most starch methods.
TABLEv. RECOVERY O F ADDEDPOTATO Product
So) beans Lima beans
O
I8
Starch Added
Starch Found
Mg. 0 49 3
Mg. 25 6 74 2 109 6 158 0 69 5 117 5
0
49 3 0 19 3
STlRCH
Recoiers
w
98 6 98 2
97 4
D
Discussion 0
4
80
90
100 TENDEROMETER
110 READING
I20
I30
FIGURE2. RELATIONSHIP OF PER CEKTSTLRCH TO TESDEROMETER READIKG IN RAWPEAS Curve d r a u n by inspection
Since the vegetables are to be analyzed for a constituent which is not in solution, great care should be taken to keep the disintegrated material well mixed as it is being weighed out. Care should also be taken not to add an excess of perchloric acid to the sample, since a fe\v tenths of a milliliter of acid will cause a considerable increase in the final molality. Perchloric acid develops some heat of solution. The rise in temperature brought about when the acid is added to extract the starch facilitates its solution. Cold 4.8 molal perchloric acid, hon ever, will dissolve starch in 15 to 30 minutes. If the time period suggested in this paper is adhered to, 72 per cent acid must be used in order readily to get a sufficient rise in temperature to dissolve the starch in 5 t o 10 minutes.
ANALYTICAL EDITION
March 15. 1943
Complete extraction of the starch is indicated by recovery experiments, the ability to duplicate results closely, and the check of results on samples reground and extracted with alcohol with those obtained directly determined without extraction. Other evidence is t h a t a reextraction of the residue on the filter paper after the extraction of starch gave a negligible starch content. A starch determination on niaterial left on a 40-rnesh screen after grinding the sample in the disintegrator and washing through the screen also gave a negligible result. Further evidence of the reliability of the method has been obtained in its application. A group of raw pea samples were graded for quality with a tenderometer and then analyzed for starch. Figure 2 shows the relationship between these two factors. Fifty samples of frozen lima beans Fere graded for quality organoleptically and by brine flotation. Starch analyses on these samples correlated very well with both the organoleptic and brine flotation grading. The relationship between proportion of sinkers in 20 per cent brine and percentage of starch in lima beans is shown as a n example in Figure 3.
Rapidity of the Method Starch in a single sample can be determined in 20 t o 30 minutes if t h e reagents have been previously prepared. Forty t o 50 samples can be analyzed for starch in a n 8-hour day if a routine is established.
Perchloric Acid Hazards There is no danger whatsoever from the 72 per cent perchloric acid itself, other than the usual hazards of strong
179
acids. The cold diluted acid is a very poor oxidizing agent, as is evidenced by the fact that it will not release iodine from iodide ion. Hot perchloric acid, however, forms unstable compounds with organic material and for this reason the treated samples should never be heated. Samples have been allowed to stand in the laboratory at room temperature and to dry on filter paper for several days without accident.
Summary A very rapid and reasonably accurate method for the determination of starch in certain vegetables has been developed. It includes grinding the fresh sample in a Waring Blendortype disintegrator, extracting the starch with 4.0 to 4.8 molal perchloric acid, and estimating b y photoelectric colorimeter the dissolved starch indicated b y the blue color produced with iodine. Alcohol extraction of t h e products studied was found to be unnecessary. The use of a red filter in the colorimeter considerably reduces the error produced by dextrins when present.
Literature Cited (1) Assoc. Official Agr. Chem., Official and Tentative Methods of Analysis, Chap. 27, p. 360, 5th ed., 1940. (2) Bonney, V. B., and Rowe, S. C., J. Assoc. Oficial Agr. Chem., 19, 607 (1936). (3) Hassid, W. Z., McCready, R. M., and Rosenfels, R. S., IND.ENG.
CHEM.,ANAL.ED., 12, 142 (1940). (4) Kertesr, Z. I., Food Industries, 6, 168 (1934). ( 5 ) Pucher, G. W., and Vickery, H. B., IND.ENG.CHEM.,h 8, 92 (1936).
. 4 ~ ED., .
OUTSIDE Publication 3691, Bureau of Agricultural Chemistry and Engineering, U. S. Department of Agriculture.
Separation of Carotenes from Xanthophylls A. J . HAAGEN-SRIIT, C. E. P. JEFFREYS, AND J. G . KIRCHNER William G. Kerckhoff Laboratories of the Biological Sciences, California Institute of Technology, Pasadena, Calif.
T
HE methods which have been most generally used for the
separation of xanthophylls from carotenes in provitamin A analysis suffer from certain disadvantages. The separation by extraction of petroleum ether solutions with 85 t o 90 per cent methanol and the separation on various adsorption columns are time-consuming. The obvious advantages of the chromatographic method for routine work are offset b y the difficulties in obtaining adsorbents of uniform performance, and by the fact that there always appears to be some loss of provitamin A material on the columns. It was known that compounds containing conjugated double bond systems such as occur in the azulenes (6) react u-ith 85 per cent orthophosphoric acid to form blue substances which are extracted from petroleum ether by the acid. It was thought that this reaction might be applied to carotene separations. Petroleum ether solutions of the mixed pigments of canned pineapple, which were obtained by saponification of the solid material with 30 per cent potassium hydroxide in methanol for 12 hours at 3" C. and extraction with petroleum ether (60 to TO"), were treated with phosphoric acid; 25-ml. portions of the petroleum ether solut,ion,which does not need to be dried, were shaken viith 4 to 5 ml. of 85 per cent phosphoric acid in a 25-m1. glassstoppered graduate. The acid layer became blue-green in color. The absorption curve for the pigment remaining in the petroleum ether solution was essentially the same as those obtained with carotene solutions which had been purified by the methanol separation and by passage through magnesia and calcium phosphate columns. Figure 1 shows a group of such curves compared uith that of pure @-carotene(S. 11.A. @-carotene),
The xanthophyll fraction obtained from a sample of these mixed pigments by the methanol separation was transferred into petroleum ether and this solution was treated with 85 per cent phosphoric acid. All the pigment was removed from the petroleum ether phase and converted into blue-green substances in the acid layer. On t'he other hand, carotene extracts, purified both by the methanol separation and by passage through magnesia or calcium phosphate columns, gave no apparent reaction when treated in the same manner with 85 per cent phosphoric acid. The absorption curves of such extracts were not appreciably altered by the phosphoric acid treatment (Figure 2). The deviation of the curve of a sample of phosphoric acid-treated 6-carotene from that of the untreated carotene is similar to that obtained with extracts of natural materials. This change in the absorption of the pigments is undoubtedly due to isomerization of the type first observed by Gillam and El Ridi (3, 4) and extensively studied by Zechmeister and his co-workers ( 5 , 7 , 8 , 9 ) , and by Beadle and Zscheile ( 2 ) . Using this shortened method, results were obtained on canned pineapple which were in satisfactory agreement with those of biological assays (Table I). I n this case the results can be expressed as units of vitamin A, since chromatographic adsorption on magnesia showed the pigment to be homogenous, and the absorption curre checked that of pcarotene which had been treated with phosphoric acid. A comparison was made of the A. 0. A. C. method ( 1 ) and the phosphoric acid method, and since carotene has a slight solubility in 90 per cent methanol the results are as might be expected (Table 11). I n each case the phosphoric acid method gave results which were slightly higher t,han those of the A. 0. A. C. method.
