Anal. Chem. 1990, 62, 2509-2512
E
2509
mechanisms involved are subject to further investigation. CONCLUSIONS We have reevaluated the analytical applicability of ICP-LEI. The results have been improved over the previous investigation (13), although the figures of merit are rather poor compared with other LEI systems (1, 3, 4). Additional progress is still needed to justify the cost of the ICP-LEI system and the complexity of the measurement. To improve the analytical performance of the present system, considerations should include the following: (a) use a higher peak irradiance pulsed laser to saturate the atomic transition, allowing a maximum number density of atoms available for ionization, improving the signal intensity and the signal precision; (b) shield the long plasma from moving air and ground and filter more effectively to minimize picking up the rf and environmental noise, thus reducing the background noise and improving the signal precision; and (c) construct specialized ICP torches and electrodes and use for optimizing the LEI approach.
A Flguro 3. Representative signal of the LEI-ICP system. The x axis is relative signal intensity and the y axis is tlme with the scale indicated as 1 min. A is for deionized dlstilled water; B is for 10 ppm Ca; and C is for 5 ppm Ca. E is the result of sample surge during sample
changing.
was 4.6%. The poorer precision obtained with the higher concentration may have been a result of the increased instability of the plasma. A higher number density of (sample) ions can increase the electron conductivity of the plasma, which will lead to a higher tendency to arc (interactions between electrodes and between the cathode and the load-coil), reducing the plasma stability; the higher dc background also adds shot noise to the detection system. Ionization Processes. The temperature of the pencil plasma (0.75 kW) measured by Long and Bolton (16)6 cm above the load-coil was about 3290 K, whereas Barnes and Schleicher (17) suggested a temperature of 2000 and 3000 K in the region well above the plasma. In the tail region of the extended plasma, a significant fraction of the analytes exists as ground-state atoms. Since the CW dye laser radiation used in this experiment does not supply sufficient energy to saturate atomic transitions, it is less likely the laser radiation is able to ionize the laser-excited atoms. The ionization of the laser-excited atoms should be dominantly a result of collisional processes. For LEI in flames, the ionization of excited atoms is usually the result of collisions between excited atoms and thermally excited molecules such as nitrogen and free oxygen. An enhanced transfer rate of excited Na atoms to the continuum state with O2was discussed by van Dijk (18). In our case, air has been entrained into the extended argon plasma; therefore metastable argon, metastable nitrogen, and free oxygen may participate in the ionization process. The detailed
ACKNOWLEDGMENT The authors thank Tom Manning for technical assistance. LITERATURE CITED (1) Travis, J. C.; Turk, G. C.; Green, R. 6. Anal. Chem. 1982, 54, 1006A. (2) Travis, J. C.; Turk, G. C.; DeVoe; Schenck, P. K.; van Dijk, C. A. Rog. Anal. At. Spectrosc. 1984, 7 , 199. (3) Green, R. 6. Analytical Applications of Lasers; Chemical Ana&& 87; Piepmeier, E. H., Ed.; Wiley & Sons: New York, 1986; Chapter 3. Act8 1989. 448,835. (4) Axner, 0.; RubinszteinDuniop, H. S p e d ” . (5) Havrilla, G. J.; Choi, K. J. Anal. Chem. 1988, 58. 3095. (6) Nlppolt, M. A.; Green, R. 6. Anal. Chem. 1983, 5 5 , 554. (7) Smith, 6. W.; Hart, L. P.; Omenetto, N. Anal. Chem. 1988, 58. 2147. (8) Hall, J. E.; Green, R. 6. Ana/. Chem. 1983, 55, 1811. (9) Magnusson, I.;Axner, 0.; Lindgren, I.; Rublnsztein-Dunlop, H. Appl. Spectrosc. 1988, 4 0 , 968. (10) Inductively Coupled Plasma Emission Spectroscopy, Parts I & 11. Chemkxl Analysis 90; Boumans, P. W. J. M., Ed.; John Wiley & Sons: New York, 1987. (11) Omenetto, N.; Winefordner, J. D. Inductivery Coupled “ a s k, Ana&ticalAtomic Spectroscopy; Montaser, A., &lightly, D. W., Eds.; VCH Publishers: Inc. 1987; Chapter 9. (12) Koppenall, D. W. Anal. Chem. 1988, 6 0 , 113R. (13) Turk, G. C.; Watters, R. L. Anal. Chem. 1985, 57. 1979. (14) Long, G. L.; Winefordner, J. D. Appl. Spectrosc. 1984, 38, 563. (15) Demers, D. R. Spectrmhim. Acta 1985, 408, 93. (16) Long, 0. L.; Bolton, J. S. Spectrochim. Acta 1987, 428, 581. (17) Barnes, R. M.; Schleicher, R. G. Spectrmhlm. Acta 1975. 308, 109. (18) Van Dijk, C. A. Two-photon Excitation of Higher Sodium Levels and Population Transfer in a Flame. Doctoral Thesis, Rijksunlverslteit te Utrecht, The Netherlands, 1978.
