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Anal. Chem. 1987, 59, 1647-1651
Direct Experimental Evidence for in Situ Graphite and Palladium Selenide Formations with Improvement on the Sensitivity of Selenium in Graphite Furnace Atomic Absorption Spectrometry Janet E. Teague-Nishimura and Takeshi Tominaga Department of Chemistry, Faculty of Science, University of Tokyo, Hongo, Bunkyo-ku, Tokyo 113,J a p a n
Tsutomu Katsura Environmental Safety Center, Waseda University, Okubo, Shinjuku-ku, Tokyo 160,J a p a n
Kazuko Matsumoto* Department of Chemistry, School of Science and Engineering, Waseda University, Shinjuku-ku, Tokyo 160, J a p a n The potential of albumin to be converted into graphlte on the surface of a pyrolltically coated graphtte tube durlng the detennlnation of selenlum by graphite furnace atomic absorption spectrometry has been discovered. Utilizing this “new graphlte surface” formed in situ, along wlth palladium as a Se retainer, has considerably Improved the selenium sensitivtty. Electron-probe mlcroanalysls has revealed for the flrst time that Se and Pd exist on the surface of the graphlte tube In a 1:l mole ratio and that no free selenlum was seen. Application to freeze-dried human serum was successfully made. The detectlon limit was 0.017 ppm.
The addition of “matrix modifiers” to produce a more accurate determination of volatile elements in graphite furnace atomic absorption spectrometry is not novel (1-10).However, if one was to make a quick survey of some of these papers, it soon becomes evident that very little experimental evidence is presented to try and explain the true mechanism behind “the matrix modification method of enhancement”. Several reports (1,5,7,lO)favoring the addition of nickel claim the formation of a high boiling selenide. h a t (10)remarked that reduction by nickel oxide of SeOz,formed by decomposition of selenite or selenate, to a less volatile form is another possibility. As an example of a different modifier Henn (11) utilized molybdenum and put forward that the formation of a high molecular weight heteropoly molybdate anion is successful a t isolating the selenium. The work presented in this paper was undertaken with the hope of being able to throw light on the mechanism for one of these matrix modifiers, i.e., Pd. From some of the papers published (11-14)on the atomization mechanism in a graphite furnace, it is known that the carbon surface of the graphite tube plays an important role in the atomization process of the analyte. Indeed, one of the is the reduction of the metal models often quoted (11,12,15) oxide by the carbon surface of the graphite tube. Botha et al. (15)reported that on the addition of ascorbic acid to solutions containing gallium, the gallium sensitivity could be improved by a factor of 2. In this sense the carbon which is assumed to be formed on pyrolysis of the ascorbic acid during the heating stages in the furnace has a reductive effect, allowing the gallium to be put into a more easily atomization form. There are many other papers (16-18)reporting a similar. carbon effect. Recently L’vov (19,20)has coined the phrase “carbothermal reactions” for those reactions in which carbon plays a reductive role. We too, are reporting an improvement on the sensitivity of selenium in graphite furnace atomic absorption spectrometry by a carbon-surface phenomenon. 0003-2700/87/0359-1647$01.50/0
That is, the selenium signal is improved by the addition of albumin which, under the conditions used, is believed to be converted into graphite. This “in situ” formation of graphite along with the use of palladium as a Se retainer has allowed us to measure selenium concentrations with much higher sensitivity. However, unlike the above, selenium not being a refractory forming element simple reaction of the metal oxide with the carbon surface is improbable. The phenomenon we describe in this paper is thought to be the first of its kind to be reported. In 1983 Verlinden et al. (21) presented a detailed review on the determination of Se by AAS. In the area of biological investigations many workers have preferred to separate the Se from its organic matrix only to find difficulties