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15.4041 19.2836 24,2430 30,7720 39,4419 51.0562 66,7531 88.1595 117.6134 %58,5155 215.8376
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( S E E F I G U R E 6).
Potentiometric Determination of Cyanide with Ion Selective Electrode Application to Cyanogenic Glycosides in Sudan Grasses W. J. Blaedel, D. B. Easty,l Laurens Anderson, and T. R. Farrell Departments of Chemistry and Biochemistry, University of Wisconsin, Madison, Wis. 53706
After accelerated hydrolysis with emulsin, cyanide may be determined in the hydrolyzate by direct potentiometric measurement with an ion selective electrode. The method was evaluated for Sudan grass samples which were also analyzed by volatilizing the cyanide away from the hydrolyzate before measurement. The root mean squared error of the procedure is around 1 ppm of HCN, or 2% relative, whichever is greater.
A BRIEF SURVEY has been made of methods of determining cyanide resulting from hydrolysis of cyanogenic glycosides in plant tissue ( I ) . The sensitivity, speed, and simplicity of the potentiometric method give it an indisputable advantage over other methods. However, despite their commercial availability, ion selective electrodes have not yet been applied to the sensitive and precise determination of cyanide in plant tissues. Gillingham and coworkers ( 2 ) have used a cyanide electrode to screen forage samples approximately for the cyanide content. Gyorgy and coworkers (3) employed an electrode for the potentiometric determination of cyanide in steam distillates from plant hydrolyzates. These authors stated without experimental confirmation that direct measurements might be made on the hydrolyzate.\ In this paper, a method is developed for the determination of cyanide in plant leaf hydrolyzates by direct potentiometric measurement with an ion selective electrode. The method is tested on Sudan grass samples which were also analyzed by
another method ( I ) involving removal of the cyanide from the hydrolyzate before measurement. THEORY
Ion selective electrodes are not completely specific, but are subject to interferences. It has been shown that the potential of an ion selective electrode is dependent not only upon the activity ( a x ) of the sought-for substance, but also upon the activities of other substances that may be present. For some electrodes,
E
=
Constant
RT + 2.303 ~log (ax + $1 zxF
+
The explicit form of is not known except for a few simple systems which do not include the cyanide electrode [Ref. 4, p 42 (G. Eisenman), p 64 (J. W. Ross), and p 98 (A. K. Covington)]. In general, may depend not only upon the activities of other substances present, but also upon the selectivity of the electrode for them. If there is interaction between the sought-for substance and interferences, may also depend upon the chemical properties of these substances. In an analysis, it is usually the concentration (Cx) which is sought. Since C, is related to ax through the activity COefficient yx, Equation 1 may be rewritten:
+
+
E = constant
RT + 2.303 log Y x + ZxF -
1 Present address, Institute of Paper Chemistry, Appleton, Wis. 54911
(1) D. B. Easty, W. J. Blaedel, and L. Anderson, ANAL.CHEM., 43, 509 (1971). (2) J. T. Gillingham, M. M. Shirer, and N. R. Page, Agron. J . , 61, 717 (1969). (3) B. Gyorgy, L. AndrC, L. Stehl, and E. Pungor, Anal. Chim. Acta, 46, 318-21 (1969). 890
ANALYTICAL CHEMISTRY, VOL. 43, NO. 7, JUNE 1971
In the methods of this paper, Equation 2 is rendered analytically useful by making all determinations in a medium of (4) R. A. Durst, Ed., “Ion Selective Electrodes,” National Bureau of Standards Special Publication 314, November 1969.