RECEIVED for review June 5,1990. Accepted August 13,1990. Research supported by NIH-5-R01-GM38434-03.
Selective Detection of Carbon-I3-Labeled Compounds by Gas Chromatography/Emision Spectroscopy Bruce D. Quimby,* P a u l C. Dryden, and James J. Sullivan
Hewlett-Packard Company, Route 41 and Starr Road, Avondale, Pennsylvania 19311 INTRODUCTION The stable isotope i3c is useful as a tracer in determining the fate of labeled compounds in reactive systems. Compounds to be studied are synthesized with 13Cincorporated into the stmctureat leveh higher than the natural abundance. The reaction producta are then analyzed for compounds with
* To whom correspondence should be addressed. 0003-2700/90/0362-2509$02.50/0
elevated 13C/12Cratios to find the parent compound and its reaction products. One area where this type of experiment is important is drug Gas chromatography/mass spectrometry (GC/MS) is often used to analyze for ‘%-labeled compounds. AS discussed by Chace and Abra”n (11, mass spedral techniques currently used in screening for labeled products suffer several shortcomings. Chromatographic overlap with unlabeled peaks can mask the presence of elevated 13C content. A more specific 0 1990 American Chemical Society
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ANALYTICAL CHEMISTRY, VOL. 62, NO. 22, NOVEMBER 15, 1990
screening method for 13C is needed in gas chromatography. To address this need, techniques involving postcolumn reactions followed by mass spectrometry have been employed (I). In this approach, the eluting compounds are converted into a common species such as C02 in a reactor such as a microwave plasma (I, 2) or a combustion furnace (3). The gases then exit the reactor and enter a mass spectrometer where the isotope distribution in the C02 is monitored. A chromatogram of the 13C02response minus a portion of the 12C02response to correct for the natural abundance is then constructed. This results in a chromatogram with response only to compounds that have excess 13C content. This paper describes a technique which also provides selective GC detection of compounds with excess 13C content. Molecular emission from CO bands in the vacuum ultraviolet region is monitored with an atomic emission detector (AED) ( 4 , 5 ) . Samples can also be analyzed for C, H, 0, N,S, P, C1, F, etc. by changing the reagent and makeup gas flows. This combination of l3C specificity with atomic information is useful in the identification of unknown compounds, especially when combined with mass spectral data, as shown by Hooker and DeZwaan (6).
EXPERIMENTAL SECTION Instrumentation. The AED used was a Hewlett-Packard 5921A and is described in detail elsewhere (4,5). All reagent and makeup gas flows are as reported previously for the carbon and sulfur chromatograms. For 13C detection, the makeup gas flow was 20 mL/min, the oxygen reagent pressure was 410 kPa, and the hydrogen reagent pressure was 70 kPa. The gas chromatograph was an HP 5890A GC with a 7673A automatic injector and a split/splitless capillary injection port operated in the split mode. An injection volume of 1pL was used throughout. The carrier gas was helium. A 25 m X 0.32 mm i.d. HP-1 methylsilicone fused silica column with a 0.17 Mm film thickness was used (Hewlett-Packard).The column flow rate was 3.3 mL/min, and the split ratio was 361. All heated zones were at 250 "C. The GC oven temperature was programmed from 60 to 180 "C at 30 "C/min for all runs except the urine extract. The urine extract was injected splitless (2 pL), and the oven temperature was held at 45 "C for 4 min and then increased 15 "C/min to 250 O C and held for 10 min. Urine Extract. A sample of human urine was collected, and a 100-mL aliquot was extracted three times with 3.3 mL of chloroform. The extracts were pooled and spiked with nitrob e n ~ e n e - ~(99 ~ Catom ~ % 13C)at a concentration of 25 ng/mL. Materials. The n i t r ~ h n z e n e - ~and ~ Cperdeuterated ~ n-decane were obtained from MSD Isotopes (Rahway, NJ), and the chloroform and isoodane from Burdick and Jackson (Muskegon, MI). The remainder of the sample reagents were purchased from Aldrich (Milwaukee,WI). Gases used were research grade, except for the nitrogen used to purge the spectrometer,and were purchased from Air Products (Allentown, PA). The helium was purified with a heated getter (VICI, Houston, TX). RESULTS AND DISCUSSION In gas chromatography/atomic emission spectroscopy (GC/AES), the atomic emission