reoccurring with the inorganic matrix into which the Se is extracted. Saeed et al. (7) have shown interferences from phosphates and iron on Se signals to occur a t wavelengths below 220 nm, leading to overcompensation by deuterium arc background correction systems. Carnrick et al. (22) claim however that, with the use of a Zeeman background correction system, this problem can be avoided. Nevertheless, there are several reports to date (5,6, 8) claiming the successful direct determination of Se by graphite furnace AAS in serum and other biological fluids. Each of these works employs a metal to ensure the retention of Se in the furnace. Again, this is another interesting area of conflict, with Ni generally coming out tops. Ping et al. (3)reported contrary to other authors (5,6)that P d could produce better sensitivities for Se when present in an organic matrix. They reported that albumin severly decreased the Se sensitivity but upon addition of Pd the sensitivity was restored to that of a simple inorganic Se(1V) solution. Our studies showed that palladium produced a 30% improvement on the sensitivity of a simple Se(1V) solution. However, a somewhat unexpected result appeared when albumin (0.1%) was added to the Se(1V) (0.05pg/mL) solution containing Pd at 100 kg/mL. Unlike Ping et al. who reported on the negative effect of albumin, it was discovered that under a carefully constructed set of conditions a doubling of the selenium signal was possible in the presence of albumin provided palladium was contained in the solution as a Se retainer. Investigation by scanning electron microscopy (SEM) has revealed, that albumin appears to be converted into graphite. Further detailed studies by electron-probe microanalysis have enabled us to report for the first time that Pd and Se exist in the ashing stage together in a 1:l mole ratio.
EXPERIMENTAL SECqION Apparatus. A Hitachi H-180-50 atomic absorption spectrometer equipped with a Hitachi GA-3 graphite furnace and deuterium background corrector was used. All measurements
when necessary were checked against possible molecular absorption by using only a deuterium lamp. All measurements were 0 1987 American Chemical Society
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ANALYTICAL CHEMISTRY, VOL. 59, NO. 13, JULY 1, 1987
made with pyrolitically coated tubes, Hitachi No. 180-7403. A Drummond microdispenser (Drummond ScientificUSA) was used to deliver 10-pL aliquots of the sample. Scanning electron microscope (SEM) micrographs were obtained with a Hitachi S570 scanning electron microscope equipped with a Horiba X-ray microanalyzer EMAX-2200. In the dark region below the pictures, the following information is displayed from left to right: SEM number, accelerating voltage in kilovolts, magnification, and scale of picture. The EMAX-2200 was equipped with a Si(Li) detector. Diffraction patterns were obtained with a Hitachi HU-11D transmission electron microscope. Instrument Setting. The 196.0-nm line with a spectral bandwidth of 1.3 nm was used for all measurements. The line source was a selenium hollow cathode lamp operated at 7.5 mA. The dry, ash, atomize, and clean steps of the Model GF-3 were experimentally optimized, and the furnace was automatically taken through its steps. The purge gas flow (argon gas 1.0 L/min) was interrupted during the atomization stage. Optimized furnace conditions were as follows: dry, 30-80 "C 30 s, 80-120 "C 60 s; ash, 300-400 "C 50 s, 400-600 "C 60 s, 600-1000 "C 60 s; atomize, 2400-2400 "C 6 S. Reagents and Standard Solutions. A Se(1V) standard was prepared by dissolving 1 g of selenium metal in 7 mL of nitric acid and 7 mL of water on heating then diluting to 1 L giving a 1000 pg/mL solution. The Pd, Pt, Cu, Al, and Ni solutions (lo00 pg/mL) as the chloride were purchased from Koisei Chemical Co. Bovine albumin was purchased from Kokusan Chemical Co. Freeze-dried human serum NIES 4 (23)was kindly supplied by K. Okamoto, National Institute for Environmental Studies, Tsukuba, Ibaraki, Japan. Reconstitution of the sample was carried out according to the instructions sent by the supplier. However in order t o obtain a clear solution, further 5-fold dilution was necessary.
