Mass spectral investigation of mechanisms of lead vaporization from a

Apr 1, 1987 - Mechanisms of lead vaporization from an oxygenated graphite surface using mass spectrometry and atomic absorption. Dean A. Bass and J. A...
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Anal. Chem. 1987, 59,974-980

974

Mass Spectral Investigation of Mechanisms of Lead Vaporization from a Graphite Surface Used in Electrothermal Atomizers Dean A. Bass and James A. Holcombe* Department of Chemistry, The University of Texas at Austin, Austin, Texas 78712

Atomic absorption/mass spectrometry was used to study vaporization mechanisms for Pb(NO,), with and without the addition of NH4H,P04. The data indicated that Pb deposited as the nitrate salt forms a surface-bound Pb(NO,), which decomposed at 550 K to a surface-bound Pbo. The rate-lmiting step for the vaporization of Pb was the reduction of PbO to form atomic Pb. With the addition of phosphate to the sample a surface-bound Pb-phosphate species was formed. X-ray photoelectron spectroscopy results indicated a 1 to 1 Pb to P ratio which would correspond to Pb2P,0,. The stabilized Pb-phosphatespecies decomposed at 1150 K to give a surface-bound PbO specles which was bnmediately reduced to atomic Pb. Vacuum atomic absorption signals coincident with CO, verified the formation of atomic Pb by a surface reaction. Correlationswith graphtle furnace atomic absorption spectroscopy are discussed.

The determination of P b is commonly performed by graphite furnace atomic absorption spectrometry (GFAAS) because of its superior sensitivity. However, P b is prone to interferences, many of which arise from gas-phase chemical reactions that attenuate the free atom density within the furnace. Consequently, matrix modifiers are routinely used in P b determinations to help alleviate interferences. Of the large number of matrix modifiers that have been employed, the phosphate salts are perhaps the most commonly employed. Matousek and Brodie (1) were the f i s t to use phosphoric acid in the determination of Pb. They clearly showed that phosphoric acid allowed for the use of higher char temperatures and gave more reproducible peak areas. In later fundamental studies of phosphate addition, Czobik and Matousek (2) suggested the formation of the metal pyrophosphate as the atomization precursor, which decomposed at a high temperature to release the free metal vapor. They also reported that none of the metals studied with appearance temperatures above that of Sn (Le., Ni and Cr) were observed a t a higher temperature with the addition of phosphate. It was postulated that the pyrophosphate decomposed to the metal oxide a t temperatures below the normal vaporization temperature observed in the absence of the phosphate; thus, the effects of phosphate addition were not observed in GFAAS experiments for these higher boiling metals. Ammonium dihydrogen phosphate, NH4H2P04,is often cited as the preferred form of the phosphate matrix modifier because of its ability not only to increase the atomization temperature of P b but also to minimize halogen interference by the low temperature volatilization of the ammonium halide (3-5). Like phosphoric acid, the addition of NH4H2P04 produces an increase in the atomization temperature and an increase in peak height. This allows for more efficient removal of the matrix as well as increased peak height sensitivity. For aqueous Pb(NO& samples without any form of phosphate addition, several mechanisms have been suggested.

Genc et al. (6) proposed the decomposition of Pb(N03)*in GFAAS to PbO(s) followed by sublimation and consequent gas-phase dissociation of the PbO to form atomic Pb. Using vacuum vaporization and mass spectral detection, Sturgeon et al. (7) noted low-temperature vaporization of PbO and the release of elemental P b at a higher temperature. They speculated, as one possibility, that the free P b could be produced from a surface reduction of lead oxide. The utility of mass spectrometry (MS) to elucidate vaporization mechanisms in GFAAS was initially demonstrated by Styris and Kay (8) in the study of Rb. Styris and coworkers also employed MS in the investigations of V205,Ba, and Se (9-11). In all cases, tube furnaces were used for atomization and the m a s spectrometer was positioned directly above the sample introduction hole. Sturgeon et al. (7) used a mass spectrometer and a CRA-90 furnace in a study of P b vaporization. While the system employed by Sturgeon et al. did not have atomic absorption (AA) capabilities during the vaporization process, that of Styris and co-workers employed AA detection concurrently with ion monitoring via MS. This study examined the vaporization mechanisms of a Pb(NO& solution deposited on a graphite surface. Mass spectral and atomic absorbance data show the P b sample vaporized as PbO a t 550 K and as atomic P b a t 800 K. The effects of the addition of a 0.1% NH4H2POIsolution were also considered. The formation of a “Pb-phosphate” compound, bound to the surface, preceding P b release into the gas phase at 1150 K, will be shown. The implications of this information to vaporization mechanisms at 1 atm pressure in GFAAS will be discussed.

