Anal. Chem. 1980, 52, 165-167
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Rapid Analysis of Complex Mixtures by Mass SpectrometryIMass Spectrometry G.
L. Glish, V. M. Shaddock, K. Harmon, and R.
G. Cooks*
Department of Chemistry, Purdue University, West La fayette, Indiana 47907
Replicate determinations have been made on cyclohexane, carbon monoxide, and blood serum for traces (1-22 ng) of pyridine, dimethyl ether, and urea, respectively. The experiment employed single reaction monitoring to characterize the analyte with repetition rates of 15-51 samples per hour. Relative standard deviations were 5 to 15%.
Double focusing mass spectrometers employ two analyzers which are coupled to achieve velocity focusing and, hence, high mass resolution. If the analyzers are uncoupled and scanned independently, they can be used to select specific reactant ions entering the inter-analyzer region and particular product ions leaving that region ( I , 2). This ability to specifically analyze for particular reactions has led to the development of the mass-analyzed ion kinetic energy spectrometer (MIKES) which records all the reactions of a particular ion in a single scan. This capability has been successfully applied to the direct analysis of specific components in complex mixtures (3, 4 ) . Implicit in the use of a mass analyzer as a separator in mass spectrometry/mass spectrometry (MS/MS) (5-11) is a time advantage over chromatographic alternatives. This paper seeks to demonstrate this feature explicitly. Speed of analysis is a particularly important consideration in the detection of trace amounts of carcinogens, pollutants, or impurities in complex mixtures where 110th high sample throughput and high sensitivity are required. T h e time required for analysis must include that used in sample preparation, viz., isolation, derivatization, and purification, as well as the requisite instrumental analysis time. Superior characteristics in this regard, and in sensitivity and compound specificity, have made gas chromatographyjmass spectrometry (GC/MS) the method of choice for trace organic analysis (12). Nevertheless, prechromatographic sample cleanup and derivatization of compounds may he complex and time-consuming, but successful application of GC/MS often demands this (12). In addition, the time required for a complete chromatographic separation of a mixture may be excessive, particularly when capillary columns are employed. A reduction in retention time may be realized by purification and isolation of the component of interest; however, because of the additional steps, overall analysis time may not decrease. Mass spectrometry/mass spectrometry (MS/MS) appears t o have two advantages in rapid mixture analysis. First, it permits t h e analysis of complex mixtures with little or no sample pretreatment ( 3 , 4, 13). Second, the primary separation performed in MS/MS is analogous to that in GC/MS but components are separated much more rapidly. Furthermore, any individual component can he selected a t any time during the analysis. This is to be contrasted with the sequential delivery of sample to the mass spectrometer in GC/MS. (This mode of separation has its own distinct advantages, including preconcentration of solutes from solvents and higher sample fluxes, albeit for shorter times.) Three chemical systems were investigated, liquid/liquid, gas/gas, and solid/liquid, t o determine the applicability of 0003-2700/80/0352-0165$01 OO/O
MIKES to rapid sampling. The liquid/liquid system consisted of trace impurities of pyridine in cyclohexane. T h e gas/gas system was a dilute mixture of dimethyl ether in carbon monoxide, and the solid/liquid system wap urea in human blood serum.
EXPERIMENTAL 'The study was performed on a mass-analyzed ion kinetic energy spectrometer which has been descrihed previously (24, 15). The only modification made specifically for this study was insertion of a septum in the reagent gas line. Isobutane was used as reagent gas at a pressure of approximately 0.3 Torr. The source temperature was 375 K and the accelerating potential was 7 kV. Nitrogen was used as collision gas at a pressure of approximately 1-2 x 10 Torr. Technical grade cyclohexane contains a small amount of pyridine. A 0.4-pL aliquot of this solution was injected directly into the reagent gas line near the ionization chamber. The analysis was by single reaction monitoring (26) for the transition 80' 53+ which corresponds to loss of HCN from protonated pyridine. In this procedure the magnet was set to pass protonated pyridine ( m / r = 80) into the collision cell, and the electric sector voltage was adjusted to pass fragment ions a t m / z 53 which result from collision-induced dissociation of the protonated molecule. This transition was monitored with respect to time and when the signal fell to base line another sample was injected. lJsing a standard addition method, it was independently shown that the cyclohexane sample contained 2.6 ng/FT, of pyridine. Dilute gas mixtures were prepared for study by injecting, via a gas syringe, an appropriate volume of dimethyl ether into a flask containing carbon monoxide. Aliquots (10 or 30 ILL)of the gas mixture were introduced into the mass spectrometer as in the previous experiment. The reaction monitored was the transition 47+ 31+, loss of methane from the protonated ether. The sampling procedure was modified slightly for the solid/ liquid system. Volumes of a one-tenth dilution of human blood serum were injected onto small lengths of solid ceramic tubing and dried under a nitrogen stream before introduction into the mass spectrometer source via a direct insertion probe. The probe was introduced into the source and heated to 460 K while monitoring the loss of ammonia from protonated urea, 61' 44+. The sample size was 1 pL of diluted blood serum which contained approximately 20 ng of urea. This method of sample introduction was found to produce higher quality spectra than those obtained by simply loading a glass tipped probe with serum. These advantages should extend to direct analysis of samples in other complex matrices, and this has already been noted for urinary acid identification.
