INDUSTRIAL A X D ENGINEERING CHEMISTRY
J i i l v . 1926
712
Separation of the Amyloses in Some Common Starches 18’
By T. Clinton Taylor and H. A. Iddles COL7lMBIA UNIVERSITY,
N E W Y O R K , N. Y .
T IS a well-established fact that the material which by Taylor and Nelson.’ The product obtained in this makes up the starch granule is not homogeneous but way was white and under the microscope still showed the presrather is composed of a t least two substances.3 The ence of granules. A small amount of hydrochloric acid still physical and chemical properties of these two amyloses are remained, as determined by drying the purified starch and markedly different and the literature records many attempts titrating with standard alkali, which showed a content of to separate them. As typical of the methods employed 0.13 per cent by weight in the case of one sample of corn there might be mentioned the work of Tanret4 on separation starch. I n order to make sure that this treatment did not by sedimentation, Sherman and Baker5 by centrifuging, and remove appreciable quantities of nonextraneous fatty maLing and Nanji6 by selective fermentation. terial, a 500-gram sample of corn starch was refluxed 8 hours Since starches of different genera exhibit such striking each day for 6 consecutive days and allowed to stand in condifferences in properties, it is desirable that a method be tact with the purification medium during the intervening tinic. found to separate quantitatively, if possible, these two The sample was then filtered and washed as in a n ordinary treatment. By this drastic or prolonged treatment the constituents from some of the common starches. Each portion can be i n v e s t i g a t e d fa-tty content as determined separately and the properb y h y d r o 1y s i s decreased ties of the different starches from 0.47 per cent to 0.28 HYare the starchesfrom different sources so differper cent in this particular studied as a function of the ent in properties that neither they nor their dextwo components and of the case. trines or other derivatives are usually interchangeable? minor n o n c a r b o h y d r a t e Can the differences be due to the presence of fatty acids Gelatinization constituents. in, for example, corn starch and the presence of phosI n this paper are given Belore s t a r c h can be phoric in potato starch? Where are these noncarbohythe results of experiments on separated into the two pordrate constituents to be found? Is the heterogeneity of the separation of the amytiom, alpha- and beta-amystarch due to them and is there any way of utilizing loses in corn, rice, and potato lose, i t is first necessary to them to effect a separation of the different components starches and a brief descripof the starch granule? Is there any chemical difference rupture the granule. This tion of the amyloses with between the amyloses after separation? This paper is rupture, or gelatinization, special regard to the nonthe first step in an attempt to attack the starch probm a y b e accomplished by carbohydrate constituents. lem from this point of view. h e a t i n g a suspension of It was for a more detailed The method given herein insures complete disintestarch in water, by heating study of these noncarbohygration of the starch, allows the use of relatively high under pressure, or by chemidrate constituents such as concentration of material, and gives some very intercal agencies such as aquefatty acids7 and phosphoresting preliminary results bearing on these questions. ous alkali or solutions of ous that the method was a m m o n i u m thiocvanat,e, sought originally and work c h l o r a l hydrate, or other is now in progress on one phase of this problem-namely, the structure of the fatty water-soluble suhstances.6 Since heating in water alone tends t’o give very highly hydrated and viscous solutions,$ acid-bearing amylose in corn starch. The nomenclature, according to Meyer, of alpha- and beta- only small quantities of starch may be handled in t,hese amylose is used throughout to designate. respectively, the types of gelatinization. Microscope examination shows slimy, less soluble, heavier constituent and the mobile, easily large quantit’ies of swollen but unrupt’ured granules in such pastes. These granules go with the slimy alpha portion and dispersed, soluble portJion. so give a fictitiously high value to this constituent. PracPurification of the Starches tically all previous attempts a t separat>ionsuffer from these defects and give rise to the discrepancies between the writers’ Since starch is separated mechanically from the other method and the former ones. To avoid these difficulties substances, principally nitrogenous, which occur with it in it was decided t,o use a water solution OC ammonium thiothe plant, it was first’ necessary to remove such impurities. cyanatelo as the active agent, to cause swelling and rupture of This was accomplished by treating the starch with an alcoholic the granules. In this way it was possible to obtain very solution of hydrogen chloride according to the direct’ionsgiven mobile starch solutions containing as high as 8 per cent dry weight of st,arch. As will be shown lat,er, the starch so treated 1 Received February 24, 1926. has undergone apparently no chemical change. 2 Contribution No. 512 from the Chemical Laboratories of Columbia University. From a dissertation presented by H. A. Iddles t o the Graduate MElErHoD-one hundred grants of dry powdered starch Faculty of the Department of Chemistry of Columbia University, in partial were treated with a solution of 40 grams of ammonium thiofulfilment of the requirements for the degree of doctor of philosophy. cyanate dissolved in a mixture of 200 cc. of water and 50 cc. 8 Nageli, “Die Starkekorner,” Zurich, 1858 ; Meyer, “Untersuchungen of alcohol. The mass was warmed to 40” C. and vigorously iiber die Starkekorner:” Maquenne and Roux, Comfit. rexd., 140, 1303 (1905); A n n . chim. phys., [8]9, 179 (1906). stirred until i t became homogeneous. By making frequent
I
w
chim., [4]17, 8 3 (1916).
