resultant photograph will be fluorescent spots, seemingly suspended in mid air, with true color rendition of the spots. This procedure is not acceptable since there is no frame of reference for the spots. In fact, when the analyst visually inspects a TLC plate in a view box, he can see the plate because of the eyes’ limited sensitivity to UV. Other UV filters are available that cut off the UV light (i.e. 2A, 2B), but are not as efficient as the 2E. The residual UV light transmitted results in a blue appearance of the print, and spot color is not true. EXPERIMENTAL Photography was carried out on a Camag MP-4 Camera system, equipped with Foto-UV lighting system using Polaroid Type 58 film. The Foto-UV system consists of two 8-W mercury short wave lamps, 8-W black lite long wave lamps, and two Corning 9863 filters. WARNING: Do not view U v lights without proper eye protection! Four 150-W photofloods Type B provide the white light source. Procedure. Place a TLC plate on the copy stand base, center, frame, and bring into proper focus. Place an 80B filter in the filter drawer, set the f-stop for 5.6, and set shutter speed at ‘/eo s. Switch on the photofloods and allow t o come to proper temperature for 5 s before the exposure is made. Trip the shutter, turn the lights off without moving anything. Replace the 80B with a 2E filter, reset the f-stop t o 4.5 and allow the long wave UV (short wave may also be used) lamps to warm up for 5 s. Make a second exposure for 1full minute by holding down the shutter release. Turn off the lamps, remove the film from the camera, and process as recommended. RESULTS A N D DISCUSSION T h e result is a photograph that has the proper color rendition and is taken using reasonable exposure times. Each part of the intentional double exposure serves a different function. The first exposure sets a frame of reference for the spots. The 80B filter provides the proper color correction to allow the use of photofloods with a daylight type film. The proper result of this exposure should be a dim plate that is gray in color. The plate should be light enough to be able to
read any notations written on the plate, but as dark as possible to retain contrast in final print. If the plate is not gray, a color correction filter may be used (this is usually unnecessary). The second exposure photographs the spots. The 2E filter is a UV cut off filter, preventing any of the UV light that is used to activate the fluorescence from exposing the film. This filter prevents the blue cast from appearing on the film, resulting in proper color of the spots. Color correction filters a t this point are unnecessary. An important benefit from this procedure is that the film speed is effectively increased for the second exposure. The first exposure serves to “preflash” the film for the second exposure. The technique of “preflashing” film to get higher effective film speeds has been used by photographers for many years. CONCLUSIONS The procedure outlined above allows the analyst to photograph UV activated fluorescent spots on TLC plates with the following three advantages: (1)The exposure times are shorter than with other techniques; (2) the use of colorcorrecting filters is usually not necessary; (3) the colors of the fluorescent spots in the final print closely match those seen by the eye. Since the prints obtained by this procedure reasonably represent what is seen by the eye, they can be valuable documents for the TLC analysis. The technique outlined above is not limited to the specific camera system indicated under Experimental. The only requirement is that the copy stand has both a UV and white light source and can accommodate filters. The first exposure can be determined experimentally by installing the proper filter for the light source, and adjusting exposure until the print shows a dark gray plate. The second exposure uses the 2E filter and exposure time is adjusted until the intensity of the fluorescent spot in the prints matches that seen by the eye.
RECEIVED for review April 11, 1977. Accepted June 6, 1977.