controlled composition, so that yx is constant. Also, +/yx is treated as a “blank” (B), contributed to by noncyanide substances in the sample and reagents that affect E. Further, if E is measured always against the same reference electrode (say the SCE) and at controlled temperature, then any junction potentials are also constant, and the voltage developed becomes quite simply dependent upon C,, and may be taken as a measure of Cx: E = E‘
+ S log (Cx + B )
(3)
The imposition of such control is simple in principle, but difficult in practice. The standard addition technique 14 (p 381), 5, 6)] has become very popular as a means of compensating for proportional error with ion selective electrodes, but it does not permit evaluation of the blank, which has been uncritically and erroneously ignored in many applications. In the following work, the blank may be determined by taking advantage of the volatility of HCN. There are many important sources of nonproportional error-electrode potential drifts and fluctuations due to temperature drifts of the sample solution or to instrument changes during warm-up or departures from steady state operating conditions, variations in geometry and electrodeto-ground capacitance between measurements, and variations in memory or time-dependent effects between measurements. Our experience has shown that these errors can be subtle and large if not properly provided for: typically, errors of the order of 5 mV (corresponding to a relative error around 20 in concentration) may exist undetected and uncompensated, even when the method of standard addition is used. To reduce such errors, measured potentials for all samples in the following work are referred to the potential of a standard solution, measured before and after each sample. EXPERIMENTAL
Equipment. The cyanide electrode (Model 94-06, Orion Research, Inc., Cambridge, Mass.) was threaded on its end to receive a ll/?-inch length of Plexiglas tubing (1-inch 0.d.). With the electrode end upwards, the Plexiglas tube formed a cup having an inside volume around 9 ml that held the sample solution. A small motor-driven stirrer (600 rpm) and the reference electrode (fiber junction calomel electrode, cat. no. 39170, Beckman Instruments, Fullerton, Calif.) dipped into the solution. (Magnetic stirring was not used because heating of the solution resulted.) Samples of about 6 ml were easily accomodated by this design. Cell potentials were read with an expanded scale pH meter in the millivolt mode (Model 12, Corning Glass Works, Medfield, Mass.). Calibration of the meter was facilitated with an adjustable bucking potential between the reference electrode and the meter, provided by a 1.5-V battery across a 5000-ohm potentiometer. A recorder (Model SR, E. H. Sargent and Co., Chicago, Ill.) for the pH meter output was useful for indicating when the potential had reached steady state. Procedure for Reading the Potential of a Solution. Before reading the potential of a solution, the meter was calibrated with a standard NaCN reference solution consisting of lOWM NaCN in a solution containing 0.05M potassium acid phthalate and 0.022M NaOH. (This composition was chosen early in the work because it represented approximately the composition of plant tissue homogenates. Other compositions would have been equally satisfactory.) With the standard NaCN reference solution, the bucking potential was adjusted to give a reading of 70 mV. The reference solution ( 5 ) A. Liberti and M. Mascini, ANAL.CHEM., 41, 676 (1969). (6) M. J. D. Brand and G. A. Rechnitz, ibid., 42, 1172 (1970).