4
Bull.
5
J. A m . Chem. SOL.,38, 1885 (1916).
SOC.
S J . Chcm. SOC.(London), 124, 2666 (1923). 7 Taylor and Nelson, J . A m . Chem. SOC., 42, 1726 (1920).
8
8 10
Reychler, Bull. SOC. chim. Belg., 29, 118 (1920). 41sherg, THIS JOURNAL, 18, 190 (1926). J . SOC.Chem. I n d . , 28, 213 (1909).
INDUSTRIAL A N D ENGINEERING CHEMISTRY
714
observations under the microscope, the point at which the granules first break and finally become completely disintegrated could be determined. When all the granules had undergone this change, 95 per cent alcohol was added to precipitate the gelatinized mass. This was then ground up with successive portions of 95 per cent alcohol, thereby removing practically all the ammonium thiocyanate and dehydrating the product. After drying in a vacuum desiccator over sulfuric acid for several days, the starch was ready for separation studies. Ultra-Filtration through Permeable Membranes
The first method employed to effect a separation was the ultra-filtration of the beta-amylose away from the suspended alpha-amylose by means of permeable collodion filters. This method was selected because suspensions of relatively high concentrations, 8 to 10 per cent, could be used, the filtrate in all cases was perfectly clear, showing a complete separation had taken place, and in most cases the process was rapid, lasting from 1 to 3 days. For the filtration it was necessary to prepare collodion membranes of varying degrees of perm e a b i l i t y , which was best accomplished by foll o w i n g the method of Nelson and Morgan." A 2 per cent collodion solut i o n was prepared by dissolving pure collodion in 79 : 25 (by w e i g h t ) alcohol-ether m i x t u r e . For each membrane 5 cc. of this solution were run on to a fogged 10.2-cm. ( 4 - i n c h ) circular glass plate which was floated o n a m e r c u r y surface. I n this way a membrane of uniform t h i c kn ess c o u l d be o b t a i n e d . When evaporation had U proceeded to the desired Figure 1-Filtration Apparatus s t a g e , t h e p l a t e and membrane were immersed in graded alcohol-water mixtures and finally allowed to soak overnight in distilled water. During this treatment or by a slight mechanical action the membranes were loosened from the plate and were ready for use. I n order to assign a final grade or determine the relative permeability, the ratio of the solvent-that is, the weight of the finished membrane less the dry weight-to the dry weight of the membrane was used. The filtration apparatus used in these experiments (Figure 1) consisted of two small bell jars, the upper one serving as the container of the suspension to be filtered and the lower one fitted with an air outlet tube, as the receiver. Between the two was placed a nichrome gauze to support the collodion membrane made as described above. The two jars were supported on a ring stand and sealed together with a melted rubber-paraffin-beeswax preparation, which hardened to a firm joint. Pressure was applied to prevent any leakage a t the joint, by means of strong rubber bands attached to two wooden amis fitted over the necks of the upper and lower containers. I n each filtration the amount of dry starch, prepared as noted under Gelatinization, was stirred with a very small 11
J . B i d . Chem., 68, 305 (1923).