Determination of Mercury in Edible Oils by Combustion and Atomic Absorption Spectrophotometry Wei-Chong Tsai and Lih-Jiuan Shiau Food Industry Research and Development Institute, P.O. Box 246, Hsinchu, Taiwan, Republic of China
In recent years, the environmental mercury contamination has been recognized as a health problem. Accurate and reliable methods of analysis of a wide variety of materials, which may contain trace amounts of mercury in both organic and inorganic forms, are needed. Alkyl or aryl forms of mercury may be determined by gas-liquid chromatography (1-3). After wet digestion or combustion, total mercury could be quantitatively determined by using various methods, of which the application of flameless atomic absorption spectrophotometry is perhaps the most common and convenient one. Although much has been studied on the occurrence of mercury in biological materials, such as fish and grain, by acid digestion followed by reduction and aeration ( 4 , 5 ) ,by amalgamation and heating (6, 7), by direct combustion (8, 9), or by various combinations of these and other less common techniques (10-14), little has been known about the mercury contamination in vegetable oils. Knauer et al. (15) determined mercury in petroleum and petroleum products by burning the sample in a Wickbold oxy-hydrogen combustion apparatus and collecting the va-
porized mercury in an acidic permanganate solution, and essentially quantitative recoveries were obtained. This paper describes a rapid and reliable method that can be carried out with equipment available in most laboratories. The method, which is a modification of the Schoniger combustion technique (8, 9), includes pretreatment of the sample by burning and collection of the vaporized mercury in an acidic permanganate solution. The closed-system combustion apparatus, in which the sample was burned with the aid of oxygen stream, is different from Wickbold’s combustion apparatus. EXPERIMENTAL Reagents. (a) Acidic potassium permanganate oxidizing solution was prepared daily by dissolving 5 g of reagent-grade potassium permanganate in 1 L of 3 N H2S04solution. (b) Stannous chloride reducing solution (16) was prepared by dissolving 1 g of reagent-grade hydrazine dichloride, 20 g of sodium chloride, 20 g of hydroxylamine hydrochloride, and 33 g of stannous chloride in 25 mL of 18 N H2S04and diluting to the volume of 1L with deionized distilled water. (c) Stock solution of HgC12 (1000 pg Hg/mL) was prepared from E. Merck standard ANALYTICAL CHEMISTRY, VOL. 49, NO. 11, SEPTEMBER 1977
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( a ) Oil
combustion system
rrkr iunK
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I
Diameter o f #iof $ oxy9mtdlo# td) Asbrstnr lining cloth
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E 32 HM
Figure 2. Combustion flask (D) and oxygenation tube (E) of Figure 1 Pi'otirucrio f i 7 d (mode by lea9ih IZ w., dla 0.5 mm of pbtinwrrr filomed)).
b
Figure 1. ( a ) Oil combustion system. (A) Mercury vapor collecting tube. (B) Connecting tube. (C) Pyrex glass stopper. (D) Combustion flask. (E) Oxygenation tube. (F) Small beaker (diameter 2 cm; height, 1.3 cm). (G) Wick. (6) Pyrex glass stopper. ( c ) Small beaker and wick. ( d )Asbestos lining cloth. (a) hole for oxygenation tube, (b) hole for observation of combustion
solution for atomic absorption spectrophotometers. The working solution was prepared by diluting the stock solution 200 times with 0.5 N HC1. The working solution was prepared immediately before use. (d) Mercury solutions for recovery studies: the inorganic mercury solution was prepared by diluting the inorganic mercury standard stock solution (1000 pg Hg/mL) 100 times with acetone; phenylmercuric acetate solution was prepared by dissolving 1.70 mg of phenylmercuric acetate in 100 mL of acetone, and the concentration of mercury is about 10 pg/mL; and methylmercuric chloride solution was prepared by dissolving 1.25 mg of methylmercuric chloride in 100 mL of acetone, and the concentration of mercury is about 10 pg/mL. The accurate mercury concentrations of the three solutions were standardized against a water solution of mercury standard and were rechecked every time they were used. Samples were pretreated by mixing with 20 mL of the acidic permanganate solution and incubated in a boiling water bath for 10 min to decompose acetone and other organic compounds. Apparatus. The combustion system is shown in Figures 1, 2, and 3 and the Mercury Analyzer, Coleman MAS-50, was used for cold vapor mercury determination. All glassware used for experiments was decontaminated from mercury by soaking in a cleaning solution overnight and then rinsing thoroughly with deionized distilled water and oven-drying. Procedure. About 2 g of edible oil was precisely weighed into a small beaker F and put into the flask D, which was encapsulated with asbestos lining cloth for insulation. A wick was set at the center of the beaker F, and a Pyrex glass stopper C, a connection 1642
ANALYTICAL CHEMISTRY, VOL. 49, NO. 11, SEPTEMBER 1977
Figure 3. Platinum lighter
tube B, and a mercury vapor collection tube A, which contained 20 mL of 0.5% KMn04-3 N H2S04solution and an aspirator were connected. The wick is made of Whatman Chromatography Paper No. 1,thickness 0.16 mm, by rolling a small sheet of the dimension 1.6 X 7.5 cm into a column of about 2 cm in diameter and 1.6 cm high, and fixing in a glass ring of 2-cm diameter and 0.3-cm height. The wick was pretreated by heating in a 100 "C oven for 30 min to prevent mercury contamination in filter paper before use. The aspirator was opened a little to produce low vacuum in the combustion system, and the wick was lighted with a platinum electric lighter (Figure 3). The oxygenation tube E was immediately attached, and the oxygen flow rate was first adjusted to about 200 mL/min and then to about 600 mL/min, and soot formation should be avoided. After 12-14 min of combustion, the sample oil and the wick would be burned out. The oxygen flow was kept for 2 more minutes to drive out the residual mercury vapor from the flask D. The mercury vapor collection tube A was put in boiling water for 10 min (this step could be omitted if the sample contains no organic mercury), and then the solution was transferred into a BOD bottle. A small amount of hydroxylamine-hydrochloride was added to reduce KMn04before 10 mL of the stannous chloride solution was added. Total mercury in the sample was determined by the mercury analyzer, Coleman MAS 50.