EXPERIMENTAL SECTION The atomic absorption/mass spectrometry (AA/MS) vacuum system is shown in Figure 1. The instrument consisted of a quadrupole mass analyzer (QMA), a UTI programmable peak selector (PPS),and an Apple 11+ microcomputer. The QMA used in this study (Uthe Technology International, Model 1OOC) was equipped with electron impact ionization and a 16-stageCu/BeO electron multiplier and had a mass range of 1-400 amu. The ionization sowce was operated at 70 eV and the electron multiplier at 3000 V. The resulting gain of the electron multiplier was approximately lo5 at m / e 28. The QMA was calibrated with trichlorotrifluoroethane. The PPS was used to control the $MA and to collect and store data. It has the capability of monitoring up to nine masses or analog signals, using a rapid sequential scanning mode of operation. In this study, variations of four individual masses and one analog signal were monitored sequentially at the rate of 8 ms per point producing a final time resolution of 40 ms per channel. When improved time resolution was required, the quadrupole was manually set on a specific mass and on an analog channel of the PPS was used to collect the data. The maximum time resolution in this mode was 1.5 ms. The 64K Apple 11+ microcomputer was interfaced to control all electronic operations of the system. Experimental parameters were entered and stored in the computer, which then controlled the various timing sequences including pumping down the chambers and controlling the time and temperature for the drying,

0003-2700/87/0359-0974$01.50/0Q 1987 American Chemical Society

ANALYTICAL CHEMISTRY, VOL. 59, NO. 7, APRIL 1, 1987

PRE CHAMBER

975

ANALYZING CHAMBER

Flgure 1. Atomic absorpthmlmass spectrometer vacuum system (AA optics not shown).

Figure 3. Atomizer assembly showing (a) electrical connectors, (b) graphite surface. (c) graphite electrodes. (d) high-tension springs, (e) oxygen-free, high conductivity copper body. and (f) black body cavity in the side of the graphite surface for temperature calibration.

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MFISS Flgure 2. 800 ng of Fb in excess NH,HpO, collected in me "scanning" mode. The major isotopes of Pb (206, 207, 208) are shown. Each scan represents 74 ms during the firing of the atomizer. A total time of 1.2 s is displayed charring, and atomization of the sample. The atomizer was heated with a computer-controlled digital power supply (12). In brief, an 8-hit number sent by the computer to the power supply unit determined the applied voltage delivered by the step-down transformer to the atomizer. With a single value on the output port of the computer, a nonlinear temperature ramp resulted. A program, recently developed in this laboratory, provided a linear temperature ramp. This program calculated the voltages required by the power supply using heat transfer properties of the system. The advantage of the linear ramp was its ability to provide good resolution of peaks a t low temperatures while permitting the attainment of high final temperatures. The computer also initiated the PPS to start collecting mass spectral data coincident with data acquisition by the 8-bit analog-bdigital converter in the Apple. The Apple collection routine, written in assembly language, collected and averaged four individual points to make one data point and was capable of a time resolution of 0.2 ms per data point. After a given period of time the computer stopped collecting data, disabled the power supply, and terminated data collection hy the PPS. The collected data were then transferred to the Apple and stored on a disk along with any data collected by the Apple itself. The data were then manipulated and plotted. In another collection mode, the Apple 11+ could also be used to obtain data from a mass spectral range. With the QMA operating in the scanning mode, the graphite surface was rapidly heated, while the MS detector output was digitized and saved in memory. In this scanning mode the mass spectrometer cyclically monitored a range of up to 50 masses for a given heating cycle. The data could be displayed in a three-dimensional plot as shown in Figure 2. These traces represent the vaporization of Ph. While the resolution of quadruples falls off at high masses, the three major isotopes of Pb (m/e 206,207, and 208) are clearly

resolved. In this figure, each sran took 74 ms, and a total recording time of 1.2 s is displayed. This scanning procedure was useful in the initial survey of a new sample and was helpful in isolating unknown or unexpected species which may have been desorbed from the surface. The vacuum chamber (Figure 1)consisted of a sample introduction chamber (prechamber) and an analyzing chamber separated hy a 4-in. gate valve. The prechamber was pumped with a 110 L/s turhomolecular pump (Balzars Corp., Model TPU 110). Besides the gate valve, which separated the analyzing chamber from the prechamber, there were two other valves in the prechamber. One valve allowed for introduction of aqueous samples with a 10-fiLHamilton syringe with a IO-cm Teflon needle extension. Another valve permitted venting or flushing of the prechamher with specific gases. The analyzing chamber, which contained the QMA, was pumped with a 750 L/s oil diffusion pump (National Research Corp., Model NHS4 No. 0161-2). A liquid nitrogen trap with baffles was located directly above the pump to minimize back-streaming of any pump oils and to assist in the removal of residual water vapor from the system. A butterfly valve separated the trap and pumping station from the analyzing chamber. Typical operating pressure in the analyzing chamber was approximately 5 x 1O-I torr, A hollow cathode lamp, operated in a de mode under manufacturers' recommended currents, was used as a light source for the vacuum AA measurements. A 0.3-m monochromator (GCA/McPherson Instruments, Model LU 700) was used to monitor the 283.3-nm line of Ph. The image transfer system employed an over-and-undermirror configurationwith onetoone imaging (13)with a 1-mm horizontal slit located in the tangential image plane. The optical path was focused immediately above the graphite surface. Translational movement of the atomizer between the prechamber and the analyzing chamber was accomplished by use of a polished, chrome-plated, stainless steel rod which pushed and pulled the atomizer on guide rails. The rod was sealed by two differentially pumped, precision made, spring-loaded, Teflon O-rings (Fluorocarbon, Los Alamitos, CA). A Delrin sleeve, encased in a hrass housing, provided support for the stainless steel rod. The atomizer assembly is shown in Figure 3. The main body of the assembly was constructed from oxygen-free, high-conductivity Cu. Two graphite electrodes were clamped in the Cu hlocks and held the graphite surface between them using pressure from springs. The base of the Cu atomizer was insulated from the top blocks by ceramic sleeves and spacers. Each hlock had independent electricalconnectors which mated with high-vacuum feedthroughs in both the sample introduction and the analyzing