Vol. 18, No. 7
amount of wat,er. When the paste thus formed was free from lumps the remainder of the water was added slowly with constant stirring until a 6 to 10 per cent suspension was obtained. This cloudy suspension was poured into the filtration apparatus. As can be seen from the tables, various concentrations of starch were used with niernbranes of different grade. A uniform air pressure of 4 em. of mercury, regulated by a manostat, was applied through the tube a t the top of the apparatus shown in Figure 1. The filtrate was perfectly clear in all cases, although the rate of filtration varied somewhat, with the permeability of the membrane. The operation was continued until washings gave no further tests for beta-amylose with iodine t,est solution. The solutions thus obtained were analyzed for solid content by drying a sample on clean, ignited sand at 105" C. for 4 hours, and by polariscope readings taken on the clear filtrate. I n Table I are recorded the results of a series of such filtrations. The yield of beta-amylose by this method is given in column 6 for three comnion stRrches. of Gelatinized Starch Solutions (4) (5) (6) (7) -FILTRATE-AYYLOSESBetaAlpha Sample Grade of Volume amylose Beta (by diff.) Cc. Grams D Per cent Per cent Grams membrane Corn starch 7.44 9.4 100 6.382 , 85.7 14.3 20.00 22.0 330 16.523 . 82.0 18.0 8.00 4.1 117 6.642 ... 83.0 17.0 13.7 143 5.247 6.07 86.4 13.6 15.00 14.3 470 12.880 18214 85.0 15.0 10.00 19.0 290 8.360 181.6 83.6 16.4 10.12 15.0 195 8.906 181.6 87.9 12.1 Av. 84.8 15.2 Rice Starch 272 8.146 81.4 18.6 10.00 20.0 Table I-Ultra-Filtration (3) (1) (2)
... .
9.06 10.00
16.0 14.0
220
270
7.346 8.284
... .... ..
Av.
10.00 10.00
81.0 82.8
81.7
19.0 17.2 18.2
Polalo Starch 9.724 181.6
97.2 2.8 97.1 2.9 Av. 97.15 2.85 a Rotations were actually determined for green mercury line, wave length 546.1 Angstriim units a t 2 5 O C . These results were multiplied b y the factor 0.8486 [Vosburgh, J . Am. Chem. Soc., 43, 219 (19Zl)l to convert them approximately to the sodium D line values given above. 13.0 13.0
259 256
9.715
...
As mentioned above, the membranes are graded on the ratio of solvent in a given film to the dry weight of collodion in it. Att,ention is called t'o the wide range of permeability of the membranes through which bet.a-amylose will pass. The lowest grade membrane used was 2, but owing to the extreme slowness of filtration its use was impract.ica1although the betaamylose will pass through it. As shown by averages in column 6, t,here is a striking similarity in the soluble or betaamylose content of the first two cereal starches as contrasted with the higher soluble content of t,he tuber starch. I n subsequent experiments, where the alpha portion was determined directly and the beta found by difference, it will be shown that the ratios as given in Table I are substantially concordant. It is interesting to note the high beta concentrations of Ohese aqueous dialysates (Columns 3 and 4). These solutions, which are perfectly clear and transparent, can be obtained containing as much as 8 per cent beta-amylose. This is believed to be about the highest concentration that has been recorded for a starch solution with these properties. On long standing, solutions of beta-amylose become cloudy and finally deposit material in the form of an insoluble hydrated mass. This has been called reversion12 or retrogradation, and studies on the phenomenon are now being carried out in this laboratory. By not allowing the solution to stand too long, retrogradation difficulties were avoided. 12 Brown and Heron, Ann. Chem. Pharm., 189, 266 (1879); Maquenne, Compt. rend., 138, 213, 375 (1904); Maquenne, Fernbach, and Wolfe, Ibid., 138, 49 (1904).
