1193
Anal. Chem. 1983, 55, 1193-1194
Effect of Sample Dissolution Procedures on X-ray Spectrometric Analysis of Biological Materials Peter A. Pella," Howard M. Klngston, and John R. Sleber Center for Analytlcai Chemistry, National Bureau of Standards, Washington, D. C. 20234
Liang-Yuan Feng Institute of Geochemistry, Academia Sinica, Guiyang, Guizhou, The People's Republic of China
A preconcentration method for the X-ray spectrometric analysis of trace elements in coal, urban particulate, and urine, together with detection and quantitation limits, has been described previously, (1). Chelex-100 was used to separate trace elements €rom allrali and alkaline earths; the separated metals were quantitatively loaded on cation exchange resin filters and subsequently determined by X-ray fluorescence spectrometry. Recently, we have applied this method to the analysis of other types olf biological samples such as NBS-SRM 1577 bovine liver and its renewal, SRM 1577a. We have measured iron, manganese, copper, and zinc but have obtained seriously low recoveries for iron (e.g., 30-60%). Jones et al. (2) have also employed the Chelex-100 separation for the determination of several trace elementel in ten NBS-SRM's including bovine liver. They reported that the nature of the acid digestion procedure strongly influenced the recoveries. They found that binary acid digestions gave about 78% iron recovery in bovine liver and pine needles. Martinie et al. (3) have identified variouti organic compounds and ammonium perchlorate remaining in the residue aftm wet oxidation with perchloric acid. Their work indicates that amino acids in the proteins of biological samples may not be completely destroyed by the wet oxidation procedure. This suggests that elements that are strongly complexed in biological tissues such as iron present in the heme moiety may remain partially complexed. We performed several radiotracer experiments to check the Chelex separation procedure for any interference of ammonium perchlorate and investigated alternate sample dissolution methods in an effort to improve the iron recovery. Because the protein content of :NBS-SRM 1575 pine needles is much lower than that of bovine liver, we analyzed the pine needle SRM for the purpose of comparison. The results obtained were compared either to NBS certificate values or to results obtained by other workers where certified values were not available.
EXPERIMENTAL SECTION Radiotracer Recovery Studies. A solution of 59?e,65Zn,and 54Mnradioisotopes was prepared from stock solutions obtained from ORNL. A 0.5-mL aliquot of a solution of these radioisotopes was added to acid-digested samples of bovine liver, pine needles, and reagent blanks. Samples weighing from 0.5 to 1.0 g were taken for analysis and dissolved according to the procedures described in ref 1. Three fraction8 were collected from the Chelex separation and measured by high-resolution y-ray spectrometry. The f i t fraction was collected after loading the sample solution on the column. Fifteen milligramH of water was then added ,and this wash solution was checked for breakthrough of the tracers. The second fraction consisted of a 30-mL volume of ammonium acetate buffer for elution of the alkali and alkaline earths, and the third fraction was the elution of the tracers with 10 mL of 2.5 M HN03. The resin was then removed from the column and measured for any retention of the tracers. In order to maintain constant geometry in the y counting of theEie solutions, the three fractions and the column resin were placed in 250-mL polyethylene bottles and brought up to 40 mL volume with dilute "0% The bottles were then placed directly on a Ge(Li) detector and counted for 20 min each. The integrated peak areas were measured for each tracer;
Table I. Percent Recovery of Tracers after Elution with Nitric Acida isotope
pine nd 1
pine nd 2
bov liv 1
*Mn 59Fe 65Zn
100 100 100
98 85 92
99 97 98
bov NH,ClO, liv 2 + spike 99 97 97
85 85 82
spike only 99 96 98
a Estimate of combined random and systematic errors in the percent recovery values is * 2 % relative.
