1504
Anal. Chem. 1981, 53, 1504-1509
LITERATURE CITED (1) (2) (3) (4) (5) (6) (7) (8) (9)
(20) (21) (22) (23)
Martin, K. J.; Shain, I. Anal. Chem. 1958, 30, 1808. Bond, A. M.; Grabaric, B. S. Anal. Chem. 1978, 48, 1624. Annino, R. Adv. Chromatogr. 1977, 15, 33. Kirmse, D. W.; Westerburg, A. W. Anal. Chem. 1971, 43, 1035. Binkley, D. P.; Dessey, R. E. Anal. Chem. 1980, 52, 1335. Perone, S.P.; Gutknecht, W. F. Anal. Chem. 1970, 42, 906. Perone, S.P.; Sybrandt, L. B. Anal. Chem. 1971, 43, 382. Boudreau, P. A.; Perone, S. P. Anal. Chem. 1979, 57, 811. Toman, J. J.; Corn, R. M.; Brown, S. D. Anal. Chlm. Acta 1981, 123,
(24) (25) (26) (27)
Benyon, P. R. Appl. Statlstlcs, 1976, 25, 97. Hill, I. D. Appl. Statistlcs 1978, 27, 380. Statham, P. J. X-Ray Spectrom. 1978, 5, 16. Hayes, J. W.; Glover, D. E.; Smith, D. E.; Overton, M. W. Anal. Chem. 1973, 45,277. Oldham, K. 6.; Spanier, J. "Fractional Calculus" (Vol. 111 in Math in Science and Engineering Serles); Academic Press: New York, 1974. Mosteller, F.; Tukey, J. W. "Data Analysls and Regression"; AddisonWesley: Reading, MA, 1977; Appendlx A. Nicholson, R. S.;Shain, I. Anal. Chem. 1984, 36, 706. Sauerwald, F. 2. Metallkunde 1950, 47, 97.
187.
(10) Imbeaux, J. C.; Saveant, J. M. J . Nectroanal. Chem. 1973, 44,169. (11) Dalrymple-Alford, P.; Goto, M.; Oldham, K. B. J . Nectroanal. Chem. 1977, 85, 1. (12) Ammar, F.; Saveant, J. M. J . Electroanel. Chem. 1973, 47, 215. (13) Grenness, M.; Oldham, K. B. Anal. Chem. 1972, 44, 1121. (14) Barendrecht, E. Electroanal. Chem. 1987, 2 . (15) Mattison, J . M. Anal. Chem. 1972, 44, 1715. (16) Brown, S.D.; Kowalskl, 8. R. Anal. Chim. Acta. 1979, 107, 13. (17) Nelder, J. A,; Mead, R. Compuf. J . 1985, 7, 308. (18) O'Nelll, R. Appl. Statlstlcs 1971, 13, 338. (19) Chambers, J. M.; Ertel, J. E. Appl. Statistics 1974, 23, 250.
R~~~~~~ for review ~~~~~b~~ 1, 1980. ~~~~~~~dA~~~~1, 1981. This work was supported by the Director, Office of Energy Research, Office of Basic Energy Sciences, Chemical Sciences Division of the U S . Department of Energy under Contract No. W-7405-ENG-48 and by Contract No. DE AC06-76R101830-ONWI between Battelle Memorial Institute and the U.S. Department of Energy.
Effect of Graphite Furnace Substrate Materials on Analyses by Furnace Atomic Absorption Spectrometry W. Slavin," D. C. Manning, and G. R. Carnrick The Perkin-Elmer Corporation, Maln A venue, Norwalk, Connecticut 06856
Recent Improvements in the graphite furnace have greatly reduced analytical interferences. Problems remain that appear to be related to the poroslty of graphite and to the fraglllty of pyrolytlc graphlte protectlve coatings. We compare graphite tubes from several sources which have been coated in several different ways for their differences In analytlcal sensitlvlty for Cu, AI, Sr, TI, and Mo. A separate set of experiments using the determlnatlon of AI In a chloride matrlx shows even greater variation between graphlte materials prepared differently. Experiments uslng the graphlte furnace at steady-state temperature, in the platform and probe variatlons, showed less than 10% signal suppression from 50 pg of CaCI, or CuCI, on 2 ng of AI.