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-
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RESULTS AND DISCUSSION For each system, the analysis of multiple samples during a short time period was achieved. Fifty-one highly reproducible samplings of pyridine-cyclohexane were completed in 1 h as shown in Figure 1. Each sample contained 1 ng of pyridine. T h e vaporization rate of the liquids was relatively slow, as seen by the fact that large volumes of sample produced peaks of similar height but of much greater width than those resulting from smaller aliquots of the same solution. A reduction in sample size therefore increased the rate of analysis. Using full width at half maximum (fwhm) as ti measure of the reproducibility of the technique, the 51 cyclohexane samples had an average width of 21.4 f 2.6 s (standard deviation). C= 1979 American Chemical Society
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ANALYTICAL CHEMISTRY, VOL. 52, NO. 1 , JANUARY 1980
G + Q 80'4 #I
53'
#3
X 2
V
tt 51
36
SAMPLE 'IME
+ PATE
TIME
SAMPLE SIZE. 0 . 4 ~ 1 2 . 6 x IO- g / u l R A T E : 51 SAMPLES /HOi.JR
---+
Flgure 1. Single reaction monitoring for trace amounts of pyridine in cyclohexane. Peak 1 is the first sample, 51 is the fitty-first, introduced 1 h after the start of the analysis
Flgure 3.
SIZE
lul
Zx10-~9/ u 5 S A M P L E S / POJP
Single reaction monitoring of urea in human blood serum
(1 ng of dimethyl ether) of the 0.005% gas mixture were analyzed, although precision diminished (Figure 2B). Because of its complexity, human blood serum proved to be a challenging substrate for the MS/MS rapid sampling technique. Samples which contained 20 ng of urea were analyzed a t a rate of 15 per hour. Reproducibility was estimated from peak height (Figure 3) as 15% relative standard deviation. This sampling rate could easily be increased with no loss in precision because of the fact that a large portion of the analysis time (-40%) is involved in loading the sample onto the probe and introducing the probe into the ion source.
CONCLUSION
TIVE
-
TIME
SAMPLE SIZE ~ C J 5 0 x lo-'og/ul RATE 2 3 SAMPLES/PFt
SAMPLE
SIZE
IOLI
l x l c - ~ og/ul
RATE
27 SAVPLESIHR
Single reaction monitoring of dimethyl ether in carbon monoxide, 15 ng of dimethyl ether per sample. (B). Same as (A) except that each sample contains 1 ng of dimethyl ether Figure 2. (A).
