Fe,"
With Secondary Amines 0
R R-N:
I + I-
0-
I
CHS-C-C1
I I +-R
CH,-C-Cl R-N
H
+
X(CH,CONRH)
O
-
H
II I
CH,-C-~+-R
-HCI
I
H O
R
II I
CH3-C-N-R O Fe"
+
II
3CH3-C-N-R
R
I
e [Fe(CH3CONRz)3]3t
Greenish Violet Complex
.
Greenish Violet Colored Complex
The confirmation of this mechanism is obtained by the results of Table 111; according to that, the complex formed is positively charged.
ACKNOWLEDGMENT The authors are grateful to W. Rahman for providing research facilities.
LITERATURE CITED
With Tertiary Amines
-
0-
- CH3-C-C1I
- RCI
A
I
R-N
FeJ+
+
'+
I
-R
3(CH3CONR,) === 1Fe(CH,-6-NR2h] Greenish Violet Complex
(1) R. A. Lanick, Anal. Chem. 35, 1760 (1963). (2) P. H. Gore and B. B. Wheals, Anal. Chim. Acta, 30, 34 (1964). (3) Vaughn Levin, 6.W. Nippoldt. and R. L. Rebertus. Anal. Chem., 39, 581 (1967). (4) M. Pesez et a/. "Partique de L'Analyse Organique Colormetrique." Messons Publishers, Paris, France. (5) Ella Leizerovici and Cristian Oprescu, Lucr. Conf. Nat. Chim. Anal. 3rd, 3, 369-74 (1971). (6) L. V. Kuritsyn and V. M. Kuritsyna, lzv. Vyssh. Ucheb. Zaved. Khim. Khim. Tekhnol. 15, 461-2 (1972). (7) V. V. Noskov and V. I. Kovotkoya. Sb. Nauch. Tr. Magnitogorsk. Gornometlnst.. 87. 30-34 (1971). (8)H. T. S. Brhton. "Hydrogen Ions." Aberdeen University Press, Aberdeen. Scotland, 1932, p 217.
RECEIVEDfor review September 16, 1974. Accepted December 3, 1974. One of us (J. P. Singh) is also thankful to C.S.I.R., India for financial assistance.
Determination of Trace Amounts of Copper and Zinc in Edible Fats and Oils by Acid Extraction and Atomic Absorption Spectrophotometry Robert A. Jacob and Leslie M. Klevay Department of Biochemistry, University of North Dakota, and United States Department of Agriculture, Agricultural Research Service, Human Nutrition Laboratory, Grand Forks, N.D. 5820 1
Commercial vegetable oils are processed to remove trace metals which are deleterious to shelf life color and odor at levels as low as 30 ppb (I, 2 ) . Methods for trace metal determination in edible fats and oils have centered on copper mainly because of the interest within the vegetable oil industry in copper hydrogenation catalysts (3-6). Nutritionists, increasingly aware of the metabolic roles of essential trace metals Cr, Mo, Mn, Fe, Co, Ni, Cu, and Zn, require rapid and sensitive methods for analysis of these metals in all types of foods. Our research concerns the association of trace metal metabolism with the genesis of coronary heart disease, the leading cause of death in t h e United States (7). Because risk of mortality due t o coronary heart disease has been related to dietary fat (€4, and the concentration of cholesterol in human plasma (9), and
because experiments with rats have shown that the concentration of cholesterol in plasma can be increased by an increase in the ratio of zinc to copper ingested ( l o ) , we required a rapid method of analysis for zinc and copper in fats and oils. Fats and oils are particularly difficult to analyze for trace metals. Many of the usual methods of wet digestion are not recommended for use with high fat material because of associated safety hazards (11-14). The convenient direct method (solvent diluted oil samples are aspirated directly into an atomic absorption flame) is not sensitive below 0.3 ppm of each metal and is not suitable for solid fat or shortening samples (15, 16). Use of flameless atomization techniques for the determination of Cu in solvent diluted oil samples gave lower detection limits than absorption with a ANALYTICAL CHEMISTRY, VOL. 47,
NO. 4 , APRIL 1975
741
Table I. Metal Recovery from Oils by Acid-EDTA Extraction Metal content of oil, PPm C alcd
R e c o ~ e r y -' Extraction '(CHIP
Found'
KO,
of
Re1 std
replicate detns
16.1
8 3 3 5 8 8 2 1 2
dcv, 'I
Copper
Soybean base oils 0.031 0.029 0.060 0.061 0.054 0.061' 0.155 0.163 0.271 0.251 0.330 0.308 0.292 0.33od 0 .430e 0.350 0.434 0.500
93.5 (108.3) 98.4 88.5 95.1 92.6 (88.6) 93.3 (93.3) 88.5 81.4
3.8 1.8 2.9
86.8 Zinc
0.031 0.065 0.065' 0.172 0.269 0.269* 0.347 0 .447E 0.505
0.029 0.062 0.062 0.158 0.240 0.218 0.322 0.385 0.424
93.5 (91.9) 95.5 94.7 91.9 89.2 (93.0) 81 .O 92.8 (94.7) 86.0 84 .O
19.2 2.2 3.1
5 .O
a Oil samples, 50 g. * CH-char-ashing, method of reference (20). Average of triplicate determinations. Samples extracted once for two hours. Samples extracted twice for one hour each time. e Peanut base oil.