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ANALYTICAL CHEMISTRY, VOL. 59, NO. 7, APRIL 1, 1987

chamber. The general design allowed for the use of various surface geometries ranging from flat plates to graphite furnaces. In this study vaporization from a flat graphite surface was employed. The graphite flats were machined from graphite (grade FE35; Schunk Carbon Technology) with dimensions of 1.5 X 0.76 X 0.17 cm. A 3 mm deep, 1 mm diameter hole was drilled in the edge of the flat for temperature calibration. All flats were purified and pyrolytically coated (Ringsdorff-Werke GmbH). A recording optical pyrometer, which was constructed in this laboratory, was located on the analyzing chamber and was positioned 45" relative to the normal of the atomizer surface. The image of the graphite flat was focused on a 1 mm diameter aperture placed directly in front of a phototransistor. A 5-mm light-limitingaperture was placed directly in front of the focusing lens to ensure linear response of the phototransistor in the temperature range of interest. The output voltage of this pyrometer was calibrated against static measurements made with a disappearing filament microoptical pyrometer (The Pyrometer Instrument Co., Model 95). A hole (fin Figure 3) was drilled in the graphite to approximate a black body cavity for temperature calibration. The hole had a radius-to-depth ratio of 6:l. Thus, with a surface emissivity between 0.6 and 1.0, the back wall of the cylindrical cavity had an effective emissivity between 0.985 and 1.0 (14). The transmission of the windows was also accounted for in the temperature determination. Solutions. ACS reagent grade NH4H2P04was used in all studies examining the effects of phosphate salts. NH,H2P04 solutions were prepared from a 1%stock solution. Pb solutions were made from a 1000 ppm stock solution which was prepared by dissolving solid Pb(N03)2in distilled, deionized water. Procedure. The experimental run consisted of several steps. First the surface was "cleaned" at a high temperature (>2500 K) for 3 s under a vacuum in the prechamber. Then the prechamber was filled with 1 atm N2gas and the atomizer allowed to cool for 5 min. This was necessary, due to the lack of water cooling, to mure consistent atomizer temperatures during sample deposition. The sample was then deposited on the flat, pyrolytic graphite surface. It was gently dried under 1 atm N2 by heating the atomizer to approximately 350 K. No char cycle was used in this study. The prechamber was then pumped for 2.5 min with a roughing pump and for another 10 min with the turbomolecular pump. At this time, the prechamber pressure was approximately lo4 torr and the gate valve, which separated the two chambers, was opened. The atomizer assembly was pushed from the prechamber to the analyzing chamber and heated. During atomization, the graphite surface was located 3 cm below the QMA ionization cage. After the atomization cycle the collected data were transferred from the PPS to the Apple and stored on a disk. The atomizer was pulled back into the prechamber, the gate valve closed, and the atomizer again cleaned. The entire cycle required approximately 25 min. Typical operating pressure for the mass spectrometer was 5 x lo-' torr. At this pressure the mean free path was approximately 50 m and the likelihood of gas phase interactions was minimal. This low pressure allowed for the observation of species as they were desorbed from the surface. The AA data were used, in part, to assign the Pb+ mass spectral signals to the release of the atomic metal at the surface or to fragmentation of Pb-containing molecules by the electron impact ionization.

RESULTS AND DISCUSSION Aqueous Lead Solutions. With the Pb+ signal monitored at mle 208, a limit of detection of 0.25 ng was achieved with this system. Because of the reduced residence time a t low pressures, the atomic absorption signal at this concentration was below detectability and higher concentrations (X1000) had to be used in order to coincidentally collect both the AA signal and that from the mass spectrometer. Except where noted in the text, general appearance times and species evolved from the surface were independent of the concentration employed for the solutions containing only dissolved Pb(NO& in water. Assignments of various ions were made to the peaks based on isotopic ratios. The coincident appearance temperature and identical peak shapes of various

90

a n

"

3 0

500

1000

1500

2000

Temperature [K)

Figure 4. Mass spectral signal for Pb+ ( m / e 208) for 400 ng of Pb as its nitrate salt. The first peak (a) is due to fragmentation of PbO in the ionizer while (b) is due to the release of atomic Pb from the

surface. Table I. Dominant Ion Peaks for Pb(N03)2n first peak

second peakb

cot KO*, coj C02' (C02) NO2+ (Nod

cot (C02, CO)

COZ' (C02)

Pb+ (Pb)

NO' (NOZ) 02' (02)