Our recent papers (1-6) have attempted to improve the analytical conditions for trace metal determination with the graphite furnace and atomic absorption spectroscopy. When the furnace is operated a t a steady-state temperature (2, 3, 5-9) during the time the analyte is vaporized and the absorbance signal is integrated, many of the interferences previously reported for graphite furnace analyses have been reduced or eliminated. Larger amounts of sample can be analyzed if Zeeman background correction is used ( 4 ) . However it is necessary that the furnace be designed in a way to take advantage of Zeeman background correction or the advantages of Zeeman correction will be more than offset by furnace deficiencies. Signal handling must be very rapid (10) to avoid errors, as recently emphasized by Siemer and Baldwin (11). When all of these factors are controlled correctly, most of the interferences reported in the literature are very small or nonexistent and standardization may not require a close match to the sample matrix. The method of additions, with its poor 0003-2700/8 1/0353-1504501.25/0
precision and discomforting extrapolation, may not be required. In this paper we show that the quality of the graphite used for the furnace tubes is an important factor still not adequately controlled. Graphite is a porous material, easily penetrated by liquids and gases. Atomic vapor passes quite freely through a 1mm thick wall of hot graphite. Coating the graphite tubes with a thin layer of pyrolytic graphite was recommended (12) more than a decade ago to reduce the effects of the porosity of the graphite. L'vov et al. (13)have recently explained quantitatively the retention properties of graphite for the analyte that has penetrated the graphite structure. The analyte can penetrate as a liquid or as a vapor and it can penetrate during the charring step as well as during the atomization step. This applies to the matrix as well as to the analyte and must be taken into account for those matrices, like halides, which interfere in the vapor phase (2, 7). In the past, the model for the conversion of a sample into an atomic vapor assumed that, at some temperature depending upon the analyte and its matrix, the analyte became an elemental vapor and survived in the vapor phase until it exited the optical system through the tube ends or the filling hole (12, 14, 15). It is more likely that the analyte is constantly volatilized and adsorbed, some of it within the structure of the graphite near where the sample is applied and some a t considerable distances along the tube. The process resembles gas chromatography. Analytical problems associated with earlier furnace designs arose because the temperature rise was slow and the graphite was very porous. Fast heating and a strong impervious barrier separating the sample vapor from the graphite have greatly improved performance. Manning and Ediger (16) found that a pyrolytic graphite coating increased the signals for several important metals by factors as 0 1981 American Chemical Society
ANALYTICAL CHEMISTRY, VOL. 53, NO. 9, AUGUST 1981
much as &fold. Fernandez and Iannarone (17) found similar improvements when the tube was heated very rapidly, especially for the more refractory metals. Szydlowski et al. (18)studied the surface of pyrolytically coated tubes with a scanning electron microscope using tubes from the Perkin-Elmer HGA-2100. They found that the temperature of deposition had to be greater than 2300 “C to achieve a pyrolytic coating. Smith and Leeds (19)reported that the substrate temperature must be between 1750 and 2250 “C to achieve a pyrolytic coating, with the pyrolytic properties improving rapidly at higher temperatures. They point out that “pyrolytic graphite is not a single invariant material but has a struicture and related properties that are completely dependent upon the manufacturing process”. This being true, pyrolytic coatings deposited in the laboratory by the procedure of Manning and Ediger (16) must produce a good pyrolytic coating, a t best, only in the center of the tube since the ends of the tube are too cool to form a satisfactory coating (20). The commercial procedure for depositing a pyrolytic coating utilizes an oven at approximately constant temperature through which flows a mixture of a hydrocarbon in an inert gas. Many workers have observed differences in analytical sensitivity or the extent of interferences when ordinary and pyrolytic graphite are compared. Volland et al. (21) observed differences in the extent of halide interferences on Fe, Ni, Mn, and Mo. Montgomery and Peterson (22) showed that pyrolytically coated graphite tubes deteriorated rapidly when NH4N03 was used for the determination of Cu in seawater. Different batches of tubes were very different. Uncoated tubes had lower peak height sensitivities than the initial results from coated tubes, but the sensitivities did