Use of the Hydrogen-Entrained-Air Flame in Atomic Fluorescence Flame Spectrometry SIR: The basis of atomic fluorescence spectrometry has been described by Winefordner and Vickers (4) and the use of a continuum source and some properties of the argon-hydrogen-entrained air flame in atomic fluorescence spectrometry have been presented by Veillon et al. (2j. In this investigation, using a more intense continuum source, improved sensitivities of analyses were obtained for magnesium, silver, zinc, and calcium. Also, the atomic fluorescence of cobalt was observed. Some properties of a new and promising analytical flame, Hz/entrained-air, are presented. In addition, a comparison was made between two total consumption atomiaerburners which differ in their standard mode of operation-Le., one was oxygensheathed, the other was sheathed by the fuel gas (hydrogen in this study).
and cobalt and 0.40 mm. for zinc. The time constant of the phase-sensitive amplifier was 3 seconds for all studies. Table I gives the pertinent data on the operation of the two burners in their different niodes of operation and for the two different types of flames (the number in parenthesis is the flow rate of the gas in litersjminute). When operating either burner with the Ha/entrainedair flame. combustion is made possible by the air surrounding the flame and entrained by the hydrogen gas. The limit of detection was chosen as that solution concentration which resulted in a signal, the magnitude of which was equal to the magnitude of the noise. Procedure. Aqueous stock solutions containing 1000 p.p.ni. of the desired metals were prepared and diluted as required. Korking curves for each metal were obtained according t o the procedure described by Kinefordner and Staab (3) using H2/02 and H2/entrained-air flames.
EXPERIMENTAL
The experimental setup developed and used in this investigation was similar to the one used by Veillon et al. (d). The differing components included a Jarrell-Ash 0.5meter grating nionochromator equipped with a grating blazed at 3000 A. and variable slits, an E.hI.1. No. 6255B photomultiplier tube (thermoelectrically cooled to -15’ C.), a phase-sensitive amplifier (E.M.C., Model RJBj, and a variable-span recorder (L&N, Speedomax H Azar). The continuum source was an Osram 450-watt xenon arc lamp enclosed in an air-cooled lamp housing (Schoeffel Instrument Co.) and powered by a regulated d.c. supply (Sola Electric Co.). The photomultiplier tube power supply and reference signal arrangement were the same as used previously (2). The optical system was similar to that described by Mansfield et al. ( I ) , except that no baffle-box was present and only a light-trap tube between the flame and the monochromator entrance slit Tvas used. All studies were carried out using a total consumption aspirator burner, either a CK2-64 (Keyes Scientific Corp.) or a V-10 (Ditric Corp.). Conditions. Experimental conditions (monochromator slit width, region of flame viewed, gas flow rate, etc.) were not optimized exhaustively for each element. Conditions were optimized for Ag and these same conditions were used for the other elements except as noted. All measurements were made using a photomultiplier tube voltage of -1450 volts and using flame heights of 6.5 cm. and 8.0 cm. above the burner tip with the Keyes and Ditric atomizer-burners, respectively. Slit widths of 0.17 mm. were used for calcium, magnesium, silver, Apparatus.
RESULTS A N D DISCUSSION
In Table I1 the limits of detection for zinc, magnesium, silver, and cobalt in the Hz/02 and H2/entrained-air flames with the Keyes and Ditric atoniizerburners are given. The results indicate that, whether oxygen-sheathed (Keyes atomizer-burner) or hydrogen-sheathed (Ditric atomizer-burner) , the mode of operation of the atomizer-burner has little influence on the limits of detection.
Table 1.
