ANALYTICAL CHEMISTRY
318
subject t o sorption and selective vaporization while the other is free of them. The separations that occur on brass surfaces strongly suggest a crude gas-chromatography column ( 6 , 11). This analogy, in turn, suggests that the versatility of the method might be much enhanced by provision for a choice of sorbing media, of eluents, and of operating temperatures.
Table I. Trace Components Found in Heart Cuts of Alkrlbenzenes Alkylbenaene 2-Phenyl-2-methylpentane 2-Phenylpentane 3-Phenyl-3-methylhexane 3-Phenyl-3-et hylpentane I-Phenylheptane
B.P.,
C./Mm. Hg 87/20 92/34 112/20 132/50 145/50
Contaminants, Volume % EthylBenzene Toluene benzene 0.07 ... ... ... ... 0.9 0.02 ... 0.01 0.02 .., ... 0.05 0.02 0.02
LITERATURE CITED
Brown, R. H., Meyerson, S., I n d . Eng. Chem. 44, 2620 (1952). Charlet, E. M., Mass Spectrometer Group Report 72, Consolidated Engineering Corp., Pasadena, Calif ., 1950. (3) (4) (5) (6)
cuts from distillations of several alkylbenzenes. Prolonged distillation at temperatures above 200’ C. has been reported to cause significant decomposition of hydrocarbons (14). The data given in Table I suggest that slight decomposition may occur even at temperatures well below 200” C.
(7j (8) (9) (10) (11) (12) (13) (14)
DISCUSSION
The results reported in Table I illustrate that components, once identified, can be determined if means are available for measuring their contributions to the sample spectrum free of sorption and vaporization effects. I n most cases, this condition would require two separate instruments or two parallel inlet systems to one instrument, such that one sample admission is
(15)
Ibid., 74,1950. Grubb, H. M., Meyerson, S., Ibid., 76, 1950. James, A. T., Martin, A. J. P., Brit.Med. Bull. 10, 170 (1954). Kellev. H. M.. ANAL.CHEM.23. 1081 (1951). Melpolder, F. ‘W., Brown, R. A:, Washall, T. .1.,Doherty, W., Young, W. S., Ibid., 26, 1904 (1954). hfeyerson, S., Ibid., 25,338 (1953). Meyerson, S., A p p l . Spectroscopy 9, 120 (1965). O’Keal, M. J., Jr., Wier, T.P., Jr., ANAL.CHEM.23, 830 (1951). Patton, H. W., Lewis, J. S.,Kaye, W. I., Ibid., 27, 170 (1955). Purdy, K. M., Harris, R. J., Ibid., 22, 1337 (1950). Rock, S. M., Ibid., 23, 261 (1951). Rossini, F. D., Mair, B. J., Streiff, 4.J., “Hydrocarbons from Petroleum,’’ p. 110, Reinhold, S e w York, 1953. Taylor, R. C., Brown, R. A,, Young, W.S., Headington, C. E., ANAL.CHEM.2 0 , 3 9 6 (1948).
RECEIVED for review August 24, 1955. Accepted December 20, 1955.
Spectrophotometric Method for Quantitative Evaluation of Early Stages of Hydrolyses of Branched Components of Starches by Alpha-Amylases JOHN
W. VAN DYK and M. L. CALDWELL
Department of Chemistry, Columbia University, New
York 27, N. Y.
A sensitive method has been developed for measurements of the initial stages of the hydrolysis of waxy maize starch by pancreatic amylase from swine. It can be used also under suitable conditions to follow the initial stages of hydrolysis of the branched components of other starches by pancreatic amylase or by other a-amylases. The method is an adaptation of the blue-value technique originally developed for linear components of starches. It makes use of the absorption spectra of the complexes of iodine with the substrate and with its early hydrolysis products. The method makes i t possible to determine the relative rates of hydrolysis of dilute solutions of branched components of starches. It is rapid, precise, and convenient for kinetic studies. By calibration of the absorption ratios against the free aldehyde groups formed, i t becomes possible to express the velocity of the hydrolysis obtained by the spectrophotometric method in terms of the glucosidic linkages hydrolyzed.