not decrease with time. The variability of graphite in porosity and density has not been understood and the difficulty in quality control from batch to batch has been underestimated. Probably the graphite under the pyrolytic coating in the Montgomery and Peterson experiments was entirely different from those tubes which were uncoated. Some compounds are particularly destructive of the pyrolytic graphite coating lby procedures that are not well understood. Montgomery and Peterson (22) claim that NaN03 is destructive and Julslhamn (23) found HC104 particularly destructive. This acid has been recommended to digest graphite and pyrolytic carbon prior to chemical analysis of these materials (24). Chromic acid is reported to exfoliate pyrolytic graphite (19),and aluminum chloride, sulfuric acid, and heat are the commercial process for exfoliating pyrolytic graphite. When any of these materials or processes cause small defects to appear in the pyrolytic coating, the adsorptive properties of the underlying graplhite become important. More than 15 papers have reported that a refractory carbide coating on graphite improved the analytical environment for some particular analyte elements. Probably these carbides perform the same function as the pyrolytic coating in providing a dense barrier between the sample and the porous graphite. These carbide coatings on top of pyrolytic graphite may seal defects in the pyrolytic graphite layer. Graphite tubes were carbide-coated by Norval et al. (25) by sputtering W or Ti3 and carbidizing the coatings in a separate furnace at high temperature. In one tungsten-coated tube described by them, the peak absorbance for a Cu solution did not change during the 3000 firings of its life. In our experience, even with the best pyrolytically coated tubes that we have seen, the peak: absorbance does change with time although the integrated absorbance using the L’vov platform often does not (6). Several papers in the literature (22, 26, 27) and our own experience indicated that changes in analytical sensitivity with
1505
-
Table I. Instrument Parameters wavelength, element nm Cu A1 Sr Ti Mo
324.7 309.3 460.7 365.3 313.3
slit, nm 0.7 0.7 1.4 0.2
0.7
char temp, “C 900 1500 1200 1400 1800
atomization temp, “C P-Epyro others 2000 2250 2400 2600 2500 2700 2700 2700 2700 2700
usage of the graphite tube might be related to the quality of the graphite. Differences in analytical sensitivity between tubes made in different ways should also be a distinguishing characteristic. Therefore we have chosen five test elements, Cu, Al, Sr, Ti, and Mo and we have run these on many different tubes. Our experience indicated that the most refractory metals were the most variable, so four of the metals that we chose required a relatively high temperature for atomization. We used Cu as an experimental control, an intermediate metal that usually has no problems. The second set of experiments relates to the study of the interferences associated with the determination of Al. This experimental protocol has revealed very wide discrepancies between furnace analyses that would have been expected to be very similar. In this paper, the effect of varying concentrations of CaC1, upon the determination of A1 is reported. The extent of the interference is shown to vary greatly with the life of the tube. Therefore the experimental protocol consisted of adding a constant amount of A1 and increasing concentrations of CaC12,up to about 1% . Different experimental environments were subjected to this protocol. These environments included sampling from the wall of a standard furnace tube, various platforms in various furnace tubes, and a variation of the wire probe technique (3, 5 ) .
EXPERIMENTAL SECTION Equipment. All tests were performed on a Model 5000 equipped with an HGA-500, AS-40 autosampler and an 056 recorder. Instrument parameters were set according to specifications in the manufacturer’s literature (28), as indicated in Table I. Some of the data were taken, recorded, and plotted by using a Perkin-Elmer Data System 10 and a Hewlett-Packard 7225A Graphics Plotter 17603A. The Ircon automatic recording optical pyrometer and the Leeds and Northrup optical pyrometer were used to measure the true temperature inside the graphite furnace tube. The indicated controller readings were calibrated against these devices, as described previously ( I ) . The Sensitivity Experiment. We incorporated into the test protocol five different standard Perkin-Elmer pyrolytically coated tubes (part no. 290-1766) and we got very homogeneous results from these even when the experimental protocol was repeated several months later. We compared these results with measurements reported several years ago on different (but similar) equipment by Fernandez and Iannarone (17). Maximum power was used for atomization. Atomization temperatures were set by manually adjusting the HGA-500 controller