lines of hydrogen and deuterium are sufficiently separated in wavelength to permit selective detection of each isotope with typical laboratory spectrometers ( 4 , 5 , 7). However most elements, including carbon, have negligible isotopic shifts in their atomic emission wavelengths. Molecular emission, however, often exhibits pronounced isotopic shifts. Ferguson and Broida measured 12C/13C ratios in an acetylene flame by measuring the relative intensities of C2 bandheads in the Swan system in the 475-nm region (8). Johanaen and Middleboe (9,101used the CO+ fist negative system B22 - P 2 transition to measure carbon isotope ratios in carbon dioxide samples. In their system, carbon dioxide gas samples are mixed with helium in a vacuum manifold and
171.4 nm
J
241.94 nm
s- . _ . . . _ _ _ _ . _ _ _ _ . _ . 170
180
190
200
210
I
220
230
240
Wavelength (nm) Figure 1. Emission spectrum scanned with nitrobenzene vapors added
to the makeup and reagent gas mixture. 171.4 nm CO Bands Second Order -0.39 nm-+
t
a
b
335
340
Wavelength
3i5
350
(nm)
Figure 2. Snapshots of bands used for '*C and '% detection: (a) taken during unlabeled dodecane peak: (b) taken durjng nitrobenzenaisC peak; (c) "recipe" used for % selective detection.
a microwave-induced discharge is ignited. The spectrum is scanned in the 210-250-nm range, and the carbon isotopic distribution is measured from the relative intensities of the molecular bandheads. Initial experiments were aimed a t adapting the above method for the selective GC detection of carbon isotopes. With the use of oxygen reagent gas (4), carbon-containing compounds were detected as 12Cand 13Cby monitoring the intensity of the 241.94-nm lzCO+and the 241.36-nm 13CO+ bands. With the appropriate "recipes" (an algorithm which takes diode array signals and combines them to produce background-corrected chromatograms (5)), separate chromatograms for each isotope could be produced. By use of this technique, 12Cand '3c can be measured with a detection limit of around 110 pg/s. Other bands were investigated by scanning the emission spectrum while a constant level of carbon-containing vapor was bled in. Figure 1shows a typical spectrum. The intense bands in the 170-190-nm region are from the fourth positive CO system A2n(IO). The most intense bandhead, the 0-3 at 171.4 nm, is approximately 100 times more intense than the CO+ band at 241.94 nm used previously.
ANALYTICAL CHEMISTRY, VOL. 62, NO. 22, NOVEMBER 15, 1990
Figure 2 contains "snapshots" (real-time spectra obtained during elution of GC peaks ( 5 ) ) of an unlabeled compound and of a compound that is 99% 13C. The spectra were measured in the second order to provide higher resolution. The isotopic shift of the bands is clearly evident. The 0-3 band maximum for 13C is shifted from the lZCby 0.39 nm. Also shown in Figure 2 is the recipe used for 13C detection. As described in ref 5 , a recipe has two matched filters, each using a different set of pixels in the photodiode array, with different gains for each pixel. Figure 2c shows a plot of the gains of the two filters used for 13Cdetection; the signal filter is shown as a positive curve, and the background filter is negative. The chromatographic output is the weighted sum, S - kB, of the time series produced by the signal filter, S, and by the background filter, B. The adjustable background amount factor, k, determines the degree of background correction. Note that the background is not measured at the apex of the 12Cbandhead. Since the 13Cband has a high intensity at that wavelength, a large portion of the 13Csignal would be canceled by background correction at this point. A better signal-to-noise ratio was achieved by measuring the 12Csignal on the low-wavelength side of the 13C band. As the overlap of the curves in Figure 2 shows, 12Cand 13C each produce a substantial response in both the signal and background fiiters. Therefore, good selectivity for 13Ccan only be achieved by setting the background amount factor exactly. Selectivity for a "normal" 13Crecipe is defined as the ratio of response to 13Cto response to an equivalent amount of 12C. In order to optimize the background amount factor and to measure the resulting normal selectivity for 13C,at least one compound made from isotopically purified l2C would be needed. Since this was not available, the measurement of normal selectivity for 13Cwas not made. On the basis of other measurements reported here, it is estimated to