Pb' (PbOj PbO+ (PbO) Probable molecular sources are shown in parentheses. * Pb atomic absorption signal also observed at this time. species were taken to imply either a common origin at the surface or the existence of one peak being a fragment of the other as a consequence of electron impact ionization. The results from the deposition of 400 ng of P b (8 p L of 50 ppm Pb(N03)2solution) are shown in Figure 4. Although the amount is greater than that used in GFAAS, it should be noted that concentrations as low as 600 pg were used with similar results. In general, two distinct processes appeared to occur on the surface which resulted in the release of detectable species. A number of different masses were monitored during the course of this study and the dominant species that were observed at the lower and higher temperature peaks are listed in Table I. The data in Figure 4 represent monitoring of m/e 208 (Pb+) and were collected by using the PPS in a single channel mode yielding a resolution of 3 ms per point. The 900data points were treated by using a 75-point quadratic smooth (15, 16). The Pb+ signal appeared as two distinct peaks although the PbO peak appeared as a single peak coincident with the f i s t P b peak as shown in Figure 4. Similar peak shapes for the PbO+ and Pb+ signals a t the first peak suggest that this P b ion signal was a fragment of the PbO. Also, there was no detectable AA signal present during the first peak, but the second peak of Figure 5 (peak b) showed a distinct P b absorption signal. These data were in agreement with those observed by Sturgeon et al. (7). The second peak also appeared to have a shoulder which is noticeable in Figure 4. The exact nature of this shoulder is uncertain but may be due to PbO reduction a t two different active sites on the graphite surface. Coincident with the PbO evolution from the surface were signals from NO2 and 02.The approximate appearance temperature at which these species were observed was 550 K. This was in agreement with the general decomposition temperature of 540 to 800 K for Pb(N03)2(17) Pb(NOJ,(s)

-

PbO

+ 2N02 + ' 1 2 0 2

(1)

ANALYTICAL CHEMISTRY, VOL. 59, NO. 7, APRIL 1, 1987

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Temperature (K) Flgure 5. Data from approximately 2 pg of solid PbO placed on a graphite surface showing (a)Pb+ (mle 208) and (b) PbO+ (rn le 224). the sguares represent the AA signal which shows a maximum absorbance of ca.0.09. These raw data were all collected from a single firing of the atomizer and have not been smoothed. However, reaction 1 does not explain the appearance of PbO in the gas phase. When approximately 2 pg of solid PbO was placed on the surface, no early PbO+ peak was observed but instead only the peak that coincided in time with the second peak of Figure 4 was realized (Figure 5). This suggests that when the Pb(N03)z crystal decomposed to PbO, with the associated crystal rearrangement and generation of gaseous produds (i.e., NOz and Ob, some of the PbO was released from the surface (fitpeak, Figure 4). Coincident with these were signals corresponding to the evolution of CO and COz. These peaks were not present when P b was absent from the solution. The appearance of these gases may result from a reaction of some of the evolved oxidants (i.e., NOz and 0,) with the surface upon decomposition of the nitrate salts or, more likely, the result of partial binding of the Pb(N03)2to the graphite and the subsequent weakening of the carbon/surface bond when the Pb(N03)zrearranges to form PbO. Sabbatini and Tessari (18) showed difference between Pb(N03), powder and Pb(NO& in HN03 solutions deposited on a graphite surface. Using electron spectroscopy for chemical analysis (ESCA) they showed a shift to lower binding energies for the P b in nitric acid solutions deposited on the graphite. They were able to resolve two P b species on the surface whose ESCA signal was attenuated after sample charring between 673 and 703 K and were almost gone with a 823 K char. It is possible that these species represent a surface-bound Pb(N03)zas the major component [Pb-ox (A) mentioned in ref 181, and perhaps a PbO species or Pb(N03)z bound at a different site as a minor component [Pb-ox (B)], which would correlate with our results. Conversely, the HN03 and potentially different surface conditions may represent a sufficiently different system to discount any extensive comparison between our work and theirs. Holcombe and Droessler (19) postulated that the P b may be bound to the surface even before drying. They momentarily deposited a P b solution on the graphite and then withdrew over 90% of the solution from the surface with the deposition syringe. While less than 10% of the solution remained on the surface, 30-40% of the original peak area was observed upon atomization. They suggested the ion exchange of Pb+ with surface species resulting in a surface-bound Pb. The magnitude of the first peak in Figure 4 for m l e 208 (originating from PbO) relative to that of the second peak (originating from elemental Pb) was dependent on the concentration of the deposited sample. Figure 6 shows the ratio of the area of the later peak to the area of the early peak appearing at mle 208. It is important to note that both peaks increased with increasing concentration, but the higher tem-