Most of this variation is attributed to variation of sample size. Following the 0.4-pL cyclohexane samples, ten 1.2-pL analyses were performed. Excellent reproducibility again was evidenced by an average fwhm of 29.1 f 2.6 s. This shows the somewhat better precision which can be achieved with larger samples owing to better S/N, while more rapid rates of analysis are possible with smaller samples. The results for the gas mixture also demonstrate that precision diminishes with decreased sample size. Two concentrations of dimethyl ether in carbon monoxide were prepared for analysis, 0.025% and 0.005%. Aliquots of 30 FL of the 0.025% gas mixture (15 ng of dimethyl ether) were analyzed a t a rate of 23 per hour, Figure 2A. Since the samples were introduced as a vapor, the measure of reproducibility was peak height (comparison of peak area improves the precision slightly). The relative standard deviation for analysis of this mixture was less than 5%. The sampling rate was increased to 27 samples per hour when 10-pL portions
These results carry the MIKES method of mixture analysis a step further by demonstrating a rapid analysis capability on small samples which include complex mixtures. The key features of this technique of analysis are now (i) ability to analyze samples with minimal prior workup (4,17); (ii) compound specificity, including distinction of isomers (5,18) and structure elucidation (19);(iii) sensitivity (10, 11, 20); (iv) applicability to biological substrates (9);(v) compatibility with pyrolysis methods (18);and (vi) microanalysis capability (4). I t is important to note that quantitative analysis is possible (15) but a demonstration of high precision quantitative analysis is still awaited. The rates of analysis achieved in this study should not be taken as optimal for the technique. We employed direct probe or septum introduction methods which themselves take time, and it is noteworthy that sample vaporization was rate-limiting in one case. In the others, sample residence time in the source is the rate-limiting factor. More sophisticated sample handling techniques, including multisample inlet systems and flash evaporation, should result in more rapid analyses. It should also be noted that for increased specificity, multiple reaction monitoring can be used although this could itself limit the maximum attainable sample rate.
ACKNOWLEDGMENT We thank Arthur B. Coddington for technical assistance and Patricia A. Snyder for providing the blood serum samples.
LITERATURE CITED (1) K. R. Jennings in "Mass Spectrometry", G. W. A. Milne, Ed., Wiley-Interscience, New York, 1971. (2) R . G. Cooks, J. H. Beynon, R. M. Caprioli, and G. R. Lester, "Metastable Ions", Elsevier, Amsterdam, 1973. (3) R. W. Kondrat and R. G. Cooks, Anal. Chem., 50, 81A (1978). (4) M. Youssefi, R. G. Cooks, and J. L. McLauahlln. J . Am. Cb8m. Soc., . . 101, 3400 (1979). (5) T. L. Kruger, J. F. Litton, R. W. Kondrat, and R. G. Cooks, Anal. Chem., 48. 2113 11976). (6) F. 'W. Mciaffe& and F. M. Bockhoff, Anal. Chem., 50, 69 (1978). (7) K. Levsen and H.R. Schuken, Bbmed. Mass Spectrom., 3 , 137 (1976). (8) J. H. McReynolds and M. Anbar, Int. J . Mass Spectrom. Ion Phys., 24, 37 (1977). (9) R . G. Cooks in "Trace Organic Analysis", H. S.Hertz, S. W. Chester, Eds., National Bureau of Standards, Washington, D.C.. 1979. ( I O ) R. A. Yost and C. G. Enke, J. Am. Chem. SOC., 100, 2274 (1978). (11) D. F. Hunt and J. Shabanowitz, paper presented at the 27th Annual Conference on Mass Spectrometry and Allied Toplcs, Seattle, Wash., June 1979.
Anal. Chem. 1 9 8 0 , 52, 167-176 (12) E. J. Millard, "Quantitative Mass spectrometry", Heyden and Sons, London. _ _ . -., 1978. . .. . . (13) R. W. Kondrat, G. A. McClusky, and R. G. Cooks, Anal. Chem., 50, 1222 (1978). (14) J. H. Beynon, R. G.Cooks, J. W. Amy, W. E. Baitinger, and T. Y. Ridley, Anal. Chem., 45, 1023A (1973). (15) R. W. Kondrat, Ph.D. Thesis, Purdue University, West Lafayette, Ind., 1978. (16) D. Zakett, R. G. A. Flynn, and R. G. Cooks. J . Phvs. Chem., 82, 2359 (1978). (17) R. W. Kondrat, R. G. Cooks, and J. L. McLaughlin, Science, 199, 978 (1978).
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(18) A. E. Schoen, R. G. Cooks, and J. L. Wiebers, Science, 203, 1249 (1979). (19) T. L. Kruger, R. G. Cooks, J. L. McLaughlin, and R. L. Ranieri, J . Org. Chem., 42, 4161 (1977). (20) G. A. McClusky, R. W. Kondrat, and R. G. Cooks, J . Am. Chem. SOC., 100, 6045 (1978).