A
LTHOCGH other methods have been suggested from time to time, quantitative studies of the action of amylases have, until recently, been based largely upon measurements of the increase in free aldehyde groups resulting from the hydrolysis of 1,4cu-~-glucosidiclinkages of the substrate. However, such measurements are not suited to precise quantitative determinations of the initial stages of hydrolyses catalyzed by a-amylases. I n the initial stages of their action, a-amylases cause a very large reduction in the average molecular weight of the substrate for each glucosidic linkage hydrolyzed; the free aldehyde groups thus formed on large fragments are difficult to detect or deter-
mine by methods developed by the use of lorn-er molecular weight reducing sugars such as glucose and maltose. I n 1943, McCready and Hassid (8) reported the blue-value method that is well adapted to the study of the early stages of amylase hydrolyses of linear components of starches. The method is based upon spectrophotometric measurements of complexes of iodine with these substrates and with certain of their hydrolysis products (6, 8). This procedure, with certain minor modifications ( 3 , 7 , IO), has been found useful by other investigators ( 7 , 10). I n the method reported here, the blue-value technique has been adapted for the study of the early stages of the hydrolysis of fat-free waxy maize starch (9, 1 2 ) by crystalline pancreatic amylase from swine (4). The procedure permits the precise determination of the course of the early stages of the action of the amylase. The method is especially useful for kinetic studies n i t h dilute solutions of the substrate. It can be employed t o determine relative rates of hydrolysis. By calibration of the absorption values against the free aldehyde groups formed, it becomes possible to express the velocity of the hydrolysis obtained by spectrophotometric measurements in terms of the glucosidic linkages hydrolyzed. Although it was developed for an investigation of the hydrolysis of defatted waxy maize starch by pancreatic amylase from swine, the method can be employed under suitable conditions to follow the hydrolysis of the branched components of other starches by pancreatic amylase or by other a-amylases. REAGENTS AND APPARATUS
Iodine reagent. Dissolve 3.10 gr ms of iodine and 31 grams of potassium iodide per liter of distified water.
V O L U M E 2 8 , NO. 3, M A R C H 1 9 5 6 Hydrolysis mixture, a solution of defatted branched starch, amylase, and appropriate buffer and salts reacting a t 30' C., a t 40' C., or a t any other appropriate temperature. Blank solution, identical with the hydrolysis mixture except that the enzyme is omitted. Iodine-starch complex solution. Dissolve 0.05 gram of starch and 0.62 gram of iodine per liter. The instrument used was a Beckman Model D U spectrcphotometer thermostated a t 30" C.
319 sorbance of the iodine blank makes a large slit 13-idth necessary. For this reason, the absorption was characterized a t a wave length of 540 mp instead of a t the maximum. The effect of iodine concentration on the spectrum of the complex of iodine with waxy maize starch is shown further in Figure 2, where the absorbance a t 540 nip is plotted against iodine concentration. An iodine concentration of 0.06270 was chosen for the analytical method.
PROCEDURE
Ten milliliters of iodine reagent are pipetted into a series of 50-ml. volumetric flasks. T o one of the flasks a suitable volume of the blank solution is added to give a final starch concentration, after dilution, equal to 0.05 gram per liter. The solution is diluted to volume when convenient. It is allowed t o come to equilibrium for a t least 30 minutes in a 30" C. water bath, then absorbance of the complex of iodine with the original substrate (OD,) is read in a Beckman spectrophotometer a t a wave length of 540 mp. The procedure is repeated a t intervals with portions of the hydrolyzate instead of the blank solution. These measurements give the absorbance of the complex of iodine with the hydrolysis products. These absorbances are designated O D , where t is the time of hydrolysis. The absorption ratios ( A R t ) then are calculated and plotted versus time of hydrolysis, t.
The relative rate of reaction is determined from the slope of this curve or from the reciprocal of the time required t o reach a given absorption ratio. RESULTS
Development of Method. Branched components of starches do not absorb iodine readily (1, 2, 8, I f , I S ) . However, the data given in Figures 1 and 2 show that solutions containing as little as 0.005~owaxy maize starch can be used as the basis for an analytical method, provided sufficiently large concentrations of iodine also are present.
'"m A
.4s
Wove Length Figure 1.