0

20

40

60

80

100

Concentration (ppm) Flgure 6. Average ratio, for an 8-pL sample, of the area of the first to the second Pb peak shown in Figure 4 as a function of concentration. perature peak increased less rapidly. Qualitativelythis agreed with the data presented by Sturgeon et al. (7)who have shown a decrease in the PbO signal at reduced concentrations. The data in this figure suggested that the amount of PbO released may approach zero as the sample concentration approaches zero. This was consistent with the hypothesis that crystal rearrangement was responsible for the evolution of PbO. It was found that the age of the platform and the type of the platform used produced significantly different ratios of Pb0:Pb. For example, when a microboat (Allied Analytical Systems, catalog no. 44119) with a pyrolytic surface was used, the Pb0:Pb ratio was significantly smaller than that observed with the graphite flats prepared in our laboratory. This difference may reflect the size and number of Pb(N03)z crystals initially located on the surface immediately after the dry process. It can be speculated that increasing the number of surface imperfections and/or active sites may foster the formation of a larger number of small crystallites which may, in turn, yield better contact with the graphite and reduce the amount of PbO released during decomposition of the Pb(N03)2 crystallites. While strict quantitation of the data was difficult, an estimate could be made regarding the amount of PbO lost around 550 K. By comparison of the combined area of the early Pb+ and PbO+ signal to the peak area of the later Pb+ peak, it was estimated that 60% of the P b leaves the surface as PbO when 8 p L of a 20 ppm was deposited on the surface. Sturgeon et al. (7) estimated that 10% of the P b sample was lost. Again, these differences could be due to alterations in the surface conditions, drying conditions, etc. Additionally, those workers employed a graphite furnace in place of the flat graphite surface used in this study. As a consequence, collisions between the evolved gases and the walls were greatly increased and much of the PbO evolved may have stuck to the wall and not been detected by the mass spectrometer. It should be noted that the significant loss of PbO under vacuum conditions does not necessarily indicate that similar amounts would be lost under atmospheric pressure since a large number of secondary collisions between the PbO(g) and the surface could result in readsorption back on the graphite surface. Thus, PbO would not be lost by diffusion from the furnace. The coincidence between the second Pb+mass spectral peak in Figure 4 with the P b AA signal and the absence of any other mass spectral signal for a Pb-containing species eliminated the possibility that sublimation of PbO accounts for the release mechanism in GFAAS. Similarly, the coincident appearance of C 0 2 with the second Pb+ signal indicated a surface reduction of the PbO. PbO-graphite

-

Pb(g)

+ CO,COz(g) + C

(2)

978

*

ANALYTICAL CHEMISTRY, VOL. 59, NO. 7, APRIL 1, 1987

An estimate of the amount of COz evolved relative to the amount of P b evolved in this time period gave a COZ:Pbratio of approximately 2.2. The relative amounts of C 0 2 and P b were obtained by using the following equation: The peak ion current, I+, was corrected for mass dependence on the quadrupole transmission, T, the electron multiplier gain, G, and the ionization efficiency, e. The values for e, G, and T were obtained from ref 20. Because COz will fragment, a fraction of the COPwill be detected as CO+, Of, and C+ (20). The P r e l for all these species were then added together and for P b to obtain the COZ:Pbratio of 2.2. divided by the Prel The appearance of COz was supportive of the mechanism proposed in reaction 2. The dissociation of PbO(s) followed by the rapid oxidation of the surface by atomic oxygen was not likely since bulk PbO does not thermally decompose a t temperatures below 800 K (the PbO melting point is 1161 K) (21),the appearance temperature of Pb. While reaction 2 was considered the rate-limiting process for the release of P b into the gas phase, it is possible that short-lived intermediate species (e.g., a Pb-graphite bound species) are also formed. As mentioned earlier, Figure 5 shows data from approximately 2 yg of solid PbO powder placed on the graphite flat. A minute amount of PbO was adhered to a Hamilton syringe, containing 8 pL of water, by touching the syringe to a polyethylene surface with a thin layer of PbO powder. I t was estimated from the mass spectral signal that 2 yg of PbO was deposited. A single Pb+ peak appeared coincident with the peak shown previously in Figure 4 that was attributed to the evolution of free atomic P b from the surface. As before the concurrent appearance of CO and COz ions was also observed. I t should be noted that a PbO+ signal did appear coincident with the atomic P b signal. This may be due to the release of PbO from the large PbO crystal during the reduction of PbO at the surface and the evolution of P b and COP This was analogous to the evolution of PbO from the nitrate decomposition for the P b ( N 0 J Z samples. The presence of an absorbance signal, as well as the relative ratios of intensities of Pb+:PbO+, indicated that the Pb+ signal was not due exclusively to the fragmentation of PbO in the ion source but, rather, originated from atomic P b released from the surface. NH4HzP0, Addition. With the addition of 0.1% NH4H z P 0 4in a 100 ppm P b solution, mass scans from 200 to 400 amu revealed that atomic P b was the only Pb-containing species released from the surface. The Pb+ signal shown in Figure 7 appeared as a single peak that was shifted significantly later in time than the peak observed previously in the absence of NH,H2P04. Coincident with the mass spectral signal was the appearance of an atomic absorption signal, which lends further support to the release of atomic P b from the surface when the phosphate was present. COP,CO, POz, and PO were all observed in this process. While PO and POz may be the actual species desorbed from the surface, they may also be fragments of larger oxyphosphorus-containingspecies which were detected during mass scanning. The COz+ and CO+ appeared coincident with the P b signal and the ratios of the ion intensities of these species to that of Pb+ indicated a common mechanism for the release of both species. This also supported a reaction between the graphite and the Pbcontaining species located on the surface immediately preceding desorption. There were a large number of peaks throughout the spectral region resulting from the phosphate addition. However, POz+ and PO+ were the dominant phosphorus-containing signals in the mass spectrum with no other easily assignable peaks present. The dotted line, in Figure 7 , corresponds to POz+ and shows two distinct mechanisms for the evolution of the phosphorus oxide species. The intensity of the first peak

90 7

Temperature (K)

