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eliminates the region of maximum absorption and the transTABLE11. MEANSLOPECONSTANTS AND AVERAGEDEVIATION mission rises uniformly and rapidly in the red. FROM THE MEAN PHOTOMETRIC ABSORPTIONSTUDY OF STARCH-IODINE. Starch Mean Slope Average Deviation On the basis of the spectral transmission data, the materials Type Constant from Mean were examined further with the photoelectric absorption Arrowroot -5.59 0.07 -4.62 0.04 Canna meter. I n order to obtain a filter combination furnishing a -4.56 0.15 Corn transmission peak in the range of maximum absorption of -4.91 Potato 0.05 -4.40 Rice 0 .04 starch-iodine, Wratten red filters Nos. 23, 25, 26, and 29 in -4.62 0.05 Rye -5.57 0.09 Sago combination with the Jena BG18 glass filter built into the -4.9s Sweet potato 0.07 instrument were examined. These provide transmission -4.50 Tapioca 0.08 peaks of 595 to 625pp, and all are applicable since their -4.84 Wheat -3.61 Corn A values approximate the maximum absorption band of starch-3.92 Corn B -3.39 Corn C iodine. Filter 29, with a peak transmission of 620pp and a -3.68 Corn D range of 590 to 65Opp, was used in further work. -4.72 Potato A 70
Potato E Potato C Tapioca A Tapioca B
1
10
500 WAVELENGTH
-
600
700
MILLIMICRONS
FIGURE2. SPECTRAL TRANSMISSION OF MODIFIED STARCHES
-5.92 -6.03 -4.50 -1.85
Since the starch-iodine complex may be regarded as a type of chemical compound, deviations from Beer’s law may be expected for some concentrations of iodine. At higher concentrations of iodine, and when suitable filters are used, Beer’s law is apparently obeyed-that is, the logarithm of the fractional transmission becomes proportional to the starch concentration (4, 6, 6). Using the conditions established, transmission curves were determined for all the raw and modified starches available (Figures 3 and 4). I n each case the transmission of the iodine solution with no starch present is assumed to be 100 per cent; that is, the microammeter is adjusted to full scale deflection. Approximately straight-line transmission curves were obtained over the range 5 to 90 per cent transmission. Each starch has its own transmission curve of a slope which is characteristic of the starch type. For more accurate evaluation of the transmission curves, the values of log TIC (where T is the fractional transmission and C the starch concentration in grams per liter) were calculated for each value of starch concentration used. The values of log T / C are not absolutely constant and the deviations are characteristically different for the different starches. This is further evidence that accurate colorimetric determination of starch must be based upon a calibration curve prepared from the same type of starch. Over the range of 5 to 90 per cent transmission the values of log T/C do not deviate far from the mean values, and the latter, together with the average deviation from the mean, are given in Table 11. The intensity of color produced by the starch-iodine reaction is dependent upon the particle size of the starch (6). Starches characterized by large particle size show a lower transmission than those of smaller particle size. The relation under the conditions of test employed is shown in Figure 5, for which the particle size data were taken from the literature (2,7,8). The relation is inapplicable to modified starches.
Effects of Extraneous Materials
I
STARCH
-
GRAMS PEP LITER
FIQURE 3. TRANSMISSION OF RAWSTARCHES
Alkalies and compounds reducing iodine must be absent. Acidity due to sulfuric and hydrochloric acids-is withOpacity due to nonreactive substances
MARCH 15, 1939
ANALYTICAL EDITION
121
such as clay may be corrected for. This may be accomplished by preparing a clay suspension of the same opacity as the paper extract as determined with the photoelectric absorption meter. The iodine solution is then added to the clay suspension and the mixture used for setting the microammeter to full scale deflection. Iodine solution is then added to the starch-containing sample and transmission measurement is made in the usual way. For a starch concentration of 0.1 gram per liter, sodium chloride, sodium sulfate, calcium chloride, and calcium sulfate have an appreciable effect on starch-iodide transmission if the concentration is greater than 0.1 gram per liter. Aluminum sulfate, ferric sulfate, and calcium thiocyanate have an initially very large effect and quantities of 0.05 gram per liter seriously affect the system. Gum arabic has no measurable effect ; glue has little effect up t o ten times the FIQURE4. TRANSMISSION OF MODIFIEDSTARCHES starch concentration. Lecithin and dupinol (a sulfonated alcohol) seriously interfere and the sodium stearate concentration must not exceed one-half that of the starch (Table 111). The addition of any extraneous materials to starch before the addition of iodine always has 8 greater effect in increasing transmission than the addition of the same material after the starch-iodide has been formed. The data given in Table I11 were obtained upon addition of iodine to the extraneous material-starch mixture. TABLE111. EFFECTOF EXTRANEOU~ MATERIALS ON TRAR-SMISSION (Cornstarch, 0 . 1 gram per liter) --Increase in Per Cent 0.05 0.10 0 . 2 5 0.60 Extraneous Material g./L g./l. gJ1. g./L Sodium chloride, NaCl 0 0.1 O.5Q 0 . 2 Sodium sulfate, NazSO4 .. .. .. 0 . 3 Calcium chloride CaClz 2.5 Calcium sulfate, b a s 0 1 0:3 0:6 1.8 Aluminum sulfate, Ah(%34)a 18HzO 1.9 3.6 5.4 4.4 Ferric sulfate, Fez(SOa)a 2.2 7.0 8.5 4.3 Calcium thiocyanate, Ca(CNS)z 2.5 4 . 5 10.9 1 6 . 6 Gum arabic 0.2a 0.4" 0.20 0 . 3 Clue 1.0 0.1 Lecithin l 2 : 2 1 4 . 2 16:7 1 9 . 2 Dupinol 1 0 . 2 11.7 1 4 . 7 1 6 . 2 Sodium stearate 0.2 3.7 5.7 12.2 a Decrease in transmission.