be in excess of 1000. In practical work, a high value of normal 13C selectivity is not directly usable, since the recipe is intended to detect compounds with enriched amounts of 13C. Nonenriched compounds would have responses that are lower by only a factor of 91, because of the natural abundance of 13C. What is needed is not a "normal" 13Crecipe that rejects 12C, but an "enriched" 13Crecipe that rejects compounds with the natural abundance of l2C and 13C. Chace and Abramson (1) constructed such a detection algorithm by correcting a mass spectral signal characteristic of 13C,by the subtraction of a portion of a signal due to l2C. In the case of the 13C recipe used here, the background signal is mostly due to 12C. Thus, an enriched 13Crecipe can be made by subtracting more of the background signal in a normal 13Crecipe. In practice, the background amount factor for an enriched 13C recipe is set empirically, by adjusting it for minimal response to a typical interfering compound containing natural abundances of carbon isotopes. Atomic nitrogen can be determined simultaneously with the carbon isotopes. The second order of the 174.2-nm nitrogen line is sufficiently close to the CO bands in wavelength to fall in the wavelength range covered by the photodiode array (5)* Figure 3 shows the chromatogram obtained from a test mixture. The carbon in the first peak is labeled with 99 atom % lac,and the rest of the peaks have natural-abundance levels of the carbon isotopes. For the lZCchannel, the background correction is adjusted to reject 13Cresponse. This is why the labeled compound shows very little response on the 12C channel. In Figure 3, the 13Cchannel is produced by an enriched 13C recipe. The only peaks that give a response are those with
2511
b
-- -
, 1.5
. . . .
,
2.0
. . . .
, 2.5
.
.
3.0
*
3.5
.
4.0
Time (min) Flgure 3. Chromatograms of isotopic test mixture: (a) '*C at 171.4 nm; (b) enriched '% at 171.0 nm; (c) N atomic Hne at 174.2 nm. Peak identities: (1) 6.8 ng of 99 atom % nitroben~ene-~~C,; (2) 166 ng of unlabeled ndodecane; (3) 16.8 ng of unlabeled n-tridecane; (4) 5.0 ng of unlabeled n-tetradecane.
excess 13Ccontent. The size of the response is proportional to the product of the amount of the compound injected and the excess fraction of 13C incorporated into the molecule. Experiments to optimize the reagent and makeup gas flows were performed. The best performance is obtained with a low makeup flow rate and a high oxygen reagent flow rate compared to the conditions used for atomic detection (4). The low makeup flow (20 mL/min) results in increased residence time in the plasma, yielding more photons per CO molecule. The high oxygen concentration (1.5%) increases the fraction of carbon present as CO. Hydrogen combined with oxygen as a reagent gas was found previously to improve the selectivity and peak shape for atomic nitrogen response (4). Since it might be advantageous to detect nitrogen simultaneouslywith 13C,this reagent was tried with 13C. It was found that the addition of a small flow of hydrogen (0.005 mL/min) to the oxygen reagent doubled the selectivity of enriched 13Cversus the natural-abundance compounds. The reason for this is unclear at present. Detection Limits, Selectivities, and Linearities. The detection limit for both the 12Cand 13Cchannels is approximately 10 pg/s. The detection limit is defined as the weight of isotope required to produce a signal twice the peak-to-peak noise divided by the peak width a t half-height. The detection limit for atomic nitrogen measured simultaneously with 13Cat the second order of the 174.2-nm atomic line is about 3 pg/s. Figure 4 shows the response of an enriched 13C recipe to a test mixture. The scale is expanded 40-fold from that in Figure 3, so that small, residual responses are evident. Peaks 2-4 are the same unlabeled hydrocarbons as in Figure 3, but have a 5 times higher concentration in Figure 4. Note that most of the peaks give a small negative or "zigzag" response. These residual responses can result from secondary spectral features overlapping the CO bands. If the secondary emission is concentrated on the signal filter, a positive interference results. If it is more prominent on the background filter, then the interfering response is negative. In the case of an excessively large peak like 2, imperfect selectivity is caused by changes in the shape of the band emission with sample concentration (5). The peaks in Figure 4 labeled with element symbols result from 10-15 ng of compounds containing the indicated elements. No significant interferences are evident from these