RESULTS AND DISCUSSION Selenium being a volatile element has been one of the more difficult elements to be determined by atomic absorption spectrometry. Determination of Se in biological fluids such as whole blood, serum, and seminal fluids, where the Se is known to associate with proteins (24,25),poses problems from not only interference of molecular species formed during the determination but also the loss of volatile Se compounds. Dimethyl selenide and diethyl selenide have very low melting points of 58 "C and 135 "C, respectively. Also formation of smoke and carbon particles causes blocking of the light path through the graphite tube (IO). However if some or all of the above could be avoided, the usefulness of the graphite furnace in the determination of Se will be improved. Optimization of the furnace conditions is as follows: dry, 30-150 "C 40 s; ash, 150-400 "C 60 s; atomize, 2400 "C 6 s on a 0.05 pg/mL Se(1V) solution. When Pd was added to the above solution, the ashing temperature was increased to 150-1200 "C. In Figure 1 these conditions were used in the measurement of signals A, B, and C. However when we applied the same set of conditions to a Se and Pd solution containing albumin (0.1%), the results were disappointing. Unlike Ping et al. (3) who found that on addition of Pd the signal from a Se-albumin solution equalized that of a Se solution, we found that the Se-Pd-albumin solution gave poorer sensitivities than the Se solution under the same conditions. On closer inspection it was found that selenium was being lost in the drying stage. On reoptimization of the furnace conditions we realized that we were able to a t least double the selenium sensitivity in the presence of both palladium and albumin. The realization of these findings is shown in Figure 1. These reoptimized conditions are shown in the Experimental Section, under instrument settings. From Figure 1D-F, it can be seen that as the period of the drying stage was gradually increased the selenium signal also increased with a corresponding decrease in the loss of Se in the drying stage. The peaks marked with an asterisk in Figure 1 correspond to this loss in the drying stage, these peaks
l 1
I *
I
I
I
I
1
I ~l
I-?
-LJ
A
B
C
D
Figure 1. Realization of albumin/graphite enhancement effect on the Se signal: (A, C, D, E, F) Se 0.05 pg/mL, Pd 100 pg/mL, albumin 0.1%; (8)Se 0.05 pg/mL, Pd 100 pg/mL. A, 6, and C show two measurements of each solution. D, E, and F show single measurements of each solution. Peaks marked with an asterisk show Se loss. Sample volume was 10 pL.
0 L,
400
1000
1600
Temperature ('C) Figure 2. Effect of ashing temperature on selenium sensitivity: 0 , 0 . 0 5 pg/mL Se 100 pg/mL Pd + 0.1 % albumin: A, 0.05 pg/mL Se + 100 pmg/mL Pd; H, 0.05 pg/mL Se. Sample volume was 10 pL.
+
appeared a t temperatures around 130 "C. It can be clearly seen from peak F of Figure 1 that when the early peak has been completely diminished, the selenium signal is approximately doubled compared to when albumin is not present, Le., peak B in Figure 1. Effect of Ashing T e m p e r a t u r e on t h e Se Signal. In graphite furnace atomic absorption spectrometry selection of the appropriate ashing temperature is particulary important when a volatile analyte occurs in an organic matrix. For most elements, as the ashing temperature is raised the sensitivity is decreased. Figure 2 shows that with the addition of Pd the Se sensitivity in the presence of albumin (0.1%) is stable until 1200 "C which is similar to that of the middle curve (Se-Pd only). Selenium present in the furnace alone shows loss of sensitivity after the ashing temperatures reaches 400 "C. Therefore it can be confidently stated that palladium successfully retains selenium in the graphite furnace. Background absorptions for both palladium and albumin were scrupulously checked. Figure 2 also shows that albumin enhances the sensitivity two times (upper curve) compared to the signal obtained with Se-Pd only (middle curve). We began to question ourselves as to the cause of this improvement in the selenium signal. If during the heating stage the albumin is rendering "new graphite", the surface being presented to the Se species may be different from that if albumin was not present. It therefore appears that this "new graphite surface" is better able to physically adsorb the sel-
ANALYTICAL CHEMISTRY. VOL. 59, NO. 13. JULY 1. 1987
100'
".