-
counting errors were about 1% relative. Sample Dissolution. A . Samples were dissolved in 150-mL PFA, FEP Teflon, and quartz beakers. The amounh of the acids added to dissolve 1g of sample were 20 mL of concentrated "OB, 10 mL of concentrated HC104,and 10 mL of concentrated HF. The samples were taken to near dryness, treated with another portion of concentrated HNOBand HC104acids, and again taken to dryness. The solutions were heated on a hot plate almost to the softening point for the FEP Teflon beakers (i.e., approximately 270 "C), but about 25-50 OC higher for the sample solutions in PFA Teflon and quartz. HF was not added to the sample contained in quartz until after treatment with HN03-HC104 acids, and the sample solution was transferred to an FEP Teflon beaker. The procedure for the removal of NH4+ by sublimation, pH adjustment of solution, Chelex separation, and preparation of SA-2 papers was carried out as described in ref 1. B. Sealed-Tubes (Modified Carius Technique). Samples of approximately 0.5 g, weighed to the nearest 0.001 g, were quantitatively transferred to 30.5 cm long, 3.0 mm thick wall borosilicate glass tubes with a 12 mm i.d. Additions of 10 g of concentrated HN03 and 2 g of concentrated HC1 were then made (4). Each tube was immersed immediately in frozen COz slushchloroform mixture, flame-sealed with an Oz-CH4 torch, allowed to warm to room temperature, and then placed in a steel shell with approximately 50 mL of solid COz surrounding the glass tube in order to equalize the pressure. The tubes were placed in an oven and heated for 15 h at 240 "C. The tubes were removed, dowed to cool, and then immersed in COzslush as before to freeze the solution prior to opening. The tubes were cracked opened by heating the flame seal with a torch. The solutions were quantitativelytransferred to Teflon beaken and heated to dryness on a hot plate. The residues were taken up in dilute nitric acid. These solutions were then ready for Chelex separation after pH adjustment and subsequent loading on SA-2 filter paper as described in ref l.
RESULTS AND DISCUSSION In the above procedure, ammonium perchlorate is the primary salt left after digesting samples of high protein content such as bovine liver. We therefore tested for the effect of this salt on the Chelex separation procedure. A spiked reagent blank was neutralized with enough ",OH to give an NH4C104salt content 10-fold greater than the amount calculated to remain in the digestion of a 1.0-g sample of bovine liver. The efficiency of the Chelex separation was followed by measuring the radiotracer breakthrough after each step. No significant breakthrough of the tracers occurred either during this column loading or after treatment with ammonium acetate to elute the alkali and alkaline earths.
This article not sublect to U S . Copyright. Published 1983 by the American Chemlcal Society
1194
Anal. Chem. 1983, 55, 1194-1197
Table 11. Summary of Results for NBS-SRM's 1575, 1577, 1577a, and 1577b sample Dine needle 1 2
obsd
Mn, i&/g cert .a
65 6 659
obsd
191 208 190 196 196 +-13
6 7 5 i 15
714
Fe, /.lg/g cerka
obsd
2 0 0 i 10
2.8 3.1 2.8 3.1 3.0 k0.3
cu, P g / g cert .a
3.0
* 0.3
obsd
Zn, P g / g cert .a
63 66 63 69 65 t4.6
64.9 i 5.5b 3 4 648 av 669 SC t48 bovine liver 1-1577a 10.5 9.9? 0.8 1 9 4 i 20 166 15' 158k 7 123t 8 2-1577bd 10.9 3-1577 10.3 +- 1.0 152 148 268+ 8 188 190 1 9 3 t 10 130i 13 4-1577 10.5 a NBS certificates of analysis 1575,1577, and 1577a, pine needles and bovine liver, respectively. Not a certified value, obtained by neutron activation analysis at NBS. Standard deviation of the mean at the 95% confidence level (Le., X t t s / n"'). Note: 1577b is intended as a renewal for 1577a and is believed to have essentially the same composition for the above elements as 1577a.
i;;
ii: i:;
Table 111. Results with Different Sample Dissolution Procedures
sample bovine liver SRM 1577 l a 2b 3b 4c SRM 1577a 5c
Fe, P g / g c u , /.lg/g obsd % obsd % values recovery values recovery 257 236 244 149
96
191 183 192
124 ~
81
77
149
a Dissolved in quartz beaker. Dissolved in sealed tubes.