until the pyrometer indicated the desired temperature on the inner wall of the tube. Argon was used as a support gas and flow was stopped during atomization. All elements were fired 10 times in each tube and the last five results were used for the statistical analyses. When the elements were changed, the tube was fired twice with no sample to reduce carry-over contamination. Several different tubes were tested in addition to the standard Perkin-Elmer pyrolytically coated tubes which were used as a reference. Two ordinary graphite tubes were Ta-coated in our lab by the process of Zatka (29) and these are identified as “Ta-soaked” in the tables. Two tubes were given to us by H. Human, CSIR, Pretoria, that had been made in his lab by the process described in their paper, Norval et al. (25). These are identified as “Ta-CSIR’. One tube was made by our colleagues
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ANALYTICAL CHEMISTRY, VOL. 53, NO. 9, AUGUST 1981 _ . _
Table 11. PrecisionC precision, % std ele- std P-E P-E TaWTament graphitea pyro“ soaked BSW CSIRb cu 1.2 2.4 0.7 1.1 A1 2.8 4.9 8.2 4.2 1.1 2.4 Sr 1.3 3.9 5.1 2.5 3.2 Ti 2.6 Mo 4.1 1.6 1.1 5.0 a Average of coefficients of variation for three tubes. Average coefficients of variation for two tubes. Values are coefficients of variation of the peak area data for each tube. Table 111. HGA-BOO Graphite Furnace Conditions Sampling from the Wall temp,’C ramp, s hold, s
dry
char
140
1500 1
1
2200
2900
0
1 1
dry temp,’C ramp, s hold, s
char
atomize
clean out
cool
1600
2400
2900
20
1
1
0
1
20
25
8
1 1
IO
Sampling from the Tungsten Wire dry temp,”C ramp, s hold, s
char
cool
450
2100
20
2200
2900
20
1
1 30
1 10
1
1 1
1
20
15
071
073
073
070
074
ABS-SEC
77
102
207
392
497
615
FIRINGS
Table IV. Sensitivity (pg/0.0044 abs) for Standard Perkin-Elmer Pyro Tubes Fern. new & no. new old new old Iann. 1 no. 2 no. 2 no. 3 no. 3
280
clean cool atomize out
063
5
Figure 1. The changes in peak shape with Increased number of flrlngs. The sample is 2 ng of AI from a platform In a pyrolytically coated tube.
atomize clean out
20 40 5 Sampling from the Platform
0.70
10
at Perkin-Elmer, Bodenseewerk, Germany, by a process similar to that described by Norval. This tube was tungsten-coated and identified as “W-BSW. Two new pyrolyticallycoated tubes were tested and they are identified as “new-pyro” and are available as Perkin-Elmer part no. B009-1504. In this work, sensitivity was defined as the amount of metal (picograms) that produced a 1%absorption signal (0.0044 absorbance). Thus the data in Tables IV and V are in pg/0.0044 abs. The concentrations of the test solutions were adjusted to be within the linear range for each test and typically produced absorbance signals between 0.15 and 0.7 abs. Peak height data were used for this work because changes in peak shape were among the results we wished to expose. Differences less than 2-fold are not significant using sensitivity as a criterion. Although the protocol of this experiment was not designed to look specifically at precision, Table I1 shows the coefficients of variation obtained for each type of tube. Aluminum in Chloride Experiments. For the A1 in CaCI, experiments, the furnace conditions for sampling from the tube wall, from the platform, and from the wire are given in Table 111. The internal argon purge gas flow was stopped during the atomization step. For the platform studies, a solid pyrolytic graphite L’vov platform (2) was used. In the experiment with CuC1, matrix, we replaced the graphite platform with a plate of tantalum. The Ta plate was 10-mm long, 3-mm wide, and 0.5-mm thick, which was smaller than the standard graphite platform. For the wire probe studies, the tee-tube extension was made of isotropic (uncoated) graphite which was pressed into a tube of pyrolytically coated graphite. The indicated char temperatures in the probe work were not the actual temperature of the probe containing the sample but rather the temperature measured on the wall at the inside center of the sample tube. The probe was considerably cooler than this. Since we are not able to measure the probe temperature, we investigated the A1 loss vs. char tem-
cu A1 Sr Ti
4
3
21
15 2
91
94
Mo
11
11
2 16 2 116 12
2 20 2
3 15 3
8 26
195
111
27
12
850 90
10
new no. 4 2 11
1.3 60 13
new no. 5 2
9 1.3 93 9
