An Improved Method for Determination of Selenium in Biological Material SIR: I n the past several years many excellent methods have been published for determining the concentration of selenium in biological material. But even with these improved methods many analytical problems remained unsolved. For example, all of the practical methods were very time consuming, requiring from 8 hours in some cases to as long as 48 hours in others. Many of these methods required expensive instruments not found in many analytical laboratories. Therefore, realizing the great demand for a rapid and accurate' quantitative method of analysis which could detect microquantities of organic-bound selenium, we developed and published a method which appeared to solve all of these problems ( 2 ) . However, after this method was published, a n improved method was devised which allows one to analyze a single sample in less than an hour or 15 samples in about 2 hours, and the results appear to be better than those obtained with our previous method. The only other practical methods that can compare with this method on a time basis and still retain their sensitivity are neutron activation analysis and atomic absorption spectrophotometry. The cost of the equipment alone makes these methods prohibitive in many laboratories. Also, neutron activation analysis becomes impractical if one wishes to analyze a large number of samples. EXPERIMENTAL
Apparatus and Reagents. STANDARD SELENIUM SOLUTION. Prepare a stock solution containing 1 mg. of selenium per ml. by dissolving 2.190 grams of carefully dried sodium selenite in distilled water t o which
Table 1.
I
TIME (MINUTES)
Figure 1 . Time required for maximum color development of the Se-DAB complex a t 60' C. 4 pg. Se
approximately 80 ml. of 48% hydrobromic acid has been added. Dilute this solution t o 1 liter. Standardize this solution gravimetrically. Prepare a solution containing 1 p.p.m. b y diluting the stock solution. DIGESTIONMIXTURE. Dissolve 10 grams of sodium molybdate in 150 ml. of distilled water. Slowly add 150 ml. of concentrated sulfuric acid to the molybdate solution. After cooling, add 200 ml. of 70-72% perchloric acid. Saturated NaOH, 90% formic acid, 4OyG hydroxylamine hydrochloride, 0.2M EDTA (disodium salt), 3,3'diaminobenzidine (DAB), (0.5% aqueous solution freshly prepared from the tetrachloride), metacresol purple, (0.1% in 0.0026N NaOH).
Selenium Content in Dried Tissue
Described method pg. Se recovd. pg. Se recovd. from 0.10 g. from 0.15 g. 3.90 4.00 4.00 4.25 4.20 3.90 4.00 4.07 4.20 3.90 4 04 f 0 13 40 4
5.95 5.70 6.10 5.95 6.18 6.25 5.95 6.37 6.37 6.25 6.11 f0.23 40.7
Neutron activation analysis p.p.m. Se recovd. from 0.36 g. 3 7 . 0 f 0.9O 35.4 f 0 . 9 39.6 f 0 . 9
Av. pg. Std. dev. pg. Av. p.p.m. 37.3 a Plus or minus values given are standard deviations estimated from counting statistics only. 430
ANALYTICAL CHEMISTRY
per
4.0 ml.
Constant temperature bath, pH meter, and a Spectronic-20. Procedure. Digest 0.1 to 0.2 gram of dried material or 0.5 to 1.0 gram of wet material in 5 ml. of the digestion mixture. Digest in either a 50ml. 'digestion tube or in a microKjeldahl flask. Add 5 t o 6 boiling stones to prevent bumping. (The boiling stones should be boiled for several minutes in t h e digestion mixture a n d thoroughly washed with distilled water prior to use. This removes trace amounts of selenium which we have found in some boiling stones obtained commercially.) Heat the tube or flask over a low flame with continuous swirling. I n about 5 to 10 minutes the solution begins to boil, and vigorous oxidation takes place soon thereafter. Most samples, such as skeletal tissues, hair, blood, and plant material are completely digested in about 15 to 20 minutes, and the solution becomes clear at that time. Upon digesting bone tissue, however, a white insoluble precipitate appears at the completion of the digestion, and it should be removed before continuing the analysis by means of a fine-porosity fritted glass filter. After digestion, allow the tube to cool and then wash its contents plus the boiling chips into a 250- ml. beaker with about 20 ml. of distilled water. Qring the p H up to about 7 with saturated NaOH using metacresol purple as an indicator. At this pH the indicator changes to a purple color. The p H is then lowered to between 2 and 3 by adding 2 ml. of 90% formic acid. A4dd4 ml. of 40% hydroxylamine to reduce any excess oxidative reagent.