flame but problems were encountered with matrix smoke interference and metal volatilization (17-19). T h e charashing method (20) is sensitive but is inordinately time consuming. Willis extracted butter and butter oil with nitric acid and analyzed the extracts for copper using atomic absorption spectrophotometry (21). Deck and Kaiser used a n acidEDTA solution t o extract sub-ppm amounts of copper from shortenings and oils prior t o colorimetric analysis (22).T h e method described here employs acid-EDTA extraction of copper and zinc followed by atomic absorption analysis. EXPERIMENTAL Reagents and Apparatus. All glassware was washed to remove metal contamination with 10%Radiacwash, Atomic Products Corporation. Hydrochloric and nitric acids used were J. T. Baker U1trex. The 18%HCI-O.Ol% EDTA extractant solution was prepared by adding 525 ml of ultrapure HC1 to 500 ml of metal free water and then adding 0.11 g of EDTA. Hydrochloric acid solutions exceeding the azeotropic composition of 20.2% HC1 will lose HC1 through the reflux condenser until the constant boiling mixture is attained. Recovery studies (Table I) were performed on samples prepared with low metal soybean or peanut base oil and National Bureau of Standards oil soluble organometallic standards No. 1080, bis(1phenyl-] ,3-butanediono)copper 11, 16.5 f 0.08% Cu, and No. 1073b, zinc cyclohexanebutyrate, 16.66 + 0.05% Zn, according to accompanying NBS directions. All oil and fat samples were commercial purchases. Beef fat was rendered at 110-120 "C (23) to extract the fat from the rind. Atomic absorption analyses were performed on a Varian Techtron Model AA-5 spectrophotometer equipped with a Varian DI30 digital indicator readout and using an air-acetylene flame and resonance lines of 324.75 and 213.86 nm for copper and zinc, respectively. Procedure. A sample of oil or melted fat (50 f 0.02 g) was refluxed in a 300 ml24/40 standard taper round bottom flask with 55 ml of acid extractant for two hours at a reflux rate of approximately one drop per second (too rapid heating resulted in violent bumping). The sample was then cooled and 50 ml of the extractant 742
9
A N A L Y T I C A L CHEMISTRY, VOL. 47, NO. 4, APRIL 1975
was pipetted out of the lower aqueous layer and into a 250-ml Erlenmeyer flask (we encountered no problems with emulsions for the oils we extracted). The sample was again refluxed for two hours with a fresh 50-ml portion of extractant. The second extractant portion was pipetted out, added to the first, and the combined extracts were diluted to 150 ml with metal-free water. The total aqueous extract solution was then pipetted out of the flask and into a 250-ml beaker. Care was taken to leave most or all of any remaining oil in the Erlenmeyer flask. Ten ml of ultrapure "03 and 2 boiling chips were added to the beaker, and the solution was boiled down to 1- to 2-ml volume with a watch glass cover on top. The solution was then diluted to an appropriate dilution volume with metal-free water prior to atomic absorption analysis. Dilution volumes were 25.0 ml for all oil analyses except for the 0.03 ppm metal oil (Table I) and the blank analyses, which were 10.0 ml. Recovery study blanks consisted of base oil samples with no added metal standard.