perature for each series of experiments to ensure that Al was not being lost in the char step. In all these experiments the amount of AI introduced in each firing was 2 ng. The sample volume for wall and platform sampling was 20 pL and for wire sampling was 5 pL. Each datum represented the average of three firings in sequence. Thus each series of about 10 different concentrations of CaC1, represented about 30 firings. The data are reported in absorbance-seconds. The peaks at the end of the run were much broader, and, therefore, not so tall since the area remained nearly constant (Figure 1). This is a good example of the advantage of using area integration. RESULTS AND DISCUSSION The Sensitivity Experiment. The sensitivities that were obtained for standard Perkin-Elmer pyrolytically coated tubes are shown in Table IV. Also included for comparison are the sensitivities reported by Fernandez and Iannarone (17). The three tubes marked new no. 1 , 2 , and 3 were run in the first series. They are very similar and agree adequately with the data of Fernandez and Iannarone. Fernandez and Iannarone did not run Sr. When the test protocol was repeated several months later, tubes 2 and 3, now old and well-used, were retested and new similar tubes, no. 4 and 5 were added. Note that the new tubes agreed very well with the earlier sets of data but that the old tubes had lost sensitivity, particularly for Mo and Ti. By contrast, AI and Cu were not very sensitive to aging. The consistency of these data on new tubes has given us confidence in the test. The data in Table V utilized the test protocol in the different tubes. The second “new pyro” tube and the second ‘“I’a-CSIR’ tube produced data which were indistinguishable from that shown in Table IV. The two “Ta-soaked” Zatka tubes produced no useful signal for the three refractory metals although the tubes worked well for Cu. The typical standard pyro tube, the Ta-CSIR tube, and the new pyro tube all agreed with the reference data and were indistinguishable by this test. The W-BSW tube did not perform satisfactorily for the refractory metals. In addition the tube appeared to be contaminated with AI.
ANALYTICAL CHEMISTRY, VOL. 53,NO. 9, AUGUST 1981
1507
Table V. Sensitivity (pg/O.0044 abs) for Several Tubes std P-E pyro
Fern. & Iann.
cu
A1 Sr
Ti Mo a
Large spurious peak.
2 7 2
3
4 21
15 2
91
94
11
11
new pyro
113 15
Ta-CSIR
W-BSW
3
1 11 2 130
3 190
16
40
a
Ta-soaked
std P-E ordinary graphite 10
7 89 a
11 4
b b
370 34
* No peak at L O X expansion.
0,30r-
0 UNUSEO TUEE 0 AFTER APPROX 100 FIRINGS
A AFTERAPPROX. ZOO FfRfNGS x AFTER APPROX 300 FIRINGS
' '
0.25
@ GARMESTAN1
ooL---
o.l
MATSUSAUf
t
01 '
Ib
a
100 I
I
1000
IO C a C I z
Sampling from the wall, the signal for 2 ng of AI in different amounts of CaCI,. Rapid heating was used in a pyrolytically coated tube. Points are included from Matsusaki et al. (30)and Garmestani et al. (37). Flgure 2.
.""
J
I
I
10
100
1000
P O CaCIz
Flgure 3. Sampling from the wall, showing change with the number of firings. Rapid heating was used in a pyrolytlcally coated graphite tube.
r
0.70
1RO FIRINGS
It is interesting to note the last column, marked "std PE ordinary graphite". Three of these tubes were run with essentially identical results. They were made from a high-quality graphite and were not plyrolytically coated. They performed particularly poorly for the refractory metals and for Cu but were fine for Al. The reference data by Fernandez and Iannarone (17) included sensitivity for the ordinary graphite tube which is in good agreernent with our results. The only significant difference was for Ti where we found the tubes to be about 3-fold more sensitive than the earlier study. Aluminum in Chloride Experiment. When the series of CaC12 solutions was atomized from the wall of a standard furnace, there was no large suppression of the analytical signal, although the data did not follow a very regular trend, as shown in Figure 2. For contrast, we have plotted points from the work of Matsusaki et al. (30)and Garmestami et al. (31),who have studied the same system. We have not seen the great suppression of signal they reported. The points were plotted by normalizing their signal for A1 with no CaC1, to our point on the chart. If the same tube was sampled repeatedly, the signal gradually declined as shown in Figure 3, but the trend remained erratic. When the analytical protocol was repeated with the standard platform and tube, Figure 4 shows (1)that the trend was much more regular, (2) the signal was larger, and (3) unfortunately, at large concentrations of CaCl,, the suppression was much greater. As the tube wore on, the suppression started at lower concentrations of CaC1,. Other experiments with other similar platforms and tubes showed similar results. The change with time evident in the data varied from tube to tube. With several tubes the rate of increase in interference was greater than shown, while for several other tubes it was significantly less, although the experimental conditions were kept constant. These variations may be caused by differences in the pyrolytic coating. When the protocol wai repeated on a platform in a standard graphite tube that was not pyrolytically coated, the results were much better (Figure 5 ) . The graphite used for the
4
..'TER 30
FIRINGS
0'30L7 0.20
pg C o C l p
Figure 4. Sampling from a pyrolytic platform in a pyrolytically coated graphite tube. TER 180 FIRINGS
TER 0 FIRINGS
I
10
100
1000
PO coc12
Flgure 5.
tube.