The solution now starts to change to a greenish color. Add 4 ml. of 0.2M E D T A to mask the interfering ions. I t is now advisable to check the p H to see that it is still between 2 and 3. If not, either add concentrated ",OH or 90% formic acid for the adjustment. Add 2 ml. of O.5ye 3,3'-diaminobenzidine, and complex in a constant temperature bath at 60" C. in subdued light for only 20 minutes. After removal from the bath, adjust the pH to between 7.0 and 7.5 with concentrated N H 4 0 H . Since the solution at this point is quite colored, the indicator change is not distinct and a pH meter must be used. Rinse the solution into a 125-ml. separatory funnel with a few milliliters of distilled water, and extract with exactly 4 ml. of toluene by shaking the mixture vigorously for 1 minute. Allow 3 t o 4 minutes for complete layer separation and discard the aqueous layer. Transfer the toluene extract to a centrifuge tube and centrifuge for a few minutes. Determine the absorbance of the extract a t 420 mp with a Soectronic-20 using a toluene blank. Preuare a standard curve bv adding 2, 4, b, 8, and 10 ml. of the"1 p.p.m: standard solution to 5 ml. of the digestion mixture and heat to boiling for about 5 minutes, then carry out the remainder of the procedure. Y
RESULTS AND DISCUSSION
The advantage of this method over our previously published method ( 2 ) is in the elimination of the ascorbic acid reduction step which was necessary to isolate the selenium from interfering ions. Gutenmann and Lisk (3) reported that the selenium-DAB complexing reaction can be carried out in the presence of EDTA. EDTA masks ions such as Fe(III), Cu(II), and V(II1) which have likewise been shown to complex with DAB (1). The elimination of the ascorbic acid reduction step reduces the analysis time by a t least 1 hour. I n addition, another 40 minutes can be saved by hastening the rate of the complexing reaction. We found that a t 60" C . maximum complexing occurs in 20 minutes (Figure 1). After about 23 minutes it appears that decomposition of the selenium-DAB complex starts to occur. T o determine the efficiency of this method 20 analyses were carried out on samples of acetone-dried liver obtained from sheep whose diets were supplemented with subtoxic levels of sodium selenate. [The preparation of this dried tissue is described in ( 2 ) . ] Some of the same dried tissue sample was sent
to General Atomics (General Dynamics General Atomic Division, .ictivation ;inalysis Service, San Diego, Calif.,) for neutron activation analysis. After comparing the results obtained from the neutron activation analysis with those obtained by this method, it must be concluded that this method is capable of recovering virtually all of the organic-bound selenium. These data are summarized in Table I. LITERATURE CITED
(1) Cheng, K. L., ANAL.CHEM.28, 1738
(1956).
( 2 ) Cummins, L. M., Slartin, J. L., Maag, Grace W., Maag, 11. I)., Zbzd., 36, 382 (1964). ( 3 ) Gutenmann, W.H., Lisk, D. L . , J . Agr. Food Chem. 9,488 (1961).
LAURENCE M.C ~ M M I N S JOHN L. ~ T A R T I K DALE1). MAAG Department of Chemistry Colorado State University Fort Collins, Colo. Division of Analytical Chemistry, 148th Meeting, ACS, Chicago, Ill., September 1964. This research was supported in part by two Public Health Service Grants: Fellowship No. 5-Fl-Chl-20,675-02 from the National Institute of General Medical Sciences and Grant No. EF-00187 from the Bureau of State Services.
Use of In Situ Reactions for Characterization of Alcohols and Glycols by Nuclear Magnetic Resonance SIR: Esterification of hydroxy compounds produces downfield shifts of nuclear magnetic resonance peaks which are caused by hydrogens alpha to hydroxy groups (2'). The magnitude of the shift, the multiplicity of the shifted peaks, and the relative areas of the shifted peaks are valuable aids in determining structures. Such shifls often separate peaks which are overlapped in the spectrum of the hydroxy compound so that multiplet patterns can be determined and their areas measured. The addition of a reactive isocyanate or ketene to an alcohol or glycol in the NMR sample tube is a simple and rapid means for effecting the desired changes in the KMR spectra. These reagents produce no by-products and the urethanes and esters which are produced do not need purification. EXPERIMENTAL
The proton magnetic resonance spectra were recorded using a Varian A-60 N M R spectrometer equipped with a V-6031 variable temperature probe. Peak positions are given in parts per million, relative to tetramethylsilane
as an internal standard. Diphenylketene was prepared by the procedure of Staudinger ( 4 ) . Trichloroacetyl isocyanate was prepared by the method described by Speziale and Smith (3). The sample of 2,2-dimethyl-5-phenyl1,3-pentanediol was synthesized in this laboratory by a method similar to that described in the literature (1). All other materials used were commercially available, reagent grade compounds and were used without further purification. RESULTS AND DISCUSSION
Reagents which were used in this work are diphenylketene, phenyl isocyanate, and trichloroacetyl isocyanate. Diphenylketene is the least desirable of the three reagents because it is not readily available, because it does not have a long shelf life, and because its reactivity toward hindered hydroxy groups is limited. Phenyl isocyanate is a satisfactory reagent but it is also slow to react with hindered hydroxy groups. The reactivity can be increased by using catalysts and elevated temperatures. Complete reaction is not always
necessary since the alpha hydrogen peaks in the spectrum of the derivative can often be seen if the isocyanate is 50Oj, reacted. Trichloroacetyl isocyanate is the most effective of the three reagents. It reacts almost instantaneously with primary or secondary hydroxy groups and requires only 2 or 3 minutes to react with tertiary hydroxy compounds and phenols. This reagent produces no N M R peaks of its own and it has a good shelf life. Its derivatives are fairly soluble. Concentrations of about 10% in common solvents are attainable a t elevated temperatures. The main disadvantage of trichloroacetyl isocyanate is that it is not readily available; however, it can be prepared without great difficulty. The most obvious method of determining whether a hydroxy group is primary or secondary is to measure the area of the peaks which shift and thereby determine the number of hydrogens alpha to the hydroxy group. If this is impossible because of overlapping of peaks, the magnitude of the shift will furnish the answer. Shitts of about VOL. 37, NO. 3, MARCH 1965
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