RESULTS A N D DISCUSSION The value of EDTA as a metal chelator in highly acid media would appear t o be dubious; however, Deck and Kaiser obtained quantitative recovery of copper from edible oils extracting with 0.01% EDTA in strong acid and only 88-90% recovery using the acid alone (22). T h e acid extraction method was evaluated from metal recovery data summarized in Table I. T h e average metal recovery for oils with 0.03-0.35 ppm of added metal standard was 94.6% for copper and 92.6% for zinc. Recoveries for oils with greater than 0.4 ppm of metal were always less than 90%. T h e direct atomic absorption method is applicable to oils with metal content greater than 0.3 ppm (15). In addition t o recovery results using the usual procedure of two 2-hour extractions, Table I gives recoveries for samples of the 0.06 ppm metal oil extracted once for two hours and for the 0.330 ppm copper and 0.269 ppm zinc oil extracted twice for one hour. Average recoveries of copper and zinc decreased by 10 and 1%, respectively, with just one extraction and by 5 and 8%, respectively, with the shorter extraction times. Hence, the time spent performing both longer and more frequent extractions is justified by increased metal recovery. Extraction recovery results for three of the oils in Table I are compared with recoveries obtained using t h e char-ashing method of Evans (20). Recoveries by extraction us. char-ashing methods averaged 93.1 us. 96.7% for copper and 91.8 us. 93.2% for zinc. We found that increased precision for the char-ashing method, to f l ppb deviation for both copper and zinc, was obtained by covering the sample bowls with watch glass covers supported on Pyrex hooks during charring. The precision or random scatter of the analytical results was evaluated from relative standard deviations (RSD), calculated for the oils on which a larger number of replicate determinations was performed as shown in Table I. T h e RSD's for oils with 0.03-0.35 ppm of metal averaged 6.2 and 7.4% for copper and zinc, respectiyely. These values are weighted high by the relatively large RSD's (15-20%) for the 0.03 ppm oil, a consequence of the low metal concentration rather than a n above average standard deviation. T h e experimental detection limits were determined as twice the blank scatter, i.e., twice the standard deviation for measurements of eight replicate base oil blanks. These values were 0.012 pg copper and 0.009 pg zinc per gram of oil. Detection limits similarly calculated from replicate measurements of the same blank extract were 0.0013 pg copper and 0.0006 pg zinc per gram of oil. Thus, the former detection limits, which represent total analytical scatter, are approximately ten times the latter, which represent instrumental measurement scatter only. T h e presence of small amounts of residual oil in the final concentrated extract very likely contributes significantly t o the total blank
scatter via small differences from sample to sample in nebulization or light interference effects. Deck and Kaiser (22) separated the aqueous extract from the oil by filtering the mixture through 18.5-cm Whatman No. 40 filter paper. We found t h a t 100 ml of fresh extractant filtered picked u p 0.6 f 0.1 pg of copper and 3.6 f 0.3 kg of zinc from the filter papers themselves. Such contamination is appreciable for analysis of oils with metal content as low as those listed in Table I. Suitable procedures t o avoid this contamination are to pre-extract the filter papers or eliminate filtering by carefully pipetting out the aqueous layer underneath the oil. A few fats and oils present unique difficulties for metal analysis by acid extraction. We found a crude menhaden oil would not reflux with the aqueous extractant without violent bumping. Extraction of inorganic salts from some oils may cause light scattering interference in the atomic absorption flame. Willis encountered this in nitric acid extracts of butter and corrected for it by subtracting background absorbance (21). The data show that the acid extraction method gives nearly quantitative recovery of added copper and zinc standard from oils in the range of 0.03-0.35 ppm of metal. Even where metal recoveries dropped to about 90% within this range, the reproducibility was good, with RSD's of 2-3. Detection limits determined from measurement of precision a t blank levels indicate the method is useful for analysis of oils with metal content down to about 0.010 ppm. The char-ashing method though sensitive below 0.010 ppm of metal, requires 3-4 days for complete analysis. Acid extraction of oils requires 4 hours; thus, several samples a day can be analyzed using simultaneous reflux extractions. As with char-ashing, the acid-EDTA extraction method is apparently applicable to the determination of a variety of trace metals in both liquid oil and solid fat or shortening samples.