INDUSTRIAL ALVDElVGINEERI-VG CHEiVIISTRY
July, 1926
Homogeneous Beta-Amylose Solutions Produced by Ultra-Filtration
I n order to determine the effect of filtration, if m y , on the composition of successive portions of the filtrate of betaamylose, polariscope studies were made on filtrations of solutions of both gelatinized starch which had been washed with alcohol until free from thiocyanate, and pure betaamylos? solutions which had been refiltered. If the high and relatively constant specific rotatory power and the pure blue color in the iodine test on beta-amylose solutionP, produced from several independent methods of pretreatment and subsequent separation, are an indication that no degradation of the starch has taken place, then the writers' results prove that they have the beta-amylose as originally present in the granule. Samples of corn starch, the weights of which are given in Table 11, were added to water to give again approximately a n 8 per cent suspension. The filtrate was collected in about 25-cc. fractions, upon which the rotatory power was detcrmined and in which the exact amount of dry substance was obtained by the procedure given under Ultra-Filtration through Permeable Membranes. These dry weights are recorded in Table I1 as per cent beta-amylose on the sample indicated. The membranes varied from grade 6 to 16 and still the results all fall in a range of specific rotations from 182 to 188 degrees which corresponds with the data of FouardI3 on the rotations of beta-amylose solutions in which he gives 181 to 190 degrees, with a n average of 186 degrees. Had there been dextrinization or saccharification during the pretreatment of the starch, one might reasonably expect some fractionation during filtration. I n this event the first portions would probably show lower rotatory power14and marked reducing action with Fehling's solution. As will be seen, the rotatory power of successive fractions is sensibly constant. Qualitative tests with Fehling's solution were negative in every instance. Table 11-Polariscope Studies on Beta-Amylose from Corn Starch Grade 1st fraction 2nd fraction 3rd fraction 4th fraction of Beta Beta Beta Beta Wt. mem- Per Grams brane cent u2: Gelatinized Starch 7.6 184.9 7.5 182.4 7.4 185.8 8 13.0 8 6.7 6 . 2 1 8 8 . 3 6.1 186.6 5.1 184.9
Et
Et ...
8 6
6.8 12.7
7.1 184.9 5.3 185.8
. ..
16.0
6.06 188.3
6.8 187.5 5.3 183.3
6.1 187.5 5.2 184.9
...
i:3
186:7 4.5 187.5
Redissolved Beta
6.2 186.6
5.3 185.8
4.8 186.7
Effect of Method of Gelatinization on Separation of Amyloses by Ultra-Filtration
To determine whether the method of gelatinization employed had any peculiar or special effect on the separation of the amyloses, a 1 per cent suspension of raw corn starch was heat-gelatinized in the autoclave under 1 atmosphere (15 pounds) pressure for one-half hour. At the end of this time i t was found to be completely gelatinized as observed under the microscope. When such a sample was ultra-filtered, the hydrated mass so clogged the pores of the collodion filter that, even after several weeks, only a few cubic centimeters could pass through. It was interesting to note, however, that in either case the beta-amylose solutions obtained from corn starch gave a distinct blue with iodine, hydrolyzed to a clear solution (no fatty acids), and gave an 0'2 of 186.7 degrees, indicating that the beta-amyloses obtained by both methods of gelatinization are the same. 18 Compt. rend., 144, 501, 1366 (1907); 146, 285, 978 (1908); 813, 931 (1908); 148, 502 (1909).
14
147,
Defren, Original Communications, 8th Intern. Cong. A p p l . C h e n . , 13,
113 (1912).
715
Samples of the alcohol-hydrogen chloride purified starch, just neutralized with sodium carbonate, were gelatinized by heating in the autoclave a t 1 atmosphere (15 pounds) pressure for one hour. When this material was ultra-filtered a clear filtrate was obtained and the percentage of beta-amylose corresponded with those obtained from the separations on the thiocyanate-gelatinized starch, as shown in Table 111. Table 111-Ultra-Filtration of Alcohol-HC1 Treated Corn Starch Neutralized a n d Heat-Gelatinized Volume of Beta-amylose BetaSample" Grade of filtrate Grams dry wt. amylose Grams membrane Cc. in filtrate Per cent
24.7 10.0
13 13
500 297
21.1 8.7
85.5 86.7
10.0 14 288 8.7 87.3 a Sample made up to approximately an 8 per cent suspension before gelatinization.
These data indicate that the same results are obtained by pressure gelatinization as by thiocyanate gelatinization, but because of ease in handling and higher concentrations obtained the latter is by far the better method. Electrodialysis
Many investigators have mentioned the migration of starch when subjected to the action of the electric current. same^.'^ in particular, has studied the conductivity of the amylopectin, or alpha-amylose, from potato starch in relation to its phosphor& c o n t e n t . Therefore, in the separation of the two amyloses on a larger scale than in the case of ultra-filtration, it was considered possible to use this property. For this purpose an apparatus (Figure 2) was assembled which w o u l d a l l o w a cataphoresis of the starch and a t the same time a d i a l y s i s of the small amount of remaining ammonium thiocyanate and any other soluble salts. These help to make the solution c o n d u c t the current. A cylinder 65 cm. long and 18 cm. in diameter, which was constricted on either end to an Figure &Apparatus for Electrodialysis opening of 6 cm. in diameter, was used. On one end an impermeable membrane, a, was prepared by dipping a stretched cheesecloth covering in collodion, drying in air, and redipping and drying until a membrane resulted which was not permeable to the amyloses but would allow the slow dialysis of inorganic ions. This cylinder was then suspended in a large battery jar which would form one electrode chamber and could be filled to the height of the solution inside, thereby lessening the strain on the membrane when the apparatus was in use. As the second electrode chamber a similar membrane, a', was prepared on one end of a glass tube 4 cm. in diameter which dipped into the solution to be electrodialyzed. Two platinum electrodes were placed in the electrode chambers and connected with the 110-volt direct current, which thus completed the circuit. 18 Kolloidchem. Bcihefte. 6, 23 (1914); 7, 137 (1915);10, 289 (1919):12, 281 (1920): 13, 272 (1921).