Flgure 7. Mass spectral signal for Pb+ ( m / e = 208) for 400 ng of Pb in 4000 ng of NH,H,PO,. The solid line corresponds to the release of atomic Pb from the surface; the dotted line is PO,' ( m l e 63) and is displayed as one-tenth its intensity relative the Pb' signal. The first PO2+ peak is from the excess NH,H2P0, while the second is due to the decomposition of the Pb-phosphate species.

changed drastically depending on the ash temperature and the duration of this heating cycle while the second peak remained constant. Similarly, this low-temperature peak was present with or without P b in the sample. It is postulated that this initial peak was due to a thermal decomposition of the excess phosphate on the surface which was not bound to Pb. In contrast, the second peak appears coincident with the Pb+ signal and was present only with P b in the sample. The intensity ratio of either PO+ or POz+to Pb+ remains constant throughout the time period during which the Pb+ signal was observed. This indicated the existence of a P b oxyphosphorus compound on the surface which thermally decomposed a t these elevated temperatures to yield elemental P b and various oxyphosphorus compound with the dominant species being PO and POz. In a similar manner the shape of the CO and COzpeaks which were again coincident with the release of Pb, PO, and POz indicated that the graphite surface was also involved in the bonding arrangement of the P b phosphate surface species. It should be noted that neither CO nor COz were evolved a t this temperature in the absence of either P b or phosphate in the sample. The proposed mechanism for the decomposition of the surface bound species is shown in eq 3. Pb(P,Oy),-graphite

PbO-graphite

I?,

+

PO, P 0 2 ( g )

(3) Pb(g)

+

CO,CO,

It is suggested that the rate-limiting step is given by kl and that the surface reduction of PbO ( k z ) ,as shown previously, occurs at a much lower temperature and hence will not likely be the rate-limiting step at this more elevated temperature. Therefore, the surface bound PbO represents a short-lived intermediate in this reaction mechanism. The existence of this intermediate was supported by the Pb+:COZ+ratio which was relatively constant and independent of the amount of phosphate initially present in the sample. It coincides with the same ratio observed in the absence of any phosphate (except the unmodified data were taken a t an earlier temperature). The most probable stoichiometry for the surface bound Pb-phosphate species was lead pyrophosphate (PbzPzO7), which was the simplest condensed phase phosphate that could be expected (22). This was partially substantiated by the appearance temperature of the P b a t 1150 K. Duval (23) noted that bulk PbzPZO7was stable to 1208 K, although it was

ANALYTICAL CHEMISTRY, VOL. 59, NO. 7, APRIL 1, 1987 979

of minimizing potential impurities introduced by concentrated phosphate modifiers.

CONCLUSION Mass spectral data indicated that Pb, deposited as a nitrate

E. 0 0

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, 400

-

n 1

800 T I H I IMYCI

I206

1600

Figure 8. Conventional Pb absorbance signal for atomization at 1 atm N2 in a CRA 90 furnace from a Pb(NO& solution (dotted line) and Pb(N03), in an excess of NH,H,PO, (solid line).

not clear in this article whether decomposition occurs at this temperature or at a substantially higher temperature. X-ray photoelectron spectroscopy (XPS) gave a ratio of the P b to P of approximately 1. Extensions to Electrothermal Atomization. In conventional GFAAS, the absorbance signal for P b is often broad and appears at low temperatures. When NH4HZPO4is added, a single peak that is shifted to a higher temperature and has a greater peak height is observed (Figure 8). The data interpretation presented in previous sections suggests the formation, during the dry, of Pb(N0JZ from a simple aqueous solution without any excess nitrate. The thermal decomposition of this species on the surface to PbO is accompanied by the release of NOz and Oz gases and some evolution of PbO. The relative amount of PbO released as a consequence of the decomposition is dependent on the concentration of P b in the original sample and possibly the character of the graphite surface on which the sample is located. Under atmospheric pressure it is probable that the evolved PbO is readsorbed on the surface and therefore not lost through diffusion. One can speculate that this readsorbed PbO may account for the small shoulder that is commonly observed on the absorbance peaks seen in a matrix-free sample (24,25). The shoulder may also be due to PbO released from two different sites on the graphite. This surface bound PbO then undergoes decomposition with the coincident evolution of free atomic Pb, CO, and COP It is this process which limits the rate of evolution of atomic Pb. It is not clear at this time whether the activation energy for release of Pb would correspond to equilibrium values for the enthalpy of reduction of bulk PbO by graphite. In the presence of phosphate, a stable lead phosphate species (e.g., Pb2Pz0,) is bound to the graphite surface. Upon being heated, this surface complex thermally decomposes to yield oxyphosphorus compounds and a short-lived PbO-graphite species which rapidly decomposes to evolve elemental Pb(g). These results support and expand upon the furnace studies done by Czobic and Matousek (2). Of particular interest in their study was the shift that occurs when the P b and phosphate were deposited at opposite ends of the furnace. The shift was nearly equal to the shift produced when the two solutions were mixed. Their conclusions of a heterogeneous gas-solid reaction was not unreasonable as this study has shown excess phosphates to decompose before the appearance of the earliest desorbed P b peak. The apparent efficiency of this heterogeneous reaction suggests the potential analytical utility of gas-phase matrix modification with a "phosphate" or some other oxyphosphorus-containing gas as opposed to solution matrix modification. This would have the promise

