1192
Anal. Chem. 1981, 5 3 , 1192-1195 31227 31227 . . r . .
A semiquantitative estimate of the amount of methanol in each sample was performed by comparing the area of the line at 31 226.70 MHz of a standard sample to that of the unknown run under the same conditions, in which the dominant line broadening mechanism is pressure broadening. The resulting Lorentzian line shape allows the area of the line to be found by multiplying the peak height by the half-width a t halfmaximum. The ratio of concentrations in the two samples is given by
c1
-=--
cz
Flgure 5. High-resolution spectra of Thunderblrd (T) and Spanada (S)
wines and a methyl alcohol standard. A semiquantitative estimate of the amount of methanol was made by comparison of the areas of the peaks. Table 11. Trace Methanol Analysis in Wine Samples wine est amt wine est amt in ppm sample sample in ppm 1 165-205 4 65-95 2 150-1 80 5 50-80 3
50-80
ponents do not cause a problem when a few lines of a dilute component can be found in which there is no interference from lines or Stark lobes of the more abundant components. Such was the case with the wines, especially since methanol is not only a much stronger absorber of microwave radiation than ethanol but its transitions are also well modulated at lower voltages than the transitions of ethanol. The line at 30 308.00 MHz showed a little interference on the shoulder from an ethanol line, but seven lines are more than sufficient to unambiguously confirm the presence of methanol in the wine samples. The strongest line in Table I proved to be the one at 31 226.70 MHz, and we believe that observation of this line alone in wine would verify the presence of methanol.
(AV/P)I (AV/P)Z
(1)
where ai is the peak absorption coefficient and is proportional to the height of the line, Av is the half-width at half-maximum, and P is the total pressure in the sample cell. The standard sample was prepared by diluting pure methanol with water until a concentration of 90 ppm methanol was obtained. The spectra of the 31 226.70-MHz line for two of the wine samples and of a methanol-ethanol-water standard are shown in Figure 5. The concentration estimates for the wine samples are given in Table 11. Interestingly enough, the amount of methanol in the wine samples tested did not seem, at least in this very limited study, to correlate with the cost or color. There was, however, one apparent correlation. It seemed that the wines that had been fortified had less methanol content than unfortified inexpensive wines.
LITERATURE CITED Lee, C. Y.; Acree, T. E.; Butts, R. M. Anal. Chem. 1975, 47, 747. Jones, G. E.; Cook, R. L. CRC Crit. Rev. Anal. Chem. 1974, 3 , 455. Scharpen, L. H.; Laurie, V. W. Anal. Chem. 1972, 44, 378R. Lovas, F. J. Anal. Instrum. 1974, 72, 103, (5) Card, M. S.; Lajko, M. S.; Peterson, J. D. “Microwave Spectral Tables”; US. Department of Commerce, Washington, DC, 1968; Vol. 5, Monograph 70.
RECEIVED for review November 17, 1980. Accepted March 9, 1981.
Preparation of Biological Materials for Determination of Selenium by Hydride Generation-Atomic Absorption Spectrometry D. C. Reamer” and Claude Veillon Beltsville Human Nutrition Research Center, U.S.D.A., Building 307, Room 2 75, Beltsville, Maryland 20705
Several wet and dry ashing procedures for biological materials for subsequent selenium determlnatlons by hydrlde generation-atomic absorption spectrometry were investigated. Samples were spiked with, or endogenously labeled with, ‘%e to monitor losses during the procedures. Dry ashlng wlth Mg(N03)2 as an ashing aid was satisfactory. Of the methods tested, wet digestion wlth H3P04,HN03, and H202was fastest, safest, and most efflclent. Undigested fat In the samples did not retain selenium. Accuracy of the methods was verified wlth materials of known selenium content and by independent means.