99 97
131 Dissolved in PFA Teflon.
Elution of the tracers with nitric acid indicated that the recovery of all the tracers was essentially quantitative for all samples except for the blank sample containing the excess NH4C104. These results are summarized in Table I. The tracer study showed that after the acid elution step, 15% of the Fe and 18% of the Zn were retained on the column. On the basis of these experiments, we increased the elution volume of nitric acid from 10 to 30 mL to overcome any effect of differing amount of perchlorate salts on the elution recovery of the trace metals. In Table I1 are summarized the results of the analysis of bovine liver and pine needles using the dissolution procedure described in ref 1in which the only modification made was the use of a larger elution volume of nitric acid in the Chelex procedure as described above. The recoveries for each element in pine needles and for Mn and Zn in bovine liver were almost 100%. The Fe recovery for bovine liver (SRM 1577) was 61%. We then modified our sample dissolution procedure in an
attempt to improve the Fe recovery. Beakers of PFA Teflon and quartz were used to digest samples of bovine liver. These beakers enabled higher temperatures to be used than did the FEP Teflon beakers used previously. Another sample dissolution method was also used for comparison and consisted of treating bovine liver samples in borosilicate glass tubes containing HN03-HC1 acids, flame-sealingthe tubes, and then heating the samples in an oven at 240 "C. These results are tabulated in Table 111. As seen from the table, the Fe recovery improved to 90-96% with the PFA Teflon and quartz vessels. The copper recovery was also more reproducible and quantitative. The recoveries for Fe and Cu using the sealed-tube procedure were only about 60%. These data indicated that the "0,-HCl mixture did not completely destroy soluble complexes of these metals. These results indicate that high temperatures of 300 "C or more are required to break up complexes of metals in biological samples when using perchloric acid digestion procedures. This is particularly important when the analytical method used depends on ion-exchange or chelation separation of the trace metals from matrix elements such as in the Chelex procedure. Registry No. Mn, 7439-96-5; Fe, 7439-89-6; Cu, 7440-50-8;Zn, 7440-66-6;nitric acid, 7697-37-2; perchloric acid, 7601-90-3; hydrofluoric acid, 7664-39-3.
LITERATURE CITED (1) Klngston, H. M.; Pella, P. A. Anal. Chem. 1981, 5 3 , 223. (2) Jones, J. W.; Capar, S. 0.; O'Haver, T. C. Ana/yst (London) 1982, 107, 353. (3) Martlnle, G. D.; Schilt, A. A. Anal. Chem. 1976. 48, 70. (4) Gordon, C. J . Res. NaN. Bur. Stand. ( U . S . ) 1944, 33, 457.
RECEIVED for review December 9,1982. Accepted February 10, 1983.
Identification of Cyclic Acetals in Polyols by Mass Spectrometry Robert A. Sanders The Procter & Gamble Company, Wlnton Hill Technical Center, 6071 Center Hill Road, Cincinnati, Ohlo 45224
The reaction of aldehydes with alcohols in the presence of anhydrous acid to form acetals is well-known (1). If the alcohol is a polyol, the resulting acetal may be cyclic. For example, the formation of 4-methyl-1,3-dioxolanes from the reaction of propylene glycol with aldehydes present in flavors was noted 0003-2700/83/0355-1194$01.50/0
by Welch and Hunter (2). In our study, a search for the cause of an off-odor detected in an emulsifier produced under atypically and deliberately stressed processing conditions led (I). to the identification of 2-ethenyl-4-methyl-1,3-dioxolane A parallel effort to determine the fate of 2-propenal (acrolein) 0 1983 Arnerlcan Chemical Soclety