salt, existed on the surface as Pb(NO& which decomposed at low temperatures to form a surface-bound PbO with the subsequent evolution of NOz and Oz. Some PbO was released to the gas phase during this decomposition process but may readsorb on to the graphite surface in atmospheric pressure conditions. The surface-bound PbO was then reduced in a rate-limiting step to elemental P b with coincident evolution of CO and COz. In conventional GFAAS, the PbO initially evolved from the nitrate decomposition that is readsorbed onto the surface may have a slightly different energy for release of the Pb(g) and could account for the shoulders observed on the P b absorbance signal. Additionally, depending on the partial pressure of oxygen in the furnace at the time that the initial PbO is released to the gas phase, thermal decomposition in the gas phase may occur and again may be responsible for the shoulders observed on the peaks. The amount of PbO released during this decomposition appears to be a function of the mass amount of Pb(N03)zin the original sample as well as the nature of the surface from which desorption occurs. It should be noted that Sedykh and Belyaev (26) saw Pbz and PbO in a graphite furnace using molecular absorbance and 2 pg of material. The appearance of Pbz is not consistent with our results and is not expected from thermodynamic calculations. The PbO may be due to large releases from the Pb(NO& decomposition or for Pb reacting with gaseous oxygen. In either case, this work merits further investigations. With the addition of phosphate to the sample a surfacebound P b phosphate species forms with a Pbphosphorus ratio of approximately 1. It is possible that the surface-bound Pb-phosphate is in the form of PbzPZO7. The stabilized species decomposes at 1150 K and releases only the atomic P b in addition to CO, COB,and oxyphosphorus compounds. A short-lived PbO-graphite species is postulated to form during this decomposition of the lead phosphate. ACKNOWLEDGMENT The authors wish to acknowledge the efforts of Gary D. Rayson in the early design of the MS system and George F. Christopher for his contribution to the scanning and linear temperature programs. Registry No. Pb, 7439-92-1;Pb(N03)2,10099-74-8;NH4HzP04, 7722-76-1; PbO, 1317-36-8; PbZP2O7, 13453-66-2; graphite, 778242-5. LITERATURE CITED (1) Matousek, J. D.; Brodie, K. G. Anal. Chem. 1973, 4 5 , 1606. (2) Czobik, E. J.; Matousek, J. P. Talanta 1977, 2 4 . 573. (3) Ediger, R. D.; Peterson, G. E.; Kerber, J. D. At. Absorpt. Newsl. 1974, 13, 61. (4) Ediger, R. D.At. Absorpt. Newsl. 1975, 74, 127. (5) Hinderberger, E. J.; Kaiser, M. L.; Koirtyohann, S. R. At. Spectrosc. 1981. 211). 1.- - - - . - % - , I

(6) Genc. 0.;Akman, S.; Ozdurai, A. R.; Ates, S.; Balkis, T. Spectrochim. Acta, Part 8 1981, 3 6 8 , 163. (7) Sturgeon, R. E.; Mitchell, D. F.; Berman, S. S. Anal. Chem. 1983, 55, 1059. (8) Styris, D. L.; Kaye, J. H. Spectrochim. Acta, Part 8 1981, 3 6 8 . 41. (9) Styris, D. L.; Kaye, J. H. Anal. Chem. 1982, 5 4 , 864. (IO) Styris, D. L. Anal. Chem. 1984, 5 6 , 1070. (11) Styris, D. L. Freseneius' 2.Anal. Chem. 1986, 323, 710. (12) Bass, D. A.; Eaton, D. K.; Holcombe, J. A. Anal. Chem. 1986, 5 8 , 1900. (13) Salmon, S . G.; Holcombe, J. A. Anal. Chem. 1978, 5 0 , 1714. (14) Sparrow, E. M.;Albers, L. U.; Eckert, E. R. G. J . Heat Transfer 1962, C84, 73. (15) Savitzky, A.; Goulet. M. J. E. Anal. Chem. 1984, 3 6 , 1627. (16) Proctor, A.; Sherwood, P. M. A. Anal. Chem. 1980, 5 2 , 2315. (17) Stern, K. H. J . Phys. Chem. Ref. Data 1972, 1 , 758. (18) Sabbatini, L.; Tessari, G. Ann. Chim. (Rome) 1984, 74, 779. (19) Holcombe, J. A.; Droessler, M. S. Fresenius' 2.Anal. Chem. 1988, 3 2 3 , 689. ( 2 0 ) Model lOOC Operating and Service Manual: Uths Technology International: Sunnyvale, CA.

980

Anal. Chern. 1987, 59,980-984

(21) Handbook of Chemistry and Physics, 53rd ed.; CRC Press: Cleveland, OH, 1972. (22) Corbridge, D. E. C. Phosphorus; Elsevier: New York, 1980; p 118. (23) Duval, C. Anal. Chim. Acta 1950,4 . 159. (24) McLaren, J. W.; Wheeler, R. C. Ana/yst (London) 1977, 702. 542. (25) Salmon, S. G.;Davis, R . H., Jr.; Holcombe, J. A. Anal. Chem. 1981, 5 3 , 324.

(26) Sedykh, E. M.; Belyaev, Yu. I . Zh. Anal. Khim. 1979, 34(10). 1984.

RECEIVED for review September 2,1986. Accepted December 1986. support for this project was provided by National Science Foundation Grant CHE-8409819.