W
pt
Cu
AI
.. Ni
Flgum 3. Comparison of ltm effects of Pd. Pt. Cu. AI, and NI on Se sensnMty. Each solutbn contams 0.05 MgImC Se and 0.1% abumln. The metals are present as W r chlorides at 100 p g l m l . The peaks
marked
wllh
an asterisk contain no albumin.
enium species. Thus with successful retainment of Se by Pd and a -new graphite surface" in the furnace tube, higher sensitivity is obtainable for the determination of Se by graphite furnace atomic absorption spectrometry (GF-AAS). Effect of Palladium Amount. Julshamn e t al. (25)reported that the amount of modifier has a significant effect on the enhancement factor. It was found in this study that after addition of 40 pg/mL of Pd the Se signal remained unchanged. However additions of Pd higher than 150 pg/mL caused the albumin in the matrix to precipitate. Addition of buffers could not prevent the precipitation. Effect of Albumin Amount. Studies were undertaken to examine the effect of the increase in albumin concentration on the selenium sensitivity. Results showed that as little as 0.01% albumin improves the Se signal. However, when concentrations exceed the 1.5% level, severe background absorptions were seen. At 3% molecular absorptions were seen in the drying and ashing stages. If albumin was, through conversion to graphite, giving this enhancement on the Se signal, then a carbon substitute for albumin should give similar results. Active carbon a t 0.1% and 1.0% replaced albumin and in the Se(IV) solution along with the presence of Pd was taken through the heating cycle. In both cases the signals were 30% higher than in the albumin case. However, due to the fact that active carbon is insoluble, the reproducibility of the signal was poor. As another example poly(viny1 alcohol) was added to a similar Pd/Se solution; examination by SEM again showed graphite formation. It therefore appears that any low volatile organic compound, if convertible into graphite, leads to improvement in the Se sensitivity in GF-AAS. Therefore
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it seems that albumin, like active carbon and poly(viny1 aicohol), is converted by pyrolysis to graphite in the furnace. From time to time a Se/Pd solution only was injected into the furnace after a previous injection of a Se/Pd/alhumin solution. However, no enhancement effect was seen. It therefore appears that a "new graphite surface" in required for each measurement. Enhancement Effect of Palladium, Platinum, Aluminum, Copper, a n d Nickel Addition in t h e Determination of Selenium i n 0.1% Albumin Solution. The retainment abilities of several other metals were suhsequently investigated. The result8 are shown in Figure 3. All metals were added as their chloride (I00 p/mL). Except for Ni all elements showed an improvement over the absorption reported in the absence of albumin marked by an asterisk. Electron Microscopy Studies. In order to get direct experimental evidence to support the alhumin/graphite theory, electron microscopy studies were carried out. If the formation of graphite was occurring, its presence should he able to be revealed by this technique. Initially three tubes were analyzed: J E T 1, no pretreatment; ,JET 2,50 injections of a Se 0.05 pg/mL + Pd 100 pg/mI, + albumin (0.1%) (10 pL); JET 3,50 injections of a Se 0.05 pg/mL + Pd 100 pg/mL + active carbon (0.1%) (10 pL). All tubes were taken through the furnace cycle after each injection, no cleaning stage was carried out. The tubes were cut with a diamond cut.ter and washed with acetone prior to examination by SEM. Photographs taken of the scanning electron micrographs are shown in Figure 4. The typical layer structure of graphite can he clearly seen. Although the buildup of the graphite did vary along the length of the tube surface, estimation of buildup depth is possible by use of the scale a t the bottom of the SEM micrograph. As an example; the tube injected with active carbon gave 39 pm of graphite J E T 3, while the tube injected with albumin gave 27.4 pm of graphite J E T 2. These SEMs were taken in the region directly below the injection port hole of the graphite tube. Figure 4 shows vertical sections of the three tubes, JET 1, JET 2, and JET 3. However. these SEMs only allow us to see information on depth of the surface being presented to the atomizing species. Figure 5 shows SEMs taken from above, the surface appearances are clearly different. The graphite formed from the albumin has a much more lumpier appearance compared to the graphite found on the original tube. Energy dispersive spectroscopy ( E D 3 results show that when Pd and Se have been injected into a graphite tube without albumin, the Se amount decreased substantially (