was then aspirated to waste, the cell was flushed and filled with the sample solution, and the potential was read. The sample solution was aspirated to waste, the cell was flushed and filled with fresh standard NaCN reference solution, and the potential was read. When the difference between the standard solutions was less than 1 mV, the sample reading was corrected by of the difference. Only rarely was the difference between standards greater than 1 mV, in which cases the readings were repeated. Procedure for Cleaning the Electrode. The manufacturer’s recommendations were followed for ordinary cleaning (7). On a few rare occasions, however, the electrode became scratched by hard usage, or fouled with glass homogenates, and response time lagged. The electrode was reconditioned by hand sanding on a flat piece of 400 mesh silicon carbide waterproof sand-paper until the scratches and foreign deposits were removed. Further grinding was done on a 23-p silicon carbide paper (Ultralap, manufactured by Pfizer’s Mineral, Pigment, and Metals Division, New York, N.Y.), followed by finishing on 8-p Ultralap with silicone oil. One moderately used electrode has been reconditioned three times in this way over a two-year period, and it still functions well. Hydrolysis and Measurement of Sudan Grasses. The hydrolysis procedure has been described ( I ) . A blank of around 3 ppm was found applicable with little error to a considerable variety of plant tissues, as described later in this paper, so blanks were not determined for each sample of grass. A 0.25-gram portion of grass was homogenized in 7.5 ml of pH 5 potassium acid phthalate buffer. The homogenate was accumulated in a 50-ml narrow-mouth glass-stoppered bottle. After homogenization, 1.50 ml of emulsin reagent were added (almond beta glucosidase from Worthington Chemical Co., Freehold, N. J., 1 mg per milliliter of pH 5 phthalate buffer). After incubation for an hour in the stoppered glass bottle at room temperature, the mixture was brought to around pH 12 with 1.10 ml of 1 M NaOH. The basic solution was permitted to stand for a half hour to hydrolyze small amounts of glycosides not hydrolyzed enzymatically. Then an additional 1.10 ml each of 1 M NaOH and of H2S04were added, and the potential of the solution was determined as described above. The total cyanide molarity was found from a working curve for grasses, prepared as described later. The cyanide content of the grass, as parts per million of HCN, was calculated: 1.33 X lo6 X
(
Total cyanide molarity from the working curve
-
(4)
RESULTS AND DISCUSSION
Response Curve for Standard Cyanide in Basic Phthalate Solutions. A stock solution was prepared which contained 0.0356M potassium acid phthalate, 0.097M NaOH, and 0.049M Na2S04, which represented closely the composition of homogenates after processing for potentiometric measurement. This was called the basic phthalate solution. A portion of this stock solution was then made up to 0.001M in NaCN, by weight from the reagent grade salt. A series of solutions down to 10-+M in NaCN was then prepared by dilution, and their potentials were measured as described in the experimental section. A plot of cell voltage us. the log of the cyanide concentration according to Equation 3 is shown in Figure 1. A least squares analysis of data for solutions having concentrations of 10-5M or greater revealed a slope of -58.4 mV, within a millivolt of the theoretical slope of -59.1 mV at 25 “C. The standard deviation of (7) Orion Research Inc., “Instruction Manual Cyanide Activity Electrode Model 94-06,” Cambridge, Mass., 1967. ANALYTICAL CHEMISTRY, VOL. 43, NO. 7, JUNE 1971
891
$-200 W’
v,
2E -150w
>
w n
ENDOGENOUS CYANIDE PLUS BLANK __--------
Lw
ADDED
g -IOOw
Z 0
F
5I-
I
I
-6
-5
I
I
-4 -3 LOG OF CYANIDE MOLARITY
B Figure 1. Response curve of the cyanide electrode for standard NaCN in basic phthalate solution*
Figure 2. Response curves for standard NaCN added to Greenleaf Sudan Grass hydrolyzates*
Slope, S, -58.4 mV per decade Intercept, E’, -360.5 mV Standard deviation, 0.34 mV
Slope, S, -57.2 mV per decade Intercept, E’, -361.5 mV Standard deviation, 0.25 mV
* All
*
potentials measured relative to a selected potential of -70 mV for a solution containing lO-5M NaCN in 0.050M potassium acid phthalate and 0.022MNaOH