Differentiation of Isotopically Labeled Nucleotides Using Fast Atom Bombardment Tandem Mass Spectrometry Larry M. Mallis, Frank M. Raushel, and David H. Russell*

Department

of

Chemistry, Texas A&M University, College Station, Texas 77843

The posittonal isotope exchange reactlon has proven to be a valuable tool In elucldatlng mechanistic pathways for enzyme-catalyzed reactlons involving phosphoryl transfer. Several examples of the analysts of phosphorylated nucleotkles by fast atom bombardment ionlzatlon mass spectrometry have been reported; however, the small number and low relative abundance of structurally slgnlflcant fragment Ions make structure elucidation dlfflcult. Recently, we reported that the dlssociatlon reactions of organo-alkall-metal Ion complexes are Influenced by the alkalhetal Mndlng &e, and this effect can enhance the relathre abundance of structurally signlficant fragment Ions In the colllslon-Induced dissociation spectrum. I n the present studles the [M Na]' Ions of [p'802,~y-"0,y-"0,]~rldlnetrlphosphate formed by fast atom bombardment ionlzatlon and analyzed by using tandem mass spectrometry are examined to determine the position of "0 atoms in the molecule.

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The positional isotope exchange (PIX) technique has proven to be a valuable tool in elucidating mechanistic pathways for enzyme-catalyzed reactions ( I ) . The PIX technique has been used with reactions involving phosphoryl transfer in nucleotides followed by analysis with 31PNMR or derivitization (to enhance volatility) followed by electron impact ionization mass spectrometry. Several studies on the analysis and structure elucidation of nucleotides and nucleosides using mass spectrometry have been reported. For example, field desorption (FD) (2, 3),desorption chemical ionization (DCI) (4),thermospray ( 5 ) ,and liquid ionization (6) have been successfully used for the analysis of nonderivatized nucleosides and nucleotides. More recently impressive results on polar, nonvolatile organic molecules have been demonstrated using a group of particle-induced desorption ionization techniques, e.g., secondary ion mass spectrometry (SIMS) (7,8),z52Cfplasma desorption mass spectrometry (PDMS) ( S I I ) , laser desorption (LD) ( I Z ) , and fast atom bombardment mass spectrometry (FAB-MS) (13-24). Although several studies concerning the analysis and structure elucidation of phosphorylated nucleotides using FAB ionization have been reported, the number and abundance of structurally significant fragment ions are low in the FAB mass spectrum. For this reason, several workers have proposed the use of tandem mass spectrometry (TMS) in combination with FAB ionization for structural characterization of polar organic molecules (25-29). Owing to the large number of reaction channels available to the collisionally activated ion,

the abundance of structurally significant fragment ions in a FAB-TMS spectrum is low (25). One factor to consider in the case of FAB ionization of nucleoties is the greater sensitivity for the negative ion spectrum. This is undoubtedly a result of the extent of dissociation of the acidic phosphate groups in the liquid matrix prior to particle bombardment (30). The lowest energy dissociation reaction available to the collisionally activated ion [M - HI- is electron detachment; consequently, it would be preferable to analyze the nucleotide by positive ion FAB ionization. Conversely, the phosphate groups of the nucleotide have relatively high alkali metal ion affinities and even trace impurities of sodium give rise to abundant [M + Na]+ ions and only weak [M H]+ ions. Molecules such as peptides, sugars, nucleotides, etc. contain highly polar functional groups which have different proton and alkali metal ion affinities. It follows, therefore, that the binding sites of protons and alkali metal ions to the organic molecule may differ. In the event that protonation and cationization (via alkali metal ions) occur at different sites in the organic molecule, the types of fragment ions obtained and the relative abundances of those ions may differ substantially for the [M + H]+and [M + Na]+ ions. Recently, we showed that the collision-induced dissociation reactions of organoalkali-metal ion complexes of peptides are influenced by the alkali metal ion binding site (31). In this study it was also demonstrated that the relative abundance of structurally significant fragment ions was enhanced in the FAB-TMS spectrum of [M + Na]+ ions (31). In the present study, the dissociation reactions of the [M Na]+ ions of nucleotides to determine the position of oxygen-18 (ls0)atoms will be examined. First, the proposed mechanism for the PIX reaction will be described with particular reference to exchange of l80into the ap bridging triphosphate (UTP). position of [p-1802,py-180,y-1803]uridine Second, the position of the l80atoms will be determined by analyzing the FAB-TMS spectrum for the [M + Na]+ ions of [~-1802,py-1s0,y-1803]UTP before and after the PIX reaction is performed.

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EXPERIMENTAL SECTION The labeled potassium dihydrogen phosphate (KHZPlsO4)used in the synthesis of [@-1802,/3y-180,y-1803]uridine triphosphate (UTP) was prepared by following the procedure of Risely and Van Etten (32). All other chemicals necessary for the synthesis and positional isotope exchange reaction of [/3-'802,/3y-180,y1E03]UTPwere purchased from Sigma Chemical Co. Dithiothreitol (no. 15,040-0)and dithioerythritol (no. 16,176-4)used as the fast atom bombardment matrix were purchased from Aldrich Chemical Co.

@ 1987I American Chemical Society 0003-2700/87/0359-0980$01.50/0