INDUSTRIAL AND ENGISEERISG CHEMISTRY
716
I n the operation of this apparatus, samples of the completely gelatinized, thiocyanate-treated starch varying from 100 to 300 grams were suspended in a sufficient quantity of distilled water to make about an 8 per cent suspension. The starch was well stirred and then placed in the cylinder. The electrode chambers were filled to the proper level with distilled water and a direct current of 110 volts was connected so that the positive pole was a t the bottom of the apparatus. This was essential since it was found that the alpha-amylose migrated to the positive pole and therefore t o take advantage of both effects, migration and gravity, this arrangement was adopted. Note-By means of a U-tube the effect of an acid or alkaline medium On the migration of the corn starch was studied Two electrode chambers with impermeable collodion membranes wcre prepared and placed so a s to d i p just into the suspension of starch in either arm Since the effect of gravity would be equal in the two arms, any boundary movement could be attributed to the effect of the applied electric current I n this way the alpha-amylose was found to move toward the positive pole whether in an alkaline or acid suspension
K h e n the current was applied a flocculation occurred in a very short time and soon a sharp boundary was seen to move in a downward direction, leaving a clear solution behind. until the flocculated material formed a loose mass a t the lower membrane. While this separation was taking place the water in the electrode chambers was periodically changed, so that all soluble inorganic material might be completely dialyzed away. After the separation was complete the upper solution containing the soluble or beta-amylose was siphoned off and total solids were determined by drying a sample in sand a t 105' C. The major part of the amylose may be recovered by precipitation with alcohol and washing with absolute alcohol, which gives a white, fine powdery product. To continue purification of the residue of alpha-amylose in the electrodialysis, distilled water was added again and a second migration allowed, after which the supernatant liquid was drawn off and the concentration determined. This process was continued until the soluble beta-amylose was washed away from the alpha-amylose, as shown by the absence of color when iodine test solution was added to the washings. The heavy paste of alpha-amylose remaining was frozen's and melted, producing a partially dehydrated and more granular mass, which could be sucked reasonably dry on a Biichner funnel. Treatment of this material with absolute alcohol until dry or desiccation in an oven for 4 hours a t 105' C. gave the alpha-amylose the weights of which are given in Table IV. This method is more convenient for the isolation of the dry alpha-amylose than the previous one. Table IV-Electrodialysis of S t a r c h (2) (3) (4) 7-Alpha-amylose-Beta-amylose Grams Per cent Per cent (by diff Corn Starch 100.00 11.45 11.5 88.5
(1) Sample Grams
100.00 187.35 200.00 273.00 242,06
14.80 25.62 27.84 37.37 37.85
Av. Rice Sfarch 100.00
85.10
100 00 100 00
15.91 14.50
14.8 14.0 13.9 13.7 15.6 14.0
85.2 86.0 86.1 86.3 84.4 86.0
15.9
84.1
17.0
Av. 16.5 Potato Starch 1 93 1 9 1 i5 1 7 Av. 1 8
)
83.0 83.5
98 1 98 3 98 2
The average results as given in column 3 show the per cent of alpha-amylose as determined by direct weight. I n Table I, column 6, the results of a direct determination of betaamylose were given. 16
Oakes. Columbia Dissertation, 1924.