Atomizerburner CK2-64 (Keyes) IT-10(Ditric)
Table I1 also shows that both atomizerburners give similar results with both flame types, the Ditric burner giving slightly better results overall. In addition, from the standpoint of convenience, a coniparison of the two atomizerburners reveals that the Ditric burner was much less likely t o clog a t all concentrations studied (thereby resulting in signals which are more reproducible and reliable), and it exhibited less of a memory effect than the Keyes burner. On the other hand, the Ditric burner was much louder, especially with the H2/ entrained-air flame, and some sorb of noise muffling device was necessary. Also, the gas consumption of the Keyes burner was much less than that of the Ditric burner. While investigating the fluorescence of silver (3280 A.) in the HzjOz flame, it was noticed that the fluorescence signal intensity increased as the 02 flow rate decreased, and that the greatest signal was obtainable when only an H2/entrained-air gas mixture was used. Similarly, zinc (2139 -4.)showed its greatest signal intensity in the H2/ entrained-air flame. The improvement in signal intensity obtained from the use of this flame and the shapes of typical curves for silver and zinc are shown in Figure 1. Over the linear regions of the curves, the fluorescence signal intensities with both atomizer-burners in the H2/entrained-air flames were about 18% and 23% greater for zinc and silver,
Burner Operating Conditions
H2/02flame Central orifice gas Sheath gas H, ( 5 . 5 ) 0 2 (4.2) 0 2 (5.0) HP(21)
HZ/entrained-air flame Central orifice gas Sheath gas Hz (5 5 ) none Hz (45) none
The number in parenthesis is the flow rate of the gas in liters/minute
Table II.
Limits of Detection of Several Metals Measured with Different Total Consumption Atomizer-Burners and Different Flame Types
Concentration (p.p.m.) Flame type Hz/Oz
Hz/entrainedair
Burner CK2-64 (oxygensheathed) 5’10 (hydrogensheathed) CK2-64
1--10 Limits of detection measured previously by atomic fluorescence flame spectrometry ( 8 )
co
Zn
Ag
0.1
0.005
Mg 1.0
20
0.1 0.05
0,003 0,003
1.0 0.01
10 1.o
0.03
0.001
0.01
0.5
0.6
0.08
0.2
...
VOL. 38, NO. 13, DECEMBER 1966
1943
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CONCENTRATION, p p.m.
Figure 1. Experimental analytical curves for silver (Ag 3 2 8 0 A,) and zinc A.) with H 2 / 0 2 and H2/entrained-air flames
A
HdOz
0 Hz/entrained
respectively, than in the Ha/Qz flames. The best limits of detection attained with the above-described experimental setup, 0,001 p,p.m. for silver and 0.03 p.p.m. for zinc, represent a considerable improvement over the best previously reported values of 0.08 p.p.m. and 0.6 p.p.m. for silver and zinc, respectively ( 2 ) . This improvement must be due in part to the use of a source of higher intensity but also in part to the use of the Hz/entrained-air flame. Because silver and zinc are metals which exhibit little tendency to forni refractory compounds (principally oxides) with flame gases, the enhancement of the fluorescence signal with the Hz/ entrained-air flanie is probably due mainly to the larger flame diameters (4) of the Hz/entrained-air flames with both atomizer burners and t o a decreased quenching of the excited atoms by collisions with molecular species present in the flame. The slopes of the zinc curves are much lower than unity. This agrees with the results previously obtained with a continuum source (b), but disagrees with theory (4) and with results obtained previously with a line source ( 3 ) . The reason for this anomalous behavior is unknown. Table I1 also shows that a vast improvement in detection limits occurs when the H2/entrained-air flame is used with cobalt (2407 A) and magnesium (2852 A , ) . The extent of the improvemerit and the &apes of typical obtained with cobalt and magnesium are shown in Figure 2. The limit of detec1944