Sampling from a pyrolytic platform in an uncoated graphite
pyrolytically coated tube is a different material from that used for the uncoated tube of Figure 5 and is believed to be more porous. We believe that when the coating begins to erode after a few firings, a larger proportion of the CaCl, matrix is retained
ANALYTICAL CHEMISTRY, VOL. 53, NO. 9, AUGUST 1981
1508
o’6m 0.5
0,s -
B ui 0.4 4
0.3
2
0.4
2
0.3
D
BE
\
To PLATFORM
-
0.2 0.1 0
A”
0
,
I
Sampling from a pyrolytic platform in new pyrolytically coated graphite tube.
100
1000
P(I CaCle
Figure 7.
I
10
100
I
io00
P O CUCl2
Figure 6.
IO
A“
Sampling from a pyrolytic platform In a graphlte tube treated
by the Zatka process (29).
through the ashing cycle, increasing the typical vapor-phase interference between A1 and chloride during the atomization step. We therefore used a similar pyrolytic platform in the new graphite tube mentioned earlier, made of a more dense graphite coated with pyrolytic graphite. The results in Figure 6 show excellent performance. In an effort to see if a carbide-coated graphite would provide useful results, we repeated the protocol in a standard graphite tube which had been Ta-carbide coated by the procedure of Zatka (29). The results in Figure 7 showed inadequate performance, although the performance improved with the age of the tube. A similar set of experiments were conducted on A1 in a CuClz matrix with results shown in Figure 8. Since the interference is the effect of chloride on Al, there should not be a major difference from the previous set of results. In fact, the improvement of sensitivity from the platform and the tungsten wire over the wall was seen. We additionally performed this experiment using a Ta plate as a platform. Discussion of A1 in Chloride Experiments. In the context of this paper, we have demonstrated that the chloride interference on A1 varied greatly with the properties of the sampling environment and with the age of the graphite tubes, which is probably the same thing. We believe that in all cases the differences in the performance of the various furnace arrangements a t steady-state temperature reflected the amount of chloride available for the vapor-phase reaction. As expected, the probe technique with tungsten wire worked well since the products of the char step were largely excluded from the furnace. The Ta platform provided a slightly better environment than the graphite platform, in similar tubes, at least for this particular analysis. The similarity of the good results from the metal probe and platform reflected the fact that some portion of the matrix survived the char step and interacted with the graphite tube, thus making the chloride available during atomization.
Figure 8. Sampling from various surfaces using 2 ng of Ai in various concentrations of CuCi,.
The data showed that the different physical environments produced different results and that the changes in the graphite with aging produced similar differences. The closer we came to an impervious surface on which to deposit the sample and an equally impervious tube to confine the atomic vapor, the smaller were the interferences. A special problem has become clear during the progress of this work. The performance of the tubes coated by the Zatka process had been puzzling. Figure 7 shows that the performance improved with the number of firings. The data in Table V showed reasonable performance for Cu but no signal for solutions of Ti or Mo. In the Zatka process, the Ta carbide is formed by using a solution containing HF. The observations are well explained by the continued volatilization of F from the body of the tube. L’vov (7) has shown that gaseous metal fluorides are even more stable than equivalent chlorides. As the tube is fired, the F is gradually depleted and the A1 sensitivity increases. The fluorides of Ti and Mo are very much more stable than that of Cu. The fact that Ta has nothing to do with the poor performance of the Zatka tubes is confirmed by the superior performance of the tubes made by the Ta-CSIR process (25) in Table V. This has led us to an explanation of another effect we have frequently observed. Sometimes a particular batch of graphite tubes will require a short “break-in” procedure. The first few firings provide low sensitivity. In the final step of the manufacturing process for graphite, a fluorinated compound (often freon) is added to the machined tubes, followed by firing at high temperature. The fluorine that is evolved scavenges residual metal impurities. Those batches of graphite tubes which require breaking in have presumably not been heated long enough to drive off the fluorine. While not fully proven, this study and other recent work in our laboratory have shown that pyrolytic coatings and metal carbide coatings produce similar, probably equivalent results. If the coatings are periodically renewed (32),the analytical performance does not degrade during the life of the tube. While the quality of the commercial graphite is variable, analytical results using these materials can often be improved in the laboratory by the procedures in the literature for pyrolyzing or carbide coating the graphite tubes.