LITERATURE CITED (1) C. D. Evans. A. W. Schwab. H. A. Moser. J. E. Hawley, and E. H. Melvin, J. Amer. OilChem. SOC.,28, 68 (1951). (2) G. R. List, C.D. Evans, and W. F. Kwolek, J. Amer. Oil. Chem. SOC.,48, 438 (1971). (3) A. J. DeJonge, W. E. Coenen, and C. Okkerse, Nature, (London), 206, 573 (1965). (4) S.Koritala and H. J. Dutton, J. Amer. Oil Chem. Soc., 43, 556 (1966). (5) S. Koritala, J. Amer. OilChem. SOC.,47, 106 (1970). (6) K. J. Moulton, D. J. Moore, and R. E. Beal, J. Amer. Oil Chem. SOC.,46, 662 (1969). (7) Anon, "Vital Statistics of the United States, 1967. Mortality." U S . Department of Health, Education, and Welfare, Washington, D.C., 1969, Vol. 2, part A, Tables 1-7. (8) A. Keys, Ed., "Coronary heart disease in seven countries," Cirwlation, 41, 42; Suppl. I, 1 (1970). (9) D. S. Fredrickson and R. I. Levy, "Familial hyperlipopro~lnemia." in "The Metabolic Basis of Inherited Disease," 3rd ed., J. B. Stanbury, J. B. Wyngaarden, and D. S. Fredrickson. Ed., McGraw-Hill, New York, N.Y., 1972, p 545. (10) L. M. Klevay, Amer. J. Clin. Nutr., 26, 1060 (1973). (11) Analytical Methods committee, Analyst, (London), 85, 649 (1960). (12) R. P. Taubinger and J. R. Wilson, Analysf, (London),90, 429 (1965). (13) Analytical Methods Committee, Analyst, (London), 98, 459 (1973). (14) C. Feldman, Anal. Chem., 46, 1609 (1974). (15) Official, J. Amer. Oil Chem. SOC.,49, 432A (1972). (16) B. Piccolo and R. T. O'Connor, J. Amer. Oil Chem. SOC.,45, 789 (1968). (17) A. Prevot, Rev. Fr. Corps Gras, 18, 655 (1971). (18) A. Prevot and M. Gents, Rev. Fr. Corps Gras, 20, 95 (1973). (19) M. K. Kundu and A. Prevot, Anal. Chem., 46, 1591 (1974). (20) C. D. Evans, G. R. List and L. T. Black, J. Amer. 01Chem. SOC.,48, 840 (1971). (21) J. B. Willis, Aust. J. Diary Tech., 1964, 70. (22) R. E. Deck and K. K. Kaiser, J. Amer. Oil Chem. SOC.,47, 126 (1970). (23) I. S. Rombauer and M. R. Becker, "Joy of Cooking," Bobbs-Merrill, Indianapolis, Ind., 1964, p 511.
RECEIVEDfor review May 16, 1974. Accepted December 5, 1974. The authors wish t o acknowledge partial support by the USDA Cooperative Agreement 12-14-100-11, 178 (61), Amend. 1. Mention of a trademark or proprietary product does not constitute a guarantee or warranty of the product by the U.S. Department of Agriculture, and does not imply its approval to the exclusion of other products that may also be suitable.
Response Surface and Atomization Mechanism in AirAcetylene Flames Kltao Fujlwara, Hlrokl Haraguchl, and Kellchlro Fuwa Department
ofAgricultural Chemistry, University of Tokyo, Tokyo 1 13, Japan
The process of atomization in the flame has been studied by many workers, and they have considered such factors as chemical properties of the analyte elements, the condition of the flame, the effect of concomitant species and so on (1-9). The geometrical distribution of the atomic absorbance in the flame together with the condition of the flame is a useful method for the investigation of the atomization mechanism. In previous papers (10, II), the present authors have investigated the atomic distribution of cobalt and of its complexes, and concluded that the pyrolysis of the bonds between the central cobalt atom and the coordinating atoms in the ligands is the rate determining process of its atomization. In order to investigate and simplify those two major factors of atomization, ie., the geometrical distribution in the flame and the chemical nature of the flame, we found that the response surface plot of the atomic and molecular absorption in the axes of flame height us. fuel rate is most
convenient (1% 13). A similar expression has been suggested and tried by other workers also (1,14). Applying this expression to simple salts of many elements, the atomization mechanisms of the fundamental aquo complexes were investigated in this paper.
EXPERIMENTAL Reagents. All reagents used in this experiment were analytical grade prepared by Wako Chemical Reagents Co. For the preparation of sample solutions, nitrates of Mn2+,Fe3+, Co2+, Ni2+,Cu2+, Zn2+, Ca2+, Cr3+, Cd2+, Ga3+, In3+, Pb'+, and chloride of Sn4+ were dissolved in distilled water containing 0.2% nitric acid. Ammonium molybdate and boric acid were dissolved in water. Concentrations of the analyte elements are listed in Table I. Apparatus. The Hitachi 207 atomic absorption spectrophotometer was used, the optics of which has been modified to the single light path with the light beam of two to three mm in diameter at the center of the flame (11). The light source for atomic absorption of the analyte element was a hollow cathode lamp made by Hitachi Ltd., except the one A N A L Y T i C A L CHEMISTRY, VOL. 47, NO. 4, APRIL 1975
743