RECEIVED for review August 23. 1!)79. Accented October 22. 1979. The work Was svupported by the National Science Foundation CHE 77-01295.
Capacitive Discharge Heating in Graphite Furnace Atomic Absorption Spectrometry C. L. Chakrabarti," H. A. Hamed, C. C. Wan, W. C. Li, P. C. Bertels, D. C. Gregoire, and S. Lee Department of Chemistry, Carleton University, Ottawa, Ontario, Canada, K7S 5B6
trothermal energy for heating graphite atomizers, and also, that rates of heating of -40 K ms-' can be achieved. However, no publication has yet appeared on the application of this technique to determination of elements by electrothermal atomization in graphite tube atomizers.
Use of an anisotropic pyrolytic graphite tube atomizer heated by a capacitive discharge comblned with malntenance of a constant temperature with an auxiliary power supply creates an isothermal condition resulting in much higher sensitivities for some elements than those available from commercial graphite furnaces and also absence of condensation of the analyte at the ends of the graphite tube and, hence, absence of memory effect. I n such a graphite tube, an isothermal environment results from 5000-fold greater resistivity across the tube than along the tube length and 225-fold greater thermal conductivity along the tube length than across the tube, and also by its thermal conductivity being about 10-fdd greater than that of regular (isotropic) graphite. The peak absorbance of all elements increases exponentially with heating rates, the low-volatility elements showing steeper increases in the peak absorbance with heating rates than the high-volatility elements. Pulse heating of an anisotropic graphite tube atomizer by capacitive discharge increases sensitivity of those elements which have, as the mechanism of atom formation, reduction of the solid metal oxide by carbon followed by vaporization of the metal atoms or solid-state decomposition of the metal oxide to form the metal atoms in the vapor phase. This technique offers promise of high sensitivity for ail elements.
THEORY Assuming, for the sake of simplicity, that analyte atoms are introduced into the analysis volume simply by the vaporization of the analyte element from the graphite surface (Le., ignoring all preceding steps of drying, ashing which may involve decomposition of and other reactions with the sample, and all intermediate steps that may be involved in the atom formation), the rate constant k for evaporation of an analyte element as a function of the absolute temperature may be expressed by:
k(T) =
(1)
where A is a frequency factor, A H is the heat of vaporization of the analyte element, R is the universal gas constant, and T i s the temperature in degree Kelvin. Since the vaporization of the analyte elements occurs simultaneously with the atomizer surface temperature increasing linearly with time, Equation 1 may be expressed as a function of time: where (To+ a t ) defines the temperature-time characteristics of the atomizer, Le., LY is the slope of the T vs. t graph and is the rate of heating of the atomizer in K ms-', t is the time in ms, and To is the intercept on the temperature axis. Equation 2 can be expressed as
Commercial electrothermal graphite tube atomizers have two limitations: they give relatively low sensitivities because of their relatively low rates of heating; their heating is nonisothermal in time and along the length of the graphite tube, resulting in condensation of the analyte vapor a t the cooler ends of the graphite tube and its subsequent re-evaporation producing unpredictable consequences (1-9). The above two limitations can be removed by using much faster heating rates with a capacitive discharge power supply together with an auxiliary power supply and anisotropic pyrolytic graphite tubes. The effect of heating rates in graphite furnace atomic absorption spectrometry has been reported in earlier publications (10, 11) from the authors' laboratory. Cresser and Mullins (12) have suggested the use of a large electrolytic capacitor for rapidly heating metal filament atomizers. A practical device for capacitive discharge heating has been recently patented (I,?). More recently L'vov (14) has reported the use of a capacitor bank as a source of elec0003-2700/80/0352-0167$01.00/0
Ae-H/RT
In h ( t ) = In A
-
m/R(T0 + a t )
(3)
Assume AH is constant and independent of the temperature T (a fairly reasonable assumption over the narrow range of temperature around the boiling point or the sublimation point). In applying Equation 2 or 3 we can recognize two cases as follows. Case 1: at >> T, This is the case with capacitive discharge heating with its high heating rates (up to 60 K ms-'); To,being the temperature a t the end of the charring cycle, is 5500 K in a typical case, at >> To, so that ( T o+ a t ) N at. With the above approximation, Equation 3 becomes In k ( t ) = In A 'C
1979 American Chemical Society
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AH/Rat
(4)