Selenium in biological materials is most often determined by one of three analytical methods: (1)chelation/extraction and fluorometric measurement of the 2,3-diaminonaphthalene
complex; (2) various atomic absorption spectrometric procedures; and (3) neutron activation analysis. All but the last require that the organic matrix be destroyed and the residue taken up into solution. Neutron activation analysis can measure the selenium directly in the sample, but special facilities and long waiting periods are usually required. Numerous methods have been used, with varying degrees of success, to destroy the organic matrix of biological samples while retaining the selenium for subsequent determination. With the oxygen flask method for sample oxidation, recoveries of added selenium ranged from 20 to 96% (1). With sealed “bomb” methods of wet digestion recoveries were less than 20%. With other wet digestion methods employing H2S04, HN03, and/or HzOz,selenium recoveries generally were less than 16% (1). Several wet digestion and dry ashing procedures have been developed that give complete recovery of added selenium. The
Thls artlcle not subject to US. Copyright. Published 1981 by the American Chemical Sociefv
ANALYTICAL CHEMISTRY, VOL. 53, NO. 8, JULY I881
most widely used wet digestion procedure for biological materials employs HNOa HzS04, and HC10, (2-7). This procedure is well suited for the destruction of the organic matrix while maintaining the selenium in an oxidized state to prevent its loss by volatilization, but requiresan exhaust h o d designed specifically for handling HCIOl fumes. In addition, careful operator attention is required during the digestion to reduce the risk of explosive reactions. Recently, added selenium was completely recovered from fish tissue that was digested with a mixture of HNO, and H.80, . . and a small amount of V90, .. as a catalyst (8). Siemer and Haeemann (9) and Tam and LaCroix (10) used a dry ashing proLedure with Mg(NO& and/or MgO’as an ashing aid with no loss of added or endogenous selenium. Recoveries of less than 100% indicate that a digestion procedure is unsatisfactory, but complete recoveries of added selenium do not per se validate the procedure unless it is known, or can be shown, that the added selenium reacts in the Same manner as the endogenous selenium. A most powerful technique in this regard is the use of endogenously radiolabeled samples (10, 11). We now describe our study of sample preparation tecbniques that can he used in conjunction with hydride generation for the determination of selenium at trace levels in biological samples. Radiotracer techniques were used with endogenously laheled tissues to examine methods for the oxidative destruction of a biological sample’s organic matrix without analyte losses. Selenium was quantitatively retained after one dry ashing and one wet digestion procedure. T h e accuracy of selenium determinations by hydride generation-atomic absorption spectrometry was verified by independent means. EXPERIMENTAL S E C T I O N Reagents. All reagents were of at least “reagent grade” purity or higher and no selenium was detected in any of the reagent blanks. Reagent grade HCI, HfiO,, and H$O, and the “Ultrex” grade HNO, were obtained from Baker Chemical Co. (Phillipsburg, NJ). Hydrogen peroxide (50%) and Mg(NO& were obtained from Fisher Scientific Co. (Fairlawn, NJ). For the SeHz generation, solutions of 5% NaBH, (No. 87658, Ventron COT. Danvers, MA) were prepared daily in 0.1 M NaOH and filtered through a 0.47-pm filter (Millipore Corp., Bedford, MA) to remove particulates and minimize decomposition. The SeHz-generatingsolution was 3.5 M in HCI and 0.9 M in HfiO,. For the radiotracer studies, carrier-free ’%e (New England Nuclear, Boston, MA) in the form of selenious acid was used. A stock solution of 1000 p g of Se/mL was prepared by dissolving elemental selenium in a minimum amount of HNOa and diluting to volume with 6 M HCI. Further dilutions with water were made as necessary. All water was demineralized by ion exchange to a resistivity of 18 MQ cm, as described previously (12). Instrumentation. Selenium was determined by hydride generation and atomic ahsorption spectrometry. Reference 11 describes the instrumentation used in this work. Selenium Hydride Generator. The modified (11) glass generator (Figure 1)was silanized several times with dimethyldichlorosilane (DMCS) to prevent the decomposition of SeHz on the glass. This step is extremely important for quantitative and reproducible results (11). The reaction chamber was a 50-mL, round-bottom flask with a stopcock attached to drain the sample and a 1819 socket added on the top side to allow introduction of the sample without disassembling the system. A syringe-fitted valve for the introduction of NaBH, was positioned in the carrier gas line so that the system could he purged first with nitrogen and then with hydrogen prior to the injection of the NaBH,. After the system was purged with hydrogen, air was introduced into the transfer line. An airlhydrogen flame was produced at the entrance to the quartz fumace, which had been electricallyheated to 900 “C. Once the flame stahilized, the NaBH, was injected into the H2gas stream, pushing the NaBH, into the acidic sample solution to be rapidly mixed. The generated SeHz quickly passed into the quartz furnace to he atomized by the flame and heated furnace and produced a sharp ahsorption signal (Figure 2).