individual measurements from the least squares straight line was only 0.34 mV, corresponding to a relative standard deviation of only 1 . 4 z in cyanide concentration throughout the range 10-3-10-5M. Points below lO-5M were not included in the least squares analysis because the response curve is known to deviate upward from the straight line at low concentrations (7). Response Curves for Standard Cyanide in Grass and Leaf Hydrolyzates. Increments of standard NaCN added to grass hydrolyzates gave closely similar response curves for all of the varieties of plant tissue studied, as shown by the following work. For each variety of grass, standard additions of cyanide were made to the hydrolyzate containing the endogenous cyanide, and also to the hydrolyzate that had been sparged with nitrogen to remove the endogenous HCN, so that the blank could be estimated. To avoid variations due to sampling, homogenization, and hydrolysis, pooled samples were used for each variety of grass. Using the techniques described previously, a 3.00-gram portion of Greenleaf Sudan grass was homogenized in 90 ml of pH 5 phthalate buffer, the homogenate being accumulated in a 250-ml narrow-mouth glass stoppered bottle. Twenty milliliters of emulsin reagent were added and the mixture was incubated in the stoppered bottle for 1 hour at room temperature. Then 15.0 ml of 1M NaOH were added to bring the mixture to a pH around 12, and another half hour at room temperature was allowed for further alkaline hydrolysis. This pool was then split into two portions. One 50-ml portion was removed from the glass-stoppered bottle, and 6.0 ml each of 1M NaOH and of 0.5MH2S04were added to give the unsparged “sample pool.” The residual 75 ml 892
ANALYTICAL CHEMISTRY, VOL. 43, NO. 7, JUNE 1971
All potentials measured relative to a selected potential of -70 mV for a solution containing 10-5M NaCN in 0.050M potassium acid phthalate and 0.022M NaOH ** Four points practically coincident
in the unstoppered bottle was acidified with 9.0 ml of 0.5M HzS04, sparged with nitrogen and slow magnetic stirring for 1 1 / 2 hours, and finally made basic with 9.0 ml of 1M NaOH to give the sparged “blank pool.” One-milliliter aliquots of standard NaCN in basic phthalate solution in the range 10-2 to 10-4M, and a I-ml aliquot of cyanide-free basic phthalate solution were added to 10-ml volumetric flasks, and the contents were made up to volume with portions of the sample pool. Similar standard cyanide additions were also made from NaCN standards in the range to lO-5M with portions of the blank pool. The voltages of these solutions were then measured as described in the experimental section. Figure 2 contains plots of cell voltage cs. the log of the added cyanide concentration, according to Equation 3. The sparged plot is curved toward the dilute end, indicating that the blank here was appreciable compared to the added cyanide. By standard addition calculations, the value of the blank was found to be 1.5 X 10-6M (indicated in Figure 2), which corresponded to an apparent 2.3 ppm of HCN in the original grass sample. This blank was added to the standard amounts of cyanide added for each aliquot, to give the total cyanide (corresponding to Cx B in Equation 3) for each aliquot, The log plot of the total cyanide is also shown in Figure 2. It may be seen that the total cyanide curve merges with the added cyanide curve in the high concentration region, where the blank is negligible compared to the added cyanide. Numerical data from which the curves are calculated are shown in Table 1. In a similar manner, standard addition calculations for the unsparged sample gave 2.9 x lO+M for the endogenous
+
Table I. Response Curve Data for Sparged Greenleaf Sudan Grass (Plotted in Figure 2) Measured Added cyanide, CX Total cyanide, CX + Ba Solution potential, mV Molarity Log Molarity Log 1 - 28 0 (blank) ... 1.5 x 1 0 - 6 5 -5.82 2 -60.5 4 x 10-8 -5.40 5 . 5 x 10-6 -5.26 3 - 79 10-6 -5.00 1.15 x 10-5 -4.94 4 - 111 4 x 10-6 -4.40 4.15 x -4.38 5 - 133 10-4 -4.00 1.02 x 10-4 -3.99 6 - 167 4 x 10-4 -3.40 4.02 x 10-4 -3.40 - 190 10-3 -3.00 1.00 x 10-3 -3.00 7 a The blank, B, may be found by extrapolating the high concentration limiting slope of the added cyanide curve down to the measured potential of the blank (- 28 mV), to give log B = - 5.82, or B = 1.5 X 10-6M. B may also be calculated using Equation 3 and the difference in potentials between solutions 1 and 2, to give 1.6 X 10-6M.