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ANALYTICAL CHEMISTRY, VOL. 62, NO. 22, NOVEMBER 15, 1990
F
,i
'
f
V
30 Pg
c13
b
1
1.5
2.0
2.5
3.0
3.5
4.0
C
Time (min) Flgure 4. Chromatogram of unlabeled multielement test mixture on enriched '% channel. Peak identities (F)12.5 ng 4-fkKKoanlsole; (Br) 13.0 ng of 1-bromohexane; (Si) 10.5 ng of tetraethylorthosilicate;(D) 9.5 ng of n-perdeuterodecane; (N) 13.5 ng of nitrobenzene; (P) 12.0 ng of triethyl phosphate; (S) 10.5 ng of tert-butyl disulfide; (Cl) 16.5 ng of 1,2,4-trichlorobenzene; (2) 850 ng ndodecane; (3) 85 ng of n-trldecane: (4) 25.5 ng n-tetradecane. Table I. Precision of Peak Areas from Replicate Injections
(element) A, nm
compd
% re1 standard dev (n = 12)
nitrobenzene-13C 1.5 nitrobenzene-13C 3.0 (12C)171 tetradecane 2.8 (13C/12C)a nitrobenzene-13C/tetradecane 0.66 "The area of 13C from nitrobenzene divided by the area of l2C from tetradecane in the same injection. Compounds and conditions are the same as in Figure 3. (N) 174 (13C)171
elements commonly encountered in GC analysis. The linear dynamic range of the 12Cand 13Cchannels is approximately 1000, and that of the nitrogen line is about 10000. Chromatographic Precision. Table I contains precision data from 12 replicate injections of the test mixture shown in Figure 3. The relative standard deviation of the raw peak areas are less than 3% on all three channels. The precision of the ratio of '?c response of the unlabeled n-tetradecane to the 13C response of the labeled nitrobenzene is 4-fold better than the individual peak areas. Spiked Urine Sample. Figure 5 shows a portion of a multielement set of chromatograms from the extract of a urine sample spiked with 'V-labeled nitrobenzene. The labeled compound is evident on both the '% channel and the nitrogen channel. The total carbon chromatogram shows significant overlap of the labeled compound with other peaks. The sulfur trace shows that at least one of the interfering compounds contains sulfur. These interferences could complicate identifying the labeled compound by mass spectrometry. Snapshots can confirm that a compound contains excess 13C. Figure 6 shows the spectrum taken a t the apex of the 13Cpeak and one from the unlabeled peak preceding it. Both have had a baseline spectrum subtracted from them. The shift of the two CO bands due to the presence of 13C is evident. The nitrogen lines in the nitrobenzene-13Cspectrum are also visible.
5.5
6.0
6.5
7.0
Time (min) Flgure 5. Chromatograms of urine extract spiked with 25 ng/mL 13C-iabelednitrobenzene: (a) C 193.lnm atomic line; (b) enriched "C 171.0 nm (second order); (c) N 174.2-nm atomic line (second order); (d) S 180.7-nm atomic line. 13C
171.0 nm
N 174.2 nm
12C 171.4 nm
Wavelength (nm) Figure 6 . Snapshots taken on peaks in spiked urine sample: (a) at apex of '3C-labeled peak; (b) at apex of un1abe)ed peak.
atomic detection, makes emission spectrometry an important complement to mass and infrared spectral detection for gas chromatography. Based on band shifts for 14Clisted in ref 9 for other band systems, it is logical to expect that, in the band system used here, the 14C0band will be shifted enough for simultaneous detection of 12C,13C,and 14Cto be carried out. Future studies will be aimed at extending the technique to the selective detection of 14C-labeledcompounds as well.
LITERATURE CITED
This technique provides a means to identify peaks as labeled
(1) Chace, D. H.; Abramson, F. P. Anal. Chem. 1989, 8 1 , 2724. (2) Heppner, R. A. Anal. Chem. 1983, 55. 2170. (3) Matthews, D. E.; Hayes, J. M. Anal. Cbem. 1978, 50,1465. (4) Quimby, B. D.;Sullivan, J. J. Anal. chem. 1990, 62, 1027. (5) Sullivan, J. J.; Quimby. 8. D. Anal. Chem. 1990, 62, 1034. (8) Hooker, D. B.; DeZwaan. J. Anal. Chem. 1989, 61, 2207. (7) McLean. W. R.; Stanton, D. L.; Penketh, 0. E. Ana/yst 1979, 98, 432. (8) Ferguson, R. E.: Broida, H. P . Anal. Chem. 1068, 28, 1436. (9) Johansen. H. S.; Mtddleboe, V. A w l . Specirosc. 1980, 34, 555. (IO) Hertzberg, G. Spectra of Diatomic Mdecdes, 2nd ed.; Molecular Spectra and Molecular Structure, Vol. I; Van Nostrand RehhoM: New York. 1950.
with 13C,even in complex matrices. The ability to screen for this important tracer isotope, together with multielement
RECEIVED for review June 12,1990. Accepted August 13,1990.
CONCLUSIONS