RESULTS AND DISCUSSION In order to decompose solvent acetone and organic forms of mercury for accurate determination of mercury concentration, the mercury solution was incubated with 20 m L of
Table I. Oxidative Decomposition Effect of 0.5% KMn0,-3 N H,SO, Solution on Acetone and Mercuric Compounds after Incubation for Different Periods of Time in a Boiling Water Bath Meter reading (pg Hg) after incubation 60 min 20 min 40 rnin 5 min 10 min Mercuric compounda 0 min 0.151 0.134 0.128 0.137 0.135 HgC4 0.141 0.137 0.142 0.141 0.043 0.134 CH,ClHg C,H,HgOCOCH, 0.071 0.098 0.096 0.096 0.097 0.100 a All three mercuric compounds were dissolved in acetone to about 1 0 k g Hg mL, and 0.01 mL of the solutions were taken for incubation with 20 mL of 0.5% KMnO,-3 N H,SO,. Each datum was the average of two determinations. Table 11. Recovery of Mercury Added in the Forms of Various Mercury the Proposed Method Mercury compound Hg added, pg/mL 0 0.033 0.068 0.041 0.074 Hg found, pg/mL 0.007 0.042 0.008 0.075 0.075 0.008 0.039 0.007 0.040 0.076 0.041 Mean, pg/mL 0.008 0.075 0.067 Corrected for blanks 0.033 Recovery, % 100 99 Table 111. Reproducibility of Mercury Analysis by the Proposed Method on Mercuric(I1) Chloride Added at Various Concentrations I I1 I11 Hg found, 0.045 0.081 0.153 pg/mL 0.046 0.080 0.160 0.044 0.081 0.163 0.045 0.080 0.160 0.045 0.080 0.160 0.046 0.084 0.165 Mean 0.045I 0.64% 0,081i: 0.90% 0.160 I 0.95% I S.D. 0.5% KMn04-3 N H2S04in a boiling water bath. Five minutes of incubation was sufficient to digest organic matters contained in the incubation mixture, and further incubation did not change the mercury analytic results (Table I). Ten minutes of incubation was taken to ensure complete decomposition of all organic matters in the sample solution. For recovery studies on mercuric(I1) chloride, methylmercuric chloride, and phenylmercuric acetate by the proposed method, known amounts of the mercury solution were added to soybean oil. The results in Table I1 showed that recoveries of HgClz in levels of 0.033 ppm and 0.068 ppm were 100% and 9970,respectively, and of methylmercuric chloride and phenylmercuric acetate in levels of 0.063 ppm and 0.052 ppm were 99% and loo%, respectively. Reproducibility of analysis by the combustion techniques was good a t low levels of mercury in 2.0g of soybean oil. Table I11 showed the results with standard deviations of 0.64%, 0.90%, and 0.95% for six repeated analyses a t levels of 0.045,0.081, and 0.160 ppm, respectively. This recovery experiment was carried out by weighing 2-g samples to which different amounts of mercuric chloride were premixed. Therefore, there were no errors caused by micropipetting variation, which was associated with the experimental results of Table I and 11. T h e temperature of flask D before burning, amounts of oxygen supply (in the range of 420 mL/min to 620 mL/min without soot formation), and the time length of burning (in the range of 11-19 min) had no significant effect on the determination of mercury in soybean oil (Table IV). Therefore, the apparatus can be continuously used by changing the small beaker F charged with a new sample. Satisfactory results of mercury determination depend on the conditions of sample burning. The soot formation during
Compounds to Soybean Oil by CH,HgCl 0 0.063 0.006 0.071 0.007 0.066 0.006 0.068 0.006 0.068 0.006 0.068 0.062 99
C,H,HgOCOCH, 0 0.052 0.007 0.060 0,008 0.058 0.006 0.059 0.008 0.059 0.007 0.059 0.052 100