Variety Greenleaf Tift Low cyanide Corn
Table 11. Response Curves for Sudan Grasses and Corn Blank Molarity ppm in Endogenous Intercept, ( X 106) grass HCN, ppm E’, mV 1.5 2.3 43.2 -361.5 2.5 3.9 213 -361.8 1.3 2.0 4.2 -360.9 2.6 2.9 12.6 -361.5
Table 111. Evaluation of Rapid Potentiometric Method for Cyanide in High Cyanide Grasses CN content of grass, Sudan in duplicate, ppm Difference, Potent.-Contin. grass variety method method in ppm rel. Sweet 61, 62 60,63 1 2 -2 -3 2 3 -1 -2 Tift 230,226 224,226 6 3 4 2 2 1 0
0
Slope, mV/decade -57.2 -57.4 -57.0 -57.6
Standard deviation, mV 0.25 0.20 0.50
0.43
m
2E -150W
> W
n
gt- -100-
ki d W
cyanide plus blank (indicated on Figure 2), which corresponded to 45.5 ppm of HCN in the original grass sample. (The endogenous cyanide content of the original grass therefore was 43.2 ppm.) When total cyanide concentrations were calculated and plotted in Figure 2, there was precise agreement with the total concentration plot for the unsparged aliquots. A least squares analysis of the total cyanide curve using points at lO-5M or above is given in the legend to Figure 2, and shows excellent linearity and low scattering. Similar measurements were made on other varieties of Sudan Grass and Corn, with results summarized in Table 11. The four response curves are remarkably similar and may be combined to give the over-all response curve shown in Figure 3. Evaluation of the Potentiometric Procedure for Sudan Grasses. The low scattering (standard deviation of 0.72 mV, corresponding to a relative standard deviation of only 1.3 %) indicated that the cyanide contents of 3 different Sudan Grass varieties and Corn could all be read from the single working curve shown in Figure 3. The method of standard addition is not necessary for precise work. Further, the low blanks (2.0-3.9 ppm in Table 11) indicated that application of a blank correction of 3 ppm would permit elimination of the blank determination for individual samples, and would cause errors of only about 1 ppm.
D -50Z
su 24 LL
t-
5 6a
oy I
I
I
I
I
-5 -4 -3 LOG OF CYANIDE MOLARITY
-6
Figure 3. Grass hydrolyzate working curve*
Slope, S, -57.43 mV per decade Intercept, E’, -361.9 rnV Standard deviation, 0.72 mV
* Composite of all curves in Table I1 (50 points) The potentiometric method was tested on Sweet and Tift Sudan grasses. Pooled hydrolyzates were prepared, potentials were measured on duplicate aliquots, and the cyanide concentrations of the hydrolyzates were found from the working curve of Figure 3. The cyanide contents were calculated and corrected for a blank of 3 ppm, to give the results shown in Table 111. In addition, aliquots of the pools were also analyzed in ANALYTICAL CHEMISTRY, VOL. 43, NO. 7, J U N E 1971
893
duplicate by the continuous procedure ( I ) , in which the HCN was volatilized away from interferences and collected in pure 0.03M NaOH for measurement. Procedure was exactly as described in the literature, except for one difference: the recovered HCN was measured potentiometrically instead of amperometrically. Results are summarized in Table 111, with individual determinations listed, rather than the averages of duplicates, so that some idea of the precision of the method may be conveyed. Not only is the precision of both methods good, but agreement between them is excellent, the root mean squared relative difference between them being around 2 %. Attempt to Apply the Potentiometric Procedure to Low Cyanide Grasses. Toward the end of this investigation, considerable interest arose in the analysis of low cyanide materials, particularly in some varieties of corn which are resistant to insects and root worm. Consequently, several materials, including low cyanide Sudan grasses, and corn and tomato leaves, were analyzed potentiometrically and also by
the volatilization procedure, as described above. The blank was determined for each material by sparging. The potentiometric method gave cyanide contents (2-13 ppm) that ranged up to twice as high as the cyanide contents determined by volatilization. Until this discrepancy is resolved, the potentiometric method is not recommended for hydrolyzates of low cyanide materials. ACKNOWLEDGMENT
Thanks are extended to L. E. Schrader of the Department of Agronomy for his cooperation in supplying the grass samples. RECEIVED for review January 7, 1971. Accepted March 2, 1971. This work was supported in part through a postdoctoral fellowship for D. B. E. provided by Hatch Funds from the U. S. Department of Agriculture, and in part through Atomic Energy Commission Grant No. AT(11-1)1082.