(mB)
iibsorption spectra of complex of iodine with waxy maize starch
I n Figure 1, the absorption spectra of 0.005% waxy maize starch in 0.062 and 0.01870 iodine solutions are shown. These spectra were measured in a Beckman Model DU spectrophotometer thermostated a t 30" C., with a 1-cm. cuvette. The appropriate iodine solution was used as the blank. For comparison, the spectrum of a 0.062% iodine solution is included. A broad absorption maximum occurs in the iodine-waxy maize starch spectrum at a wave length of about 525 mp. However, the spectrum is distorted at loyer wave lengths, because the large ab-
, .02
.04
.06
.00
GO N CENTRAT ION,% I OD1N E Figure 2. Effect of iodine concentration on absorption spectrum of t h e complex of iodine with waxy maize starch at 540 mp 0.006% starch present in each case
Calibration Curve for Conversion of Absorption Ratio to Per Cent Theoretical Glucose. The method as outlined is useful for the determination of relative rates of hydrolysis. However, when it is desirable t o express the velocity of hydrolysis in terms of the glucosidic linkages broken, it is necessary t o calibrate the absorption ratios against the free aldehyde groups formed. Such calibrations were carried out in this work by removing two samples simultaneously a t intervals from a hydrolysis mixture containing swine pancreatic amylase and waxy maize starch and determining the absorption ratio ( A R ) with one sample and the per cent theoretical glucose ( 5 , 1 4 ) n-ith the other. The per cent theoretical glucose was determined by an iodometric method (6) that had been modified to give greater sensitivity (14). Waxy maize starch concentrations of 0.05 and 0.025% and hydrolysis temperatures of 30" and 40" C. m-ere used. The relationship between the absorption ratio and the per cent theoretical glucose was found to be linear Tithin experimental error. It m-aa not influenced by the changes studied either in the concentration of the substrate or in the temperature of hydrolysis. The best 0.975 - .4R straight line by least squares gives 0.0T6 - % theoretical glucose. This particular relationship is valid only for the hydrolysis of waxy maize starch by swine pancreatic amylase, which differs in its action from that of a number of other a-amylases ( 7 ) . I n addition, the accuracy of calibration curve used here is limited by any deviation of the iodometric method from stoichiometry. Precision. The precision of the spectrophotometric method was established by determining the absorption ratios ( A R )a t 10minute intervals for two identical hydrolysis mixtures containing
ANALYTICAL CHEMISTRY
320 Table I. Determination of Absorption Ratios' for Duplicate Hydrolyzates*of Waxy Maize Starch and Crystalline Swine Pancreatic Amylase Time of Hydrolysis, Minutes 0 10
20
Absorption Ratiob 1.00 1.00
0.932
0.803
0.689 0.572 0.500
30 40 50
0.945 0.786 0.675 0.591 0.493
Deviation from Mean, d e d X 105 6.5 8.5
adapted for a study of the inhibiting effects of these low molecular weight products on the initial velocity of the hydrolysis of branched components of starches by a-amylases. ACKNOWLEDGX EYT
The authors wish to thank the Corn Industries Research Foundation for generous grants in aid of this investigation.
7 0 9.5
LITERATURE CITED
3.5
Baldwin, R. R., Bear, R. S., Rundle, R. E., J . Am. Chem. SOC.
where ODt = absorbance of hydrolyzate a t 540 mu and absorbance of original waxy maize starch solution a t 540 mu. b Wa& maize starch 0.00625%, 0.02.W NaCl. 0.01M phosphate; p H 7.2. C Standard deviation = 10.007 A R unit.
a AR = Y at time t OD0
66, 1 1 1 (1944).