Table IV. Effects of Temperature of Combustion Flask before Burning, Amounts of Oxygen Supply, and Time Length of Burning on the Determination of Mercury in Soybean Oil Temperature of Oxygen flask befoore Time length Hg found, flow rate burning, Ca of burning Pg 620 mL/min 26 14 min, 20 s 0.090 30 1 3 min, 28 s 0.090 70 12 min, 21 s 0.090 10 min, 45 s 0,090 85 420 mL/min 24 15 min, 56 s 0.087 36 18 min, 44 s 0.090 70 17 min, 28 s 0.090 121 18 min, 15 s 0.085 a Temperature of outside bottom of flask D was measured with a Type J Iron Constantan Thermocouple. sample burning caused low recoveries. T o prevent soot formation, the diameter of the tip and the angle or slope of the oxygen flow tube, as well as the distance between the tip of oxygen flow tube and the wick should be kept in such a fashion as shown in Figure 2,and the oxygen flow rate should be kept a t about 600 mL/min in order to avoid overoxygenation, which will cause soot formation. Such a special alignment is very important for obtaining ideal combustion without soot formation. A few times of practice was needed to find the best conditions. It is essential to insulate the flask D with asbestos lining cloth. Partial insulation by putting some glass fiber underneath the flask D resulted in recoveries in a range of about 70-80%, which were dependent on burning conditions. For a sample containing organic mercuric compounds, after collecting mercury vapor in 0.5% KMn04--3 N H#04 in tube A, it is advisable to incubate the solution in tube A in boiling water for 10 min in order to decompose the residual organic mercury compounds possibly remained unburned. Without further incubation in boiling water, the recoveries of inorganic mercury, phenylmercuric acetate, and and 8070, methylmercuric chloride were about 10070,9070, respectively, while they were all about 100% with further incubation. LITERATURE CITED (1) (2) (3) (4)
G. Westoo, Acta Chem. Scand., 22, 2277 (1968). S. Nlshi and Y. Horimoto, Bunsski Kagaku, 17, 75 (1968). P. Zarnegar and P. Mushak, Anal. Cbim. Acta, 69, 389 (1974). W. R. Hatch and W. L. Ott, Anal. Cbem., 40, 2085 (1968).
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(5) R. K. Munns and D. C. Holland, J . Assoc. Off. Anal. Chem., 54, 202 (1971). (6) M. J. Fishman, Anal. Chem., 42, 1462 (1970). (7) I. Okuno, R. A. Wilson, and R. E. White, J . Assoc. Off.Anal. Chem., 55, 96 (1972). (8) W. J. Herrmnn,Jr., J. W. Butler, and R. G. Smith, in "Laboratory Dhgnosis of Diseases Caused by Toxic Agents", F. W. Sunderman and F. W. Sunderman,Jr., Ed., Warren H. Green, Inc., St. Louis, Mo., 1970, p 379. (9) R. J. Thomas. R. A. Haastrom, and E. Kuchar. Anal. Chem., 44, 512 (1972). (IO) G. Thiiliez, Chem. Anal. Part., 50, 226 (1966). (11) V. Lidums and U. Ulfvarson, Acta Chem. Scand., 22, 2150 (1968). (12) 0. W. Kalb, At. Absorp. News/., 9, 84 (1970).
(13) T. Ukita, T. Osawa, N. Imura, M. Tonomura, Y. Sayato, K. Nakamura, S. Kanno, S. Fudui, M. Kaneko, S. Ishlkura, M. Yonaba, and T. Nakamura, J . Hyg. Cbem., 18, 258 (1970). (14) 0. I. Joensuu, Appl. Spectrosc., 25, 526 (1971). (15) H. E. Knauer and 0. E. Mllliman, Anal. Chem., 47, 1263 (1975). (16) W. L. Hoover, J. R. Mehon, and R. A. Howard, J. Ass@. Off. Anal. Chem., 54, 860 (1971).
RECEIVED for review February 15, 1977. Accepted June 1, 1977* This research was supported by the National of Science (Republic of China) NSC-65B-0409-18(02).