Vol. 18, No. 7
Properties of the Amyloses
The two amyloses differ greatly in their physical and chemical properties. Alpha-amylose from each starch investigat'ed is insoluble in water and when dried either by alcohol treatment or in the air forms a light brown, hard material, whereas the beta-amylose is soluble up to 8 per cent by weight and may be precipitated with alcohol giving a fine, pure white powder. The beta-amylose solution serves as a good indicator in iodometry, giving a clear, deep blue color, whereas the alpha-amylose gives a reddish violet or purplish color when in suspension. In the electrodialysis the alpha-amylose moves when a potential difference is applied, whereas the beta-amylose does not appear to move. The beta-amylose leaves no weighable ash when burned, which would indicate the absence of any inorganic constituents; t,he alpha-amylose leaves a slight residue on ignition. When subjected to hydrolysis using 10 per cent hydrochloric acid the corn alpha-amylose is attacked slowly, but when completely disintegrated leaves a flocculent residue much the same as that produced by the original starch when it is hydrolyzed. In contrast, the beta-amylose hydrolyzes readily and gives a clear, colorless solution with the characteristic reducing properties of a sugar. This condit.ion upon hydrolysis may serve well as a criterion of purity of the betaamylose together with its characteristic rotatory power of 186 degrees, its color with iodine, its solubility, it3 nonreducing properties before hydrolysis, and the absence of ash upon burning. Distribution of Noncarbohydrate Constituents
The flocculent residue from the hydrolysis of corn alphaamylose upon extraction with ether yielded a fatty residue, a property which has been shown to be characteristic of whole corn starch by Taylor and N e l ~ o n . ~I n order to make sure that the fatty material was not absorbed as free fat, a sample of alpha-amylose was extracted in a Soxhlet for 18 hours, using carbon tetrachloride as the solvent. By this treatment 17.353 grams of alpha-amylose yielded 0.066 gram extractable residue. Upon hydrolysis this sample yielded 0.2042 gram, or 1.18 per cent, of fatty material, which would indicate that the fatty material was not liberated until hydrolysis had taken place. Samples of this material were found to have an iodine number of 90.6 and 91.2, which is the same as those of the fatty acids which may be extracted from commercial refinery mud. However, during each successive treatment of the corn and rice starch, preparatory to the actual separation of the alphaamylose, a small amount of fat was unavoidably lost, so that the actual percentages decreased slightly a t each stage. T a b l e V-Fat F a t t y acids in raw starch Per cent
Extraneous fat Per cent
0.17
0.82 0.79
0.12
0.61 0.59
0.04 0.03
S t u d i e s on C o r n S t a r c h -FATTY ACIDS, PER CENT--On HCI- On NHICNSIn treated treated alphastarch starch amylose Old Starch 0.47 0.30 1.6 0.47 0.30 1.5 New Starch 0.,55 0.36 2.3 0.54 0.35 2.3
The first column of Table V shows the percentages of fatty material in the raw corn starch from both old and new preparations. The second column shows the small amount of extraneous fat as determined directly on a sample of untreated starch. This amount would be expected to be lost during the alcohol-hydrogen chloride treatment. A small amount is also lost during gelatinization, but the alpha-amylose is
IiVDUSTRI-AL A N D ENGINEERING CHEMISTRY
July, 1926
found to be much higher in fatty material, which indicates that the combined fatty acids stay in the alpha-amylose during separations, which is corroborated by the lack of any fatty material in the beta-amylose. That the amount of fat carried by the corn and rice alphaamylose approximates the total carried by the thiocyanatetreated starch, of which the alpha is a part, is evident from the results shown in Table VI. For instance, as shown in the corn-starch sample, it was possible to recover from 60 to 90 per cent of the fatty material after the separation of the alpha-amylose. The rice starch bears a slightly higher percentage of fatty acids and of alpha-amylose, and in this case * also the recovery averaged about 85 per cent. T a b l e VI-Fat SHaCNStreated starch Grams ~
Recovery S t u d i e s
~
100 100 187 200 273
100 85
AlphaF a t t y acids F a t t y acids amylose F a t t y acids recovered Per cent Grams Per cent Per cent Old Corn Starch 0.30 11.6 1.6 60 1.5 73 0.30 14.8 New Corn Stavch 2.3 90 0.36 25.6 0 . .36 27.8 2.3 91 0.36 37.4 2.3 90 Rice Starch 0.54 15.9 2.7 82 0.40 14.5 2.1 86
717
A Vacuum Regulator’ By G. Vincent Scofield CARBIDE& CARBON CHEMICALS CORP., CLENDENIN~ W. V A .