e
ANALYTICAL CHEMISTRY
(Zn 2 1 3 9
air
tion attained with magnesium, 0.01 p.p.m., is again substantially better than the previous value of 0.2 pap.m. obtained with a weaker source and different flame ( 2 ) . In this work the fluorescence signal intensities with both atomizerburners with the Hz/entrained-air flame were about 15 and 19 tinies greater for
cobalt and magnesium, respectively, than in the Hz/Qzflame. Because these metals frequently form oxides with the flame gases, the major factor responsible for the great increase in signal intensity for these metals is most likely a substantial decrease of this phenomenon in the Hz/entrained-air flame. In fact, it appears that the greatest usefulness of this new flame in atomic fluorescence may lie with those elements exhibiting a tendency to form oxides in the commonly used oxygen-containing Rames. Calcium was also studied, and it was found that the type of signal obtained was dependent on the flame type. Whereas in the Hz/entrained-air flame calcium exhibited the usual line emission signal, in the H2j02flame it displayed the molecular scattering phenomenon described by Veillon et al. ( 8 ) . This latter phenomenon results in a band-like scattering signal and is due t o the scattering of the incident radiation by molecules of compounds formed with the gases (')* The line signal obtained with the H2/entrainedair flame was about 16 times more intense, and is much preferred because it eliminates the potential interferences present with band emi-7' -sion. The limits of detection for calcium were found to be 0.1 p.p.m. and 15 p.p,m. with the HL/entrained-air and Hz/O2 flames, respectively. The previously reported value for calcium was 1.5 p,p.ni. obtained from its molecular scattering signal ( 2 ) . In general, all metals previously detected with a continuum source in atomic fluorescence
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Figure 2. Experimental analytical curvesfor magnesium ( M g 2 8 5 2 A,) and cobalt (Co 2 4 0 7 A.) with Hz/02 and %/entrained-air flames
A
HdOz
0 Hz/entrained air
spectrometry would probably be detectable a t a concentration a t least one order of magnitude lower with the above-described experimental setup. Finally, it was noticed that the properties manifested by the argon-hydrogenentrained air flame studied by Veillon et al. ( 2 ) , with respect to kind and degree, were not appreciably different from those manifested by the Hz/entrainedair flame in this investigation. I n view of its greater experimental simplicity, the H2/entrained-air flame may prove to be the more desirable flame. Work is currently in progress in this laboratory t o further develop the analyt-
ical usefulness of this flame in atomic fluorescence flame spectrometry. ACKNOWLEDGMENT
I t is a pleasure to acknowledge the nunieroue discussions with Janies D. Rinefordner, whose earlier work stimulated us to undertake this investigation. LITERATURE CITED
(1) Mansfield, J. AI., Winefordner, J. D., Veillon, C., ANAL. CHEM. 37, 1049
(1965).
( 2 ) Yeillon, Claude, Mansfield, J. AI.,
Parsons, AI. L., Winefordner, J. D. Ibid., 38, 204 (1966). (3) Winefordner, J. D., Staab, R. A., Ibtd., 36, 165 (1964). (4) Winefordner, J. D., Tickers, T. J., Ibid., 36, 161 (1964). D. W. ELLIS D. R . DEMERS Department of Chemistry University of New Hampshire Durham, N. H. 03824 WORKsupported in part by funds provided by the LT. S. Department of the Interior as authorized under the JT7ater Resources Research Act of 1964, Public Law 88-379 through the Water Resources Research Center of the University of Kew Hampshire.