z t Y Y
Bates, F. L., French, D.. Rundle, R. E., Ibid., 65, 142 (1943). Beckmann, C. O., Roger, 31.,J . Bid. Chem. 190,467 (1951). Caldwell, 32. L., .4dams, Mildred, Kung, J. T., Toralballa, G. C., J . A m . Chem. Soc. 74, 4033 (1952). Caldwell, M. L., Doebbeling, S. E., Manian, 5. H., ISD. ENG. CHEX,ANAL.ED. 8, 181 (1936). Hanes, C . S., Cattle, 31.,Proc. Roll. SOC.(London) B125, 387
=
0.0625 gram of defatted (9, 12) waxy maize starch per liter. The data summarized in Table I show that the standard deviation for a single determination is f0.007 A R units. From the relationship given above, this value would be equivalent to a standard deviation of 1 0 . 0 9 % theoretical glucose or 1 3 . 5 X 10-7 mole of aldehyde groups per liter. Limits of Concentration of Waxy Maize Starch. The lowest concentration of waxy maize starch that can be subjected conveniently to this technique is 0.0062570. It should be possible to study still lower concentrations by wing a cuvette with a longer optical path. The upper limit of substrate concentration is fixed only by its solubility. Inhibition Studies. Because iodine does not produce colored complexes with the l o m r molecular weight products of amylase action, the spectrophotometric method reported here can be
(1933). \ - - - - ,
Kung, J. T., Hanrahan, V. 3T.,Caldwell, 31.L., J . A m . Chem. SOC.75, 5548 (1953) McCready, R. AI.. Hassid. W.Z., Ibid., 65, 1154 (1943). Mindell, F. hI., Agnew, -4. L., Caldwell, 31. L., Ibid., 71, 1779 (1949).
Phillips, L. L.. Caldwell. 31. L., Ibid., 73, 3863 (1951). Rundle, K. E , Foster, J. F., Baldwin, R. R., Ibid., 66,
2116
(1944).
Schoch, T. J., Ibid., 64, 2954 (1942). Swanson, M. A., J . Bid. Chem. 172,805, 825 (1948). Van Dyk, J. W., dissertation, Columbia University, Yew York. 1984. RECErVLO for review Aiigiist 5 , 1955 Accepted December 14, 1955. Data taken from a dissertation submitted by John W. Van D y k in partial fulfillment of requirements for degree of doctor of philosophy in chemistry under the Faculty of Pure Science of Columbia Vniversity.
Activation Analysis of Trace Impurities in Germanium Using Scintillation Spectrometry GEORGE H. MORRISON and JAMES F. COSGROVE Chemistry Laboratory, Sylvania Electric Products, Inc., flushing,
A method has been developed, based on the use of neutron activation analysis, for the quantitative determination of trace impurities in germanium. Gamma scintillation spectrometry has been employed to identify and measure the amounts of the various impurities present. The method employs a minimum of chemical separation. A sensitivity of 0.001 to 1 y is attained for most elements using this technique.
THF.
, e ectrical properties of semiconductors, and the characteristics of devices in which they are used, are profoundly influenced by the presence of trace impurity atoms. I n the case of germanium used in transistors, the concentrations of impurities are so minute that previously available methods of detection have proved ineffective. Seutron activation analysis in conjunction with gamma scintillation spectrometry has been applied successfully t o the analysis of trace impurities in silicon (e), and the present study concerns the development of a similar method for the analysis of trace impurities in germanium and some of its compounds. While methods using activation analysis have been developed in the past for the determination of traces of arsenic ( 4 ) and copper (6) in germanium, the present study takes advantage of the resolution
N. Y.
of the gamma scintillation spectrometer, which permits the identification and measurement of many impurities in the same analysis with a minimum of chemical separation. The method possesses a sensitivity of 0.001 to l y for the majority of the elements. NUCLE4R REACTIOYS
When a sample is irradiated with thermal neutrons in a pile, many elements undergo an ( n , y ) reaction n-ith the formation of the corresponding radioisotopes. The amount of the radioisotope produced is proportional to the flux of neutrons involved, the reaction cross section, the neight of the element being investigated, and the duration of the irradiation. It is possible for other nuclear reactions such as (n,p)and (n,.) to occur in the case of a few elements, but it is accepted that the ( n , y ) reaction will predominate when a sample is irradiated in a pile. When germanium is irradiated, the ( n , ~reactions ) summarized in Table I occur. Three radioisotopes of germanium-germanium$1, germanium-75, and germanium-ii-are formed, as well as radioactive arsenic-77-a product of the decay of germanium-ii. Similarly, radioisotopes are formed from impurities present in the sample. hlany of the nuclides formed from the impurities are gamma emitters. Consequently, gamma scintillation spectrometry is used t o identify these impurities; it is based on the characteristic energies of the gamma photonsof the respectivenuclides.