1193
I
FURNACEHEllTEDTO900.C
Flgure 1. Modified hydride
generation system and quartz furnace.
n
1n,sct
Figure 2. Characteristics of the background-corrected absorpt‘wn signal due to selenium (5 mL, 40 ppb).
Tracer Procedure. Samples were spiked with “Se (20pL, -100ooO counta/min), digested by a dry ashing or wet oxidation method, and dissolved in 6 M HCI. Duplicate aqueous solutions were prepared and both were counted in the y spectrometer. Recovery of selenium was calculated by comparison with the duplicate aqueous standards, omitting the digestion procedure. Half of the digest was then subjected to the hydride generation procedure, and both aliquots were counted. The efficiency of hydride generation was then calculated. The generated gaseous 73SeHzwas either absorbed on coconut charcoal and counted to obtain m a s balances or passed into the quartz furnace for analysis. Endogeneously labeled liver tissues were obtained 3 and 24 h after adult male CD Sprague-Dawley rats (Charles River Breeding Laboratories, Boston, MA) had heen intraperitoneally injected with ‘%e. The samples were ultrasonically homogenized, lyophilized, and ground and thoroughly mixed with no loss of the radiolabel. Aliquots of these samples were treated in the same manner as the 73Se-spikedsamples. Matrix Oxidation Procedures. Three sample preparation procedures were investigated: high-temperature, dry asbing with Mg(NO& as an ashing aid; wet digestion with H$O,, HN03 and HZOz;and low-temperature, oxygen plasma ashing with and without Mg(NO&. For the Mg(NO&-aided muffle furnace ashing, unlabeled sample aliquots of 0.5 to 1g were added to a 50mL crucible and then spiked with %e and 2 mL of a saturated solution of Mg(NO,), was added. The samples were then mixed, dried, and ashed in a 450 “C muffle furnace for several hours. If the samples still had any dark particles, 1 mL of 50% HZO2 was added, and the samples were dried and ashed again. When ashing was complete, 5 mL of water and 5 mL of 6 M HCI were added and the samples were boiled for 5 min to reduce any
1194
ANALYTICAL CHEMISTRY, VOL.
53, NO. 8, JULY 1981
Table I. Recovery and Hydride Formation Efficiency of 75SeSpiked Samples Dry Ashed with Mg(NO,), sample NBS wheat flour NBS wheat flour brewers yeast brewers yeast wheat bran wheat bran freeze-dried pork
% 75Se % 75Seformed recovered hydride
97.7 95.4 99.2 100.0 100.5 99.4 99.2
85.1 82.3 88.9 79.6 50.5 68.8 79.2
selenate to selenite. The samples were diluted with the hydride-generating solution and analyzed and/or counted. The samples were counted after each step to monitor any selenium losses and again after the hydride generation. We treated endogenously labeled samples in the same manner, omitting the %e spike. When H3P04and HN03 were used for the wet digestion, 0.5-1 g of unlabeled samples was placed in a 50-mL Kjeldahl flask with a '%e spike and 1 mL of H3P04 and 5-10 mL of HN03 were added. Samples were allowed to stand overnight to prevent foaming when heated. After the predigestion, the samples were boiled until the dark HN03 fumes had subsided. Fifty percent H202was added dropwise until the solution cleared. The samples were boiled further until several milliliters of fluid remained. The digest also contained some undigested fat, which had no detectable selenium. After digestion, 5 mL of 6 M HCl was added and the digest boiled for 5 min to reduce any selenate to selenite. The samples were diluted with the generating solution and analyzed and/or counted. Again, endogenously labeled samples were processed likewise, without the %e spike. Freeze-dried rat livers containing endogenous %e were ashed in an RF oxygen-plasma, low-temperature asher (Model 505, LFE Corp., Waltham, MA). The samples were ashed with and without added Mg(N03)2,and recoveries were measured.