ACKNOWLEDGMENT We thank our colleagues, Sabina Slavin, W. B. Barnett, F. J. Fernandez, B. Welz, and Z. Grobenski for many useful discussions. W. Ulsamer, H. Kirch, and B. Hutsch of Ringsdorff-Werke have been most helpful. LITERATURE CITED (1) Manning, D. C.; Slavin, W. Anal. Chem. 1978, 50, 1234-1238. (2) Slavin, W.; Mannlng, D. C. Anal. Chem. 1979, 51, 261-265. (3) Manning, D. C.; Slavin, W.; Myers, S. Anal. Chem. 1979, 51, 2375-2378.
Anal. Chem. 1981, (4) Fernandez, F. J.; Myers, S. A.; Slavln, W. Anal. Chem. 1080, 52, 74 1-746. (5) Manning D. C.; Slavin, W. Anal. Chim. Acta 1080, 118, 301-306. (6) Slavin, W.; Manning, E). C. Specfrochim. Acfa, Part 8 1080, 358, 701-714. (7) L’vov, B. V. Spectrochim. Acfa, Pafl B 1078, 338, 153-193. (8) Hageman, L. R.; Nlchols, J. A.; Viswanedham, P.; Woodriff, R. Anal. Chem. 1070, 51, 1406-1412. (9) Chakrabartt, C. L.; Wain, C. C.; Hamed, H. A.; Bartels, P. C. Anal. Chem. 1081, 53, 444-450. (IO) Barnard, T. Anal. Chern. 1070, 51, 1172A-1178A. (11) Siemer, D. D.; Baldwln, J. M. Anal. Chem. 1080, 52, 295-300. (12) L’vov, B. V. “Atomic ,Absorption Spectrochemical Analysis”; Hllger: London, 1970. (13) L’vov, B. V.; Bayunov, P. A.; Ryabchuk, G. N. Specfrochlm. Acta. In press. (14) Fuller, C. W. “Electrothermal Atomization for Atomic Absorption Spectroscopy”; Chemloal Soclety: London, 1977. (15) van den Broek, W. M. G. T.; de Galan, L. Anal. Chem: 1077, 49, 2176-2186. (16) Mannlng, D. C.; Ediger, R. D. At. Abs. Newsl. 1076, 15. 42-44. (17) Fernandez, F. J.; Iannarone, J. At. Abs. Newsl. 1078, 17, 117-120. (18) Szydlowski, F. J.; Peck, E.; Bax, B. Appl. Specfrosc. 1078, 32, 402-404. (19) Smith, W. H.; Leeds, D. H. Mod. Mater. 1070, 7 , 139-218.
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53, 1509-1512
(20) Slavln, W.; Myers, S. A.; Manning, D. C. Anal. Chlm. Acfa 1080, 117, 267-273. (21) Volland, 0.; Kolblin, G.; Tschopel, P.; Tblg, G. 2.Anal. Chem. 1077, 284, 1-12. (22) Montgomery, J. R.; Peterson, G. N. Anal. C h h . Acta 1080, 717, 397-40 I . (23) Julshamn, K. At. Abs. Newsl. 1077, 76, 149-151. (24) Buzzelll, G.;Mosen, A. W. Talanfa 1077, 24, 383-385. (25) Norval, E.; Human, H. G. C.; Butler, L. R. P. Anal. Chem. 1070, 51, 2045-2048. (26) Eames, J. C.; Matousek, J. P. Anal. Chem. 1080, 52, 1248-1251. (27) Carrondo, M. J. T.; Lester, J. N.; Perry, R. Anal. Chlm. Acfa 1070, 111, 291-295. (28) Manual for the Graphfie Furnace, Analytical Procedures, 1979, Bodenseewerk, Perkin-Elmer. (29) Zatka, V. J. Anal. Chem. 1078, 50, 538-541. (30) Matsusaki, K.; Yoshimo, T.; Yamamoto, Y. Talanta 1070, 26, 377-380. (31) Garmestaml, K.; Blotcky, A. J.; Rack, E. P. Anal. Chem. 1078, 50, 144- 147. (32) Clyburn, S. A.; Bartschmldt, B. R.; Velllon, C. Anal. Chem. 1074, 46, 2201-2204.