Rapid Micromethod for Gas Chromatographic Determination of Blood Barbiturates SIR: Studies on the gas chromatographic behavior of barbiturates have been reported (2, 3, 7 , I S ) . These experiments have shown that, in general, the gas chromatography of barbiturates presents no special problems although tailing occurs with certain members of the group. Techniques designed t o reduce tailing have been described. These include the use of mixed liquid phases (19) as well as the incorporation of high molecular veight organic acids into the liquid phase ( 6 ) . Martin and Driscoll (11) minimized tailing by reducing the polarity of the barbiturate nucleus. This was accomplished by methylation with dimethyl sulfate to form the less polar 1,3-dimethyl barbituric acid derivatives. BrochmannHanssen and Svendsen (4) found that while it was not possible to separate all nienibers of a series of 21 barbiturates on a single column, all compounds could be distinguished by using 2 columns. one v-ith a nonpolar liquid phase and another with a moderately polar polyester phase. Reith, van der Heide, and Zwaal (14) too found that Xpiezon L would not adequately resolve all members of a series of 16 barbiturates. The formation of derivatives, however. permitted differentiation of unresolved pairs. Gas chromatography has been employed for the determination of barbiturates in blood ( 1 1 , 13, 14). The methods described are suitable for qualitative and quantitative analysis if adequate sample, 1 to 5 ml., is available. All, however, involve solvent extraction, filtration, and evaporation and thus are somewhat time consuming. Jain, Fontan, and Kirk ( I O ) have described a rapid micromethod for the deterniination of blood barbiturates that minimizes the preparative methodology required. Although detailed recovery studies were not reported by
these workers it appeared that recoveries of S5-9070 were obtained. In studying the metabolism of barbiturates, rapid, sensitive, and specific micromethods are required. Such methods must also be quantitatively reliable, The present paper describes such a method based on the gas chromatographic analysis of chloroforni extracts of small quantities of blood. Because of the small sample size required, the method can be used for the determination of barbiturate half-lives in single rats. In addition the method should be useful for screening toxicological specimens for the presence of barbiturates. EXPERIMENTAL
Apparatus. A Barber - Colman Series 5000 gas chromatograph equipped with a flame ionization detector was used. The electrometer (Xodel 5040) was modified as described by Gutenmann and Lisk (8) to provide additional and 3 X ranges of 3 X IO+, ampere. The electrometer mas operated at 3 x ampere with an attenuation of 1. The column was a C-shaped borosilicate glass tube, 3.5 nim. i.d. and 5 feet long, packed with 3.4% silicone fluid DC 200 (12,500 cstks.) on Gas-Chroni Q. The column packing was prepared by the solution technique (9) using 100 ml. of a 2% (w,/v.) solution of DC 200 in toluene per 25 grams of Gas-Chrom Q. The Gas-Chrom Q was used as obtained from the supplier. The packed column was conditioned overnight a t 250" C. with the carrier gas flowing. The column temperature was adjusted to give a retention time of 7 to 9 min. and ranged from 153" to 179' C. (Table I). The detector bath and injector temperatures were 250" C. and 275" C., respectively. The nitrogen, hydrogen, and air pressures were 15, 22, and 40 p.s.i.g., respectively. Extractions were performed in a 5-ml. centrifugal separator (Kontes KO. K-
41445). An aluminum disk, punched from household aluininuni foil with a size 2 cork borer, was inserted above the septum before each use t o prevent extraction of interfering materials from the septum. Procedure. Before experimental samples were injected into the gas chromatograph 10 p1. of a 0.1% (w./v.) solution of the respective barbiturate in chloroform was injected t o saturate the column. A standard curve prepared by injecting 1 to 3 pl. of a 0.01% (w./v.) chloroform solution of the respective barbiturate, measuring the peak height and computing the number of peak height units per 0.1 pg. of barbiturate injected mas run both before and after experimental samples mere analyzed. Recovery Studies. Barbiturates, either as aqueous solutions of the sodium salts or as concentrated methanolic solutions of the free acids, were added t o heparinized rat blood obtained by aortic puncture under light ether anesthesia. Standards consisting of 10, 20, 30, 40, and 50 pg./ ml. were employed in the recovery studies. Extractions. Method A. Heparinized blood, 100 pl., was extracted for 1 niin. with 100 pl. of chloroform in the centrifugal separator using a vortex mixer. After centrifugation for 5 min., 2 t o 10 pl, of the chloroform layer, was withdrawn using a 10-pl. microsyringe and injected into the gas chromatograph. If a single barbiturate was present, as in half-life studies, a second aliquot was immediately withdrawn and injected into the gas chromatograph exactly 2 min. later and duplicate determinations were made. After the compounds were eluted the peak heights were measured and the quantity of barbiturate present in the injected aliquot computed by dividing the observed peak height by the peak height units per 0.1 pg. of the respective barbiturate as determined from the standard curve. Method B. Heparinized blood, 100 pl,, was niixed with 10 pl. of 4.OM VOL. 38, NO. 13, DECEMBER 1966
0
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