Dry Ashing of Animal Tissues for Atomic Absorption Spectrometric Determination of Zinc, Copper, Cadmium, Lead, Iron, Manganese, Magnesium and Calcium E. E. Menden, D. Brockman, H. Choudhury, and H. G. Petering" Kettering Laboratory, Department of Environmental Health, Universily of Cincinnati College of Medicine, Cincinnati, Ohlo 45267
In the course of a study of the toxicity of heavy metal ingestion to the offspring of pregnant rats, we were faced with the task of analyzing a single sample by flame atomic absorption for zinc, copper, iron, manganese, magnesium, calcium, and cadmium or lead in a difficult matrix containing bone in addition to other tissues. Wet ashing was initially employed. It was observed then that a large part of the sample residue resulting from ashing and evaporation of the remaining acid could not be dissolved in hot 10% or concentrated nitric acids prior to dilution of the samples for atomic absorption spectrophotometry. This prompted a decision to investigate dry ashing as a suitable alternate method of sample preparation. A survey of literature indicated that there were several dry ashing methods with possible application to the kind of samples being investigated, although none described the determination of all of the eight metals of interest to us ( I ) . It was also evident that the recovery of metals, such as cadmium (2), lead ( 3 ) ,iron and calcium ( 4 ) and to a lesser degree zinc and copper, could be affected by sample matrix composition, temperature of ashing, interaction with the sample vessel and by incomplete solubility of the ash. Therefore, in the dry ashing method which was subsequently developed and which is described in this publication it was necessary to incorporate optimal dry ashing conditions and an effective solubilization procedure so that these obstacles were overcome and maximum recoveries of all metals were achieved.
EXPERIMENTAL Apparatus. A Perkin-Elmer Model 403 atomic absorption spectrophotometer was used as equipped with a strip chart recorder, air-acetylene and nitrous oxide-acetylene burners, and a deuterium arc lamp for background correction. A large hot plate with regulated temperature settings was used for evaporating and dissolving. Dry ashing took place in a Thermolyne Model F-6020muffle furnace equipped with a close tolerance temperature controller (Furnatrol 133). Reagents and Standards. Reagent grade Fisher Scientific hydrochloric and perchloric acids and potassium sulfate, and J. T. Baker nitric acid were sufficiently pure for direct use. Ash-aid (6) consisted of potassium sulfate dissolved in hot concentrated nitric acid (0.25 g/mL). Atomic absorption mixed metal standards, covering the range of 0.01 to 100 ppm were made in a Bolution consisting of 10% nitric acid, 0.85% hydrochloric acid, and 2.5 g of potassium sulfate per liter. 1644
ANALYTICAL CHEMISTRY, VOL. 49, NO. 11, SEPTEMBER 1977
Single metal standards, from which aliquots were added to tissue samples for recovery tests, were prepared in a solution containing 2.4% nitric acid, 0.18%hydrochloric acid, and 0.05% perchloric acid. An equal volume of this blank solution was added to each tissue serving as control. Procedure. Bodies of day-old rat pups of 5-7 g fresh weight, one-half of which were from cadmium-exposedmothers, were used in the experiments. Variable surface contamination of the bodies was removed by soaking them for several minutes in 1% EDTA (6)solution and rinsing with deionized water. Chromium plated surgical instruments also immersed in the EDTA solution, were used to cut up and mince the tissues. Each tissue sample was then distributed in equal weights between two 50-mL metal-free Pyrex beakers, one serving as the control and the other as the recovery test sample. The duplicate samples were oven dried until constant weight was attained and weighed. Standard aliquots containing 70 pg of zinc, 26 pg of copper, 3 pg of cadmium and lead, 300 pg of iron, 10 Ng of manganese, loo0 pg of magnesium, and 8OOO pg of calcium were added to the test samples. An equal volume of the solution in which single standards were prepared was added to each control sample and all samples were taken to dryness at ca. 120 OC. The added metals amounted to between 40 and 200% of the metal levels usually contained by such tissue samples (except for manganese, which was in 9-fold excess). The dried tissue was subjected to charring in the furnace at 300 "C for 5 h. The samples were placed in a cold furnace and the charring temperature was reached in approximately 40 min. One milliliter of ash-aid and 3 mL of concentrated nitric acid were added to each sample and the char was broken up and roughly ground with a thick glass rod while in contact with the liquid. The samples were taken to dryness at the ca. 120 "C temperature, which was used for all evaporation and dissolution treatments on the hot plate. The ashing took place at 400 "C in the furnace, over 20-24 h. Then the samples were treated with 3 mL of concentrated nitric acid and the acid was evaporated. Ashing was completed at the same temperature over 2-4 h, resulting in a white to yellow ash. At this point the samples were divided into groups of 5 test/control pairs per group, each group including 2-3 pairs of tissue samples from cadmium exposed animals. A different ash solubilization method was tested on each group. One method which produced good results for all metals and two other ones which gave poor or inconsistent recoveries of calcium only, are further described. Method A consisted of successive additions and evaporations of two 5-mL volumes of aqua regia. The ash residue was then dissolved by adding a third volume of aqua regia, heating until vapors appeared, agitating the beaker contents, adding 5 mL of water, reheating and again mixing the contents. Water was