RECEIVED for review February 2,1981. Accepted May 20,1981.
Determination of Insulin in Serum by Enzyme Immunoassay with FIuorimletric Detection William
D. Hlnsberg, 111, Kristin H.
Mllby, and Richard N. Zare”
Depadment of Chemistry, Stanford University, Stanford, California 94305
An enzyme immunoassay for the determination of insulin in human serum is described, based on fluorimetric detection of enzyme activity. A sandwich method using horseradish peroxidase as the label is employed; the enzyme activity is measured by fiuoresceince using the substrate, p-hydroxyphenylacetic acid. The fluorescence measurements can be carried out by liquid chromatography-laser fluorimetry or by conventional cuvette spectrofiuorimetry. The procedure ailows the rapid and sensitive quantitation of insulin with a 45mln total incubation period and a 7.9 pM or 46 pg/mL detection limit. There Is high correlation between the insulin concentrations In serum samples determined by this method and by radioimmunoassay (correlation coefficient r = 0.97).
The technique of enzyme immunoassay (EIA) has undergone rapid development during the past decade (1). EIA has attracted considerable attention as a replacement for radioimmunoassay (RIA), since it avoids difficulties associated with the radioisotopic method (such as special handling and disposal procedures, and limited reagent shelf life). Moreover, EIA is potentially the rnore sensitive approach because the enzyme label may in principle be detected at the singlemolecule level (2). The aim of the present study is to assess the extent to which an EL4 method may be improved through utilization of a more sensitive system for the measurement of enzyme activity. Earlier work in our laboratory has shown that application of laser fluorimetric techniques to the quantitation of enzyme reaction products allowgi more sensitive determinations than are possible with conveintional methods ( 3 , 4 ) . Recently we demonstrated that laser fluorimetry may be successfully incorporated into a practical EIA procedure (5) for the analysis 0003-270018 1/0353-1509$01.25/0
of insulin in human serum. In this paper, we describe an extension and improvement of this approach. We also discuss the potential advantages of EIA-laser fluorimetry and its limitations in the current state of development. Figure 1 shows a schematic representation of the “sandwich” EIA method used in this work. During step 1, the antigen, Ag (insulin), is allowed to complex with solidphase-bound antibody (Ab). In step 2, Ab labeled with the enzyme horseradish peroxidase (HRP) is added to the sample. The labeled antibody complexes the Ag that is bound to the solid phase. The amount of solid-phase-boundenzyme is thus directly related to the amount of insulin in the original sample. After a washing step, bound enzyme activity is measured. In our case this is accomplished by using the nonfluorescent substrate, p-hydroxyphenylacetic acid (HPA), which is converted upon enzyme catalysis to a fluorescent product identified (6,7)as 6,6’-dihydroxy- [l,l’-biphenyl]-3,3’-diacetic acid (DBDA), as shown in Figure 2. For quantitation of DBDA, a high-pressure liquid chromatography (HPLC)-laser fluorimeter is employed (5).
EXPERIMENTAL SECTION Apparatus. The equipment and chromatography conditions used in this work have been described in detail (5) and included a reversed-phase high-pressure liquid chromatography system. For enhancement of DBDA fluorescence, the effluent pH was adjusted by postcolumn mixing with 0.075 M NaOH in 70% CHaOH/30% H 2 0 (v/v). Both this NaOH solution and the column eluent were pumped at flow rates of 1.3 mL/min. The 325-nm line of a helium-cadmium ion laser (Liconix Model 4050 UV) was focused into a 4-hL volume of the effluent in a flowing droplet detector. The fluorescence signal was isolated by liquid filters (3) and focused onto a photomultiplier tube. Ib signal was displayed directly on a strip-chart recorder. In those analyses carried out with conventional spectrofluorimetry, a Perkin-Elmer MPF-2A instrument was employed, using 0 1981 American Chemlcal Society