RESULTS AND DISCUSSION Oxygen Plasma Ashing. Endogenously labeled liver samples were placed in the low-temperature asher and ashed at 300-W RF power at an oxygen pressure of 1torr. After only 4 h, 93-97% of the endogenous I5Se had been lost. When the samples were mixed with 1 mL of saturated Mg(NOJ2, lyophilized, and ashed at the same power level and pressure, no 75Se was lost. The major limitation of this procedure is that it takes about 48 h to completely ash a 125-mg liver sample. Dry Ashing with Mg(N03)2.As a check on the steps of the dry ashing procedure, recoveries of inorganic 75Sespikes added to samples were determined. Three methods of sample drying after the addition of aqueous Mg(NO& were evaluated: direct heating on a hot plate, lyophilization, and drying in a vacuum oven, Samples could be dried quickly on a hot plate at 110 "C, but great care was required to prevent charring and selenium loss. With this method of drying, the heating rate was difficult to control reproducibly, and selenium losses were common. Samples could be lyophilized without any loss of selenium, but the sample had to be well frozen initially to prevent foaming. The best, but slowest, drying method was in a vacuum oven at 60 "C and a pressure of 125 torr, which required about 48 h. Once dried, the samples were ashed a t 450 OC in a muffle furnace. If the samples were not completely ashed, 50% HzOzwas added, and the samples were dried and ashed again without any loss of selenium. Samples were boiled in 6 M HC1 with no selenium losses. After dilution with the generating solution, a 5-mL aliquot was removed and the 75Se counted and compared to the original spike and to the aliquot subjected to the hydride generation. These samples were counted to determine the efficiency of the hydride generation reaction for a given matrix. The quantity of 75Serecovered after digestion and the efficiency of the hydride formation are shown in Table I. Recoveries of 75Seranged from 95.4 to
Table 11. Efficiency of Recovery and Hydride Formation from Spiked Samples Digested with H,PO,, HNO,, and H,O, % 75Se
sample
recovered
freeze-dried pork 98.2 i- 1.4 cereal 101.8 t 4.7 NBS bovine liver 99.5 ?: 2.4 rat livera 100.5 f 1.2 NBS wheat flour 100.5 + 1.5 a Endogenous 75Selabel,
'
% 75Seformed no. of hydride samples
91.5 t 92.4 * 80.0 t 89.5 i84.1 t
5.7 3.2
6.5 9.2 11.5
7 13 12 16 15
Table 111. Analysis of NBS Standard Reference Materials Following Wet Digestion with H,PO,, HNO,, and H,O, sample wheat flour bovine liver rice flour
pg
of Selg
1.0 t 0.2 1.2 i- 0.16
0.39
i-
0.07
certified value
no. of samples
1.1 t 0.1 1.1?: 0.1 0.4 * 0.1
5 8 3
100.5%, and the efficiency of hydride formation from 50.5 to 88.9%. To obtain quantitative results with these erratic hydride formation efficiencies, we had to use the method of additions. Two unspiked aliquots of each sample were analyzed and two aliquots, each spiked with different known quantities of selenium, were analyzed along with a reagent blank. A linear regression analysis was preformed to determine the selenium content. We also tested standards to determine whether the large excess of Mg(N03)2interfered with the atomic absorption analysis generation procedure. At a selenium level of 50 ppb in a 10-mL sample, Mg(NO& at the concentration found in digested samples showed no interference. Wet Digestion with H3P04/HN03/H202.This digestion procedure was the most