::
Transmission1.0 2.5 5 gJ1. g,/l. g./l. 0.3 2 . 8 10 5 4.5 1.0 2.0 6 . 4 9 . 2 10.7 3.6 .. .. 4.4 2.5
4.4 2.5
4.4 2.5
21.7a 3 1 . 7 37.7 0.2 0,4a 0.3 1.3 32.1 23.7 2 $ : 7 ,. 18.2 ,. , , 23.6 , .
..
Accuracy Provided the kind of starch and its previous treatment are known, the photocolorimetric procedure is capable of high accuracy. The nature of the starch itself is the limiting factor, and if raw starches are used, the deviation from the mean of the highest and lowest transmission curves of the starches examined may amount to * 12 per cent. The data relating transmission to particle size (Figure 5 ) suggest that the latter factor is very important in determining differences in transmission of the various starches. Similarly, for modified starches (not including tapioca B dextrin), the possible error due to starch type may amount to k 2 5 per cent. The influence of starch type on colorimetric methods seems not to have been generally given the consideration deserved, although Paloheimo and his associates (6) have indicated for a number of starches the error which may be introduced by
FIGURE5. EFFECTOF PARTICLE SIZEOF RAW STARCHES ON TRANSMISSION neglect of this factor. I n the presence of dextrins, which may be present in paper together with raw or modified starches, the colorimetric method becomes totally unreliable. In addition to alkalies and reducing materials, which must be completely absent, many other extraneous materials commonly present in paper must be in limited amount if reasonable accuracy is to be obtained in the starch determination. Methods which might be employed for the separation of starch from larger quantities of such extraneous materials (6) have not been generally applied to paper. TABLEIV. ERRORIN STARCH DETERMINATION DUE TO EXTRANEOUS MATERIALS
Extraneous Material Sodium chloride Sodium sulfate Calcium chloride Calcium sulfate Aluminum sulfate Ferric sulfate Calcium thiocyanate Gum arabic Glue Lecithin Dupinol Sodium stearate
Maximum Maximum Permissible Permissible Concentration in Paper for Errors for Errors Indicated Indicated For 5 % For 10% For 5% For 10% error error error error Grans/liter % % 1.0 2.5 20 50 1.0 2.5 20 50 0.5 .. 10 10 20 0: i, 1.0 0.05 0.10 1 2 .. 0.05 .. 1 .* 0.05 5 YO0 IO:, 1.0 i.0 20 20 0.05 causes more 1 7 causes more than 10% error t%an 10%error 0 . 0 5 causes more 1% cauries more t h a n 10% error than 10% error 0.05 0.10 1 2
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INDUSTRIAL AND ENGINEERING CHEMISTRY
Table IV gives the concentration of extraneous materials in the extract which may cause errors of 5 and of 10 per cent in the measured starch concentration, assuming a cornstarch concentration of 0.1 gram per liter. The data are derived from Tables I1 and 111. Columns 2 and 3 are calculated on the assumption that cornstarch a t a concentration of 0.1 gam per liter is and are on the assumption that a 5-gram Of paper is used, that the cornstarch content is 2 Der cent, and that the extract is diluted to 1 liter. Extraneous materials which must be present in quantities less than to per cent Of the paper IV)may serious error in the starch determination. Normally these materials are present to an extent of less than 1 per cent, but this is known to be true’ the accuracy Of the starch determination within the indicated limits cannot be assumed. Considerable error may be expected if the paper
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contains acid-soluble fillers and the starch is removed by acid extraction. (I) (2)
Literature Cited Euler, H., and Bergman, s., Ko&&%Z., 31, 81-9 (1922). Hanausek, T. F.,“Microscopy of Technical Products,” New
York, John Wiley & Sons, 1907. (3) Hardy, A. C., J. Optical sot. Am., 18, 96-117 (1929). (4) Miiller, R. H., and McKenna, M. K., J. Am. Chem. Soo., 58, 1017-20 (1936). ( 5 ) Paloheimo,’L., and Antila, I., Biochem. Z., 238, 401-7 (1931). (6) Pucher, G . and Vickew, H. B.3 IXD. ENG.CHEM.,Anal. Ed., 8, 92-7 (1936). (7) Reichert, E. T., Caarnegie Inst. Wash. Pub.173 (1913). (8) Sjostrom, 0. A,. IND. ENQ.CHEM.,28, 63-74 (1936). (9) Taylor, T. C., and Iddles, H. A., Ibid., 18, 713-7 (1926).
w.,
R E C E I V ~September D 21, 1938. Presented before the Division of Physical and ~~~~~~~i~Chemistry at the 96th Meeting of the Amerioan Chemical Society, Milwaukee, Wis., September 6 t o 9, 1938.