Analysis of Acrylic Polymers Using Combined Zeisel Reaction-Gas Chromatography and Infrared Spectrometry D. G. Anderson, K . E. Isakson, D. L. Snow, D. J. Tessari, and J. T. Vandeberg DeSoto, Inc., Administratiue and Research Center, 1700 S . Mt. Prospect Road, Des Plaines, Ill,
A quantitative method for the analysis of acrylic polymers was developed using the Zeisel reaction and gas chromatography. The rate of the cleavage reaction was studied and found to depend on the monomer composition and/or the stereoconfiguration of the polymer. Also, the reaction products from hydroxylcontaining monomers were identified. Infrared techniques were developed for the quantitative determination of styrene and the qualitative identification of acrylic acid, methacrylic acid, and the half ester of maleic acid.
THESUPERIOR FILM properties and durability of thermosetting acrylic polymers make them extremely popular as vehicles in coatings compositions. These resins have diverse applications in automobile, container, and home appliance finishes. With increased acceptance by the coatings industry, it has been necessary to develop an accurate and reliable method for the analysis of thermosetting acrylic resins. A survey of the literature indicates that no comprehensive and reliable technique exists for the analysis of acrylic polymers. Previous workers have proposed pyrolysis-gas chromatography for the chracterization of polymers (1-8). This (1) J. Strassburger, G. M. Brauer, M. Tyron, and A. F. Forziati, ANAL.CHEM., 32, 454 (1960). (2) E. A. Radell and H. C. Strutz, ibid., 31, 1890 (1959). (3) R. S. Lehrle and J. C. Robb, Nature, 183, 1671 (1959). (4) S . Straus and S. L. Madorsky, J . Res. Nut. Bur. Stand., A., 50, 165 (1953). (5) F. A. Lehrnann and G. M. Brauer, ANAL.CHEM.,33, 673 (1961). (6) K. Ettre and P. F. Varadi, ibid., 34, 752 (1962). (7) Ibid., 35, 69 (1963). (8) G. G. Esposito and M. H. Swann, J . Gas Chromatogr., 3,282 (1965).
894
ANALYTICAL CHEMISTRY, VOL. 43, NO. 7, JUNE 1971
procedure is good for qualitative polymer analysis but has limited quantitative application. Determination of maleate and acrylate esters in polymers by Zeisel cleavage of the acrylate ester linkages, followed by gas chromatographic analysis of the reaction products, has also been reported (9-18). We chose the Zeisel reaction (Figure 1) for the cleavage of the acrylic esters and gas chromatography for the analysis of the alkyl iodides formed. Using this procedure, the recovery of alkyl iodides is greater than 95 for polymers containing between 10 and 90% of the methyl, ethyl, and butyl esters of acrylic and methacrylic acid. In addition, the use of isopropylbenzene as the trapping solvent allows the determination of all C1 to Cd alkyl iodides. Quantitative cleavage of the acrylate and methacrylate esters is also observed in the presence of modifying monomer units such as styrene. In order to achieve specific properties, acrylic polymers are often modified by copolymerization with monomers such as styrene, vinyl acetate, vinyl chloride, and acrylamide. The Zeisel-gas chromatographic procedure does not permit the (9) J. Haslarn, J. B. Hamilton, and A. R. Jeffs, Analyst,83, 66 (1958). (10) D. L. Miller, E. P. Sarnsel, and J. G. Cobler, ANAL.CHEM., 33, 677 (1961). (11) R. Kretz, 2. Anal. Chem., 176, 421 (1960). (12) A. Steyerrnark, J. Ass. Ofic. Agr. Chem., 38, 367 (1955). (13) S. Ehrlich-Rogozinsky and A. Patchornik, ANAL.CHEM.,36, 840 (1964). (14) W. C. Easterbrook and J. B. Hamilton, Analyst, 78, 551 (1953). (15) W. Kirsten and S. Ehrlich-Rogozinsky, Mikrochim. Acta., 4, 786(1955). (16) S. Vertalier and F. Martin, Chim. Anal. (Paris), 40, 80 (1958). (17) K. F. Sporek and M. D. Danyi, ANAL.CHEM., 34,1527 (1962). (18) G. Castello, G. D’Arnato, and E. Biagini, J. Chromatogr., 41, 313 (1959).