satisfactory in terms of speed, safety, and efficiency. Following the overnight predigestion, the samples could be processed ready for analysis in about 15-20 min. The radiotracer techniques allowed clear demonstration of the fact that the fat in the samples does not have to be digested. This is of major importance in that fat digestion is usually very tedious in most wet ashing procedures. As a check on the wet digestion procedure, recovery was studied with inorganic 75Se-spiked samples as described. Recovery also was studied in rat liver containing endogenous 75Se. Recoveries (Table 11) ranged from 98.2 f 1.4 to 100.5 f 1.5%. Apparently the (presumably) organically bound endogenous selenium was quantitatively recovered from the rat liver samples. Recovery did not differ between the 3- and 24-h endogenously labeled rat liver samples. The efficiency of hydride formation ranged from 80 f 6.5 to 92.4 f 3.2%, considerably better than that of the dry ashing procedure. Efficiencies were similar for spiked and endogenously labeled samples. However, since the efficiencies were still below loo%, samples were also analyzed by the standard addition method. In the development of a method for quantitative analysis, the accuracy must be established, usually by analysis of either similar materials of known analyte content or a single material by two or more independent means. When we, for example, digested NBS standard reference materials using the H3PO4/HNO3/HzO2procedure and determined selenium by the hydride generation atomic absorption procedure, the values agreed closely with those certified (Table 111). We also analyzed diet composite samples from a human study. These diets had been previously analyzed independently by the standard HC104 digestion and 2,3-diaminonaphthalene
Anal. Chem. 1981, 53, 1195-1199 Table IV. Determination of Se in Food Composite Samples by Two Methods amt of S e , pg/g --hydride fluorescence sample methoda methoda diet 3 diet 60 diet 57 a
0.026 0.025 0.024
0.020 0.032 0.023
1195
(3) Fiorino, J. A.; Jones, J. W.; Capar, S. G. Anal. Chem. 1978, 48, 120-1 25. (4) Ihnat, M. J. Assoc. Off. Anal. Chem. 1977, 60, 813-825. (5) Clinton, 0. E. Analyst(London)1977, 102, 187-192. (6) Knechtel, J. R.; Fraser, J. L. Analyst(London) 1978, 103, 104-105. (7) Flanjak, J. J. Assoc. Off. Anal, Chem. 1978, 61, 1299-1303. (8) Egaas, E.; Julshamm, K. At. Abs. News/. 1978, 17, 135-138. (9) Siemer, D. D.; Hagemann, L. Anal. Left. 1975, 8, 323-337. (10) Tam, G. K. H.; LaCroix, G. J. Environ. Scl. Health, Part 8 1979, 814, 515-516. (11) Reamer, D. C.; Veillon, C.; Tokousbalides, P. T. Anal. Chem. 1981, 53, 245-248. (12) Veilion, C.; Vallee, B. L. Meth. Enzymol. 1978, 54, 446-484.
Wet weight.
fluorescence method. The data (Table IV) show that agreement was good consideiring these low concentrations of selenium. (1) (2)
LITERATURE CITED Anawical Methods Cornmittee Analyst(London)1979, 104, 778-787. Ihnat, M. J. Assoc. Off. Anal. Chem. 1974, 57, 368-372.
RECEIVED for review January 30, 1981. Accepted March 25, 1981. D.C.R. is a Research Associate, Children’s Hospital, Boston, MA, and is supported in part by General Cooperative Agreement No. 58-32U4-0-127 with the U.S. Department of Agriculture. Specific manufacturer’s products are mentioned herein solely to reflect the personal experiences of the authors and do not constitute their endorsement nor that of the Department of Agriculture.