HE vacuum regulator shown in the accompanying sketch is a device to hold constant any pressure, from the lowest the vacuum pump will pull to atmospheric. With this regulator it is possible to hold a constant pressure, say of 25 mm., while conducting a 100-ml. distillation test. It is useful in preparing vapor pressure curves (below atmospheric pressure) by the dynamical method of determining boiling points under a series of pressures. It may be used when it is desirable to filter liquids under a iicontrolled” vacuumthat is, in cases where the filtrate may evaporate seriously at a low pressure, or the filtering device or the paper will not stand less than a certain pressure. A
C l 3
In the case of potato starch the percentage of ether-extractable material in the raw product was found to be only 0.02 per cent. However, the phosphorus content16j17of potato starch is quite appreciable, so i t was of interest to see if this method would likewise separate the phosphorus-bearing constituent entirely in the alpha-amylose. This was found not to be the case, for the percentages of phosphorus in raw starch and the beta-amylose were approximately the same. The phosphorus as determined by the alkaline fusion method was found to be 0.087 per cent and 0.090 per cent in the raw starch, 0.086 per cent in the beta-amylose, and 0.049 per cent in the alpha-amylose. This result might be anticipated, since in the work of Kelson and Northiop18 on phosphorus in potato starch a compound bearing the phosphorus in greater percentage than the original starch was not produced until the starch had been broken down partially by acid hydrolysis and no precipitate with alcohol could be obtained.
\
F
/ Key t o I l l u s t r a t i o n
P,
Standard iron pipe, 122 cm. long, 38 mm. diameter, closed at bottom with C1, Iron cap, and a t the top with C2. Iron cap, drilled centrally and fitted with SI, Short piece of 13-mm. iron pipe, and X2. Iron nioale. 3.2 mm. X 38 mm. long S1. No. 2 Fubber stopper, bored t o fit-^T , Glass tube, 4 mm. 0. D., 2 mm. I. D., 136cm. long, so t h a t it will hold a vacuum and yet so t h a t T will slide easily in S1 when in use .M. Mercury filled in P to the level L. About 79 cm. from bottom of P F. 5-liter Pyrex round-bottom flask R,R,Rubber vacuum tubing, 6.4 mm. I. D. X 1.6 mm. wall D , Glass tubing, to be connected with the system for which the constant low pressure i b rlpSired - -. ..- -
S2, G,
V,
A,
Rubber stopper Vacuum gage Vacuum pump Air inlet
Summary Two methods are given for the separation of the slimy or alpha from the more mobile or beta component of three common starches. By these methods it is possible to work with relatively high concentrations of starch, obtain complete disintegration of the granule, and effect a clean-cut separation. The yields of the amyloses by the two methods are concordant, but until it is possible to define an amylose the results are not absolute. Analysis for the minor noncarbohydrate constituents shows that in the case of corn starch the associated fatty acids go almost quantitatively with the alpha portion, whereas in potato starch, where phosphoric acid is prominent, it can be found in both amyloses. A more detailed study of the separated amyloses with regard to the associated noncarbohydrate substances may lead to a more precise definition of the components of the starch granule. Such work is now in progress in this laboratory. Acknowledgment The authors wish to thank the Corn Products Refining Company for the supply of refined corn starch used in this work. 1’
18
Thomas, Biorhem Bu2l , 3, 403 (1914). , 38, 472 (1916).
J . A m . Chem Soc
The glass tube T is adjusted until the desired pressure is shown by the vacuum gage G. As the pump V pulls a greater vacuum, air is automatically let in a t A . The reservoir F is necessary to absorb pressure fluctuations in the system due to intermittent admittance of air through the mercury column and to the rising and splashing mercury. The spam above the mercury (approx. 43 cm.) is to prevent it from splashing out of the pipe P. The seemingly large diameter -clef the pipe P (38 mm.) is to prevent the incoming a from causing too much “rumbline”-and consequently volume change, and therefore pressure changeon the system as it passes up through the mercury. A large rubber stopper can be used a t the top of the pipe and fitted to take the place of C2, 81,N1, and N 2 , but it is not so satisfactory. I n the regulator that the writer is using all joints between the cap C2, pipe Nl, nipple N2, and the pipe P have been brazed to insure against leaks. The cap C1 at the bottom is fitted with a rubber gasket, and the threads are shellacked, but not brazed, so that if necessary it can be opened.
-
Y
1
Received Decemher 16, 1924.
u