Carotenoids in Yellow Corn LORAN 0. BUXTON, National Oil Products Co., Harrison, N. J.
I
T IS WELL known that the vitamin A activity of yellow corn is due to the presence of carotene and cryptoxanthin, and not to the presence of the free alcohol or ester forms of vitamin A. As early as 1919, Steenbock (6) demonstrated that yellow corn was a better source of provitamin A than white corn. The experiments of Coward (3) in 1923 showed that large quantities of yellow corn are required to produce growth in rats which have been depleted of the fat-soluble vitamin A. Karrer and eo-workers (4) isolated zeaxanthin, a xanthophyll, which was found to be devoid of vitamin A activity when fed to rats. Kuhn and Grundmann (6) associated the vitamin A potency of yellow corn chiefly with cryptoxanthin. The present investigation was undertaken primarily to develop a suitable quantitative method for determining the provitamin A content of commercial samples of yellow corn. Various biological as well as colorimetric and spectrophotometric methods have been described by numerous investigators; however, none has proved entirely satisfactory as a simple, rapid, and accurate method giving reproducible results. One of the latest proposed methods is that of Clark and Gring (a), who published data on the spectrophotometric estimation of carotenoids in yellow corn. Their samples were prepared for carotene-cryptoxanthin analysis by extraction of the pigments from the ground corn with methanol, followed by saponification. The carotene and cryptoxanthin were then separated from the xanthophylls by distribution between low-boiling petroleum ether and 90 per cent methanol. The concentration of carotene and cryptoxanthin in the epiphase was detezmined by means of optical density measurements a t 4500 A. Spectrophotometric Apparatus The modified Bausch and Lomb visual spectrophotometer used by Buxton and Dombrow (1) was employed for determining the carotene-cryptoxanthin content of the unsaponifiable fraction of yellow corn samples. The concentration of carotene and cryptoxanthin was determined from the intensities of the absorption band a t 4500 A. by taking the mean of several readings. The kxtinction coefficient, E: Fm., at 4500 A. for pure @-carotene in heptane is 2380 (1). A
typical absorption curve (Figure I) for the nonsaponifiable portion of yellow corn, determined in heptane on a sample of Reid’s Yellow Dent, was photographed with a Bausch & Lomb medium-sized quartz spectrophotometer equipped with a Hilger rotating sector disk and a quartz biprism. The light source was a hydrogen-discharge tube. The wave length 4500 A. was found most desirable for determining the extinction coefficients on the carotene and cryptoxanthin fractions. Since the extinction coefficients for p-carotene and/or cryptoxanthin a t 4500 8. in heptane are essentially the same (Figure l ) , i t is possible to use the following formula for calculating the carotene and/or cryptoxanthin for a l per cent solution: (S X FIR X C ) = gamma of carotene and/or cryptoxanthin for a 1 per cent solution
where
S = the screen factor
F = the extinction coefficient for pure p-carotene or cryp-
toxanthin in heptane R = the reading expressed in centimeters C = the concentration
Experimental Procedure Weigh accurately into a digestion flask 20 grams of finely ground yellow corn and add 200 ml. of 5 per cent methanolic potassium hydroxide. Reflux on a hot plate or steam bath for at least one hour. Agitate occasionallyt o facilitate thorough digestion. Cool, allow the sediment t o settle, and decant the supernatant liquid into a separatory funnel containing 50 ml. of water. Extract the residual sediment until the washings are colorless (usually five or six extractions are sufficient) with 50-ml. portions of purified technical heptane (1). Combine the heptane and alcoholic fractions and shake thoroughly. Remove the alcoholic layer and re-extract with 50 ml. of heptane. Combine the heptane extracts and wash free from xanthophylls and alkali by shaking thoroughly with 100-ml. portions of 90 per cent methanol; reextract the first 90 per cent methanol wash with 50 ml. of heptane. To ensure thorough washing, examine the last methanol wash for free alkali by testing a few milliliters with phenol hthalein. Distill the heptane portion to a small volume un&r reduced pressure in the presence of nitrogen gas. Add a few milliliters of is0 ropanol to the heptane solution before distillation t o facilitate t i e removal of water. Make up the concentrated carotenecyptoxanthin solution t o volume (50 ml.) with heptane and determine the intensity of absorption at 4500 A. with the visual spectrophotometer.
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