Humic Acid Fractionation Using a Nearly Linear pH Gradient Michael A. Curtls, Alllan F. Witt,’ Steven B. Schram,2 and L. B. Rogers’
Department of Chemistry, University of Georgia, Athens, Georgia 30602
Model compounds and humlc acids from commerlcal sources have been eluted from Amberllte XAD-8 resin using a nearly llnear pH gradient generated by mixing a universal buffer with a solutlon of sodlum hydroxlde. Model compounds eluted In order of decreaslng acld strength. Two humlc acid samples could readlly be dlstinguished from one another by their elution proflles. Due to thie retention of buffer components by the column, the shape of the pH gradient was found to depend upon both the flow rate and buffer concentratlon.
Humic acids have been the subject of intense research because of their significance in agricultural and environmental processes (1-5). Due to their complexity, they must first be fractionated into a series of less complex mixtures. Gjessing (6-9) has used gel permeation chromatography to fractionate humic acids while others have adsorbed them onto hydrophobic resins and eluted fractions using buffers at various pH values. As the p H was increased, components having progressively higher pKa values were ionized and, thus, desorbed. Mantoura and Riley (10) used XAD-2 resin and four buffer solutions of progressively higher pH to obtain four fractions. Malcolm et al. (11)demonstrated the superior properties of XAD-8, a methyl methacrylate resin. MacCarthy et al. (12) and Thurman and Malcolm (13) generated a continuously changing pH gradient by titrating phosphoric acid with sodium hydroxide and separated humic acids into two fractions. Those fractions eluted from XAD-8 resin a t the inflection points in the titration curve of phosphoric acid. In the above approaches, the pH gradient had sharp inflections at one or more points. It seemed desirable to generate a linear p H gradient so as to better separate any mixtures of ‘Present address: Monsanto Chemical Co., St. Louis, MO 63116. 2Present address: Sid E. Williams Research Center, Life Chiropractic College, Atlanta, GA 30060.
acids having many different pK, values. Our approach was to use a universal buffer (14) which, when titrated with strong base, gave a fairly linear titration curve without sharp inflections. The universal buffer chosen was that of Prideaux (15), an equimolar mixture of phosphoric, acetic, and boric acids. This buffer does not absorb significantly at 254 nm. Hence, a mixture of aromatic test acids covering a wide range of pKa values could be used to test the feasibility of the technique. Two commercial humic acid samples and a synthetic humic acid, prepared from hydroquinone, were chosen, not with high purity in mind, but to see if differences, due to the acids themselves or to the impurities, could be found.
EXPERIMENTAL SECTION Apparatus. Two Altex Model 110-A solvent metering pumps (Altex ScientificInc., Berkeley, CA) were used to generate solvent gradients. They were controlled by an LSI-11 microcomputer (Digital Equipment Corp., Maynard, MA) having 32K words of memory and a 12-bit, 4-channel digital-to-analog converter. Samples were introduced by means of an air-actuated 6-port Valco valve, Model AC V-6UHPA (Valco Instrument Co., Houston, TX), having a 25-pL sample loop. Two detectors were used in the study: a Laboratory Data Control UV Monitor I11 254-nm fixed-wavelengthdetector (Division of Milton Roy Co., Riviera Beach, FL) and a Hitachi Model 100-10 single-beam UV-visible spectrophotometer (Hitachi, LM. Tokyo, Japan) equipped with an Altex flow cell. A Markson flow-through pH electrode, Model 737 (Markson Science Inc., Delmar, CA), and a Corning Model 130 pH meter (Corning Scientific Instruments, Medfield, MA) were used to measure the pH of the column effluent. A Corning pH electrode (No. 476022) and a calomel reference electrode (No. 476002) were used for test titrations. Optical absorbance and pH data were recorded on a Linear Model 585 dual-channel recorder (Linear Instruments Corp., Irvine, CA) and also recorded by computer for storage on floppy disk. Reagents. Water, used to dissolve samples and to prepare the eluents, was purified by a two-stage deionization followed by
0003-2700/81/0353-1195$01.25/00 1981 American Chemical Society
