Anal. Chem. 1988, 60,2338-2346
2338
ever, is that because of its small size, only a very small fraction of the entire eluate is sampled and thus the microvoltammetric electrode is much less sensitive than conventional bulk detectors. Experiments designed to remove this disadvantage are currently being pursued.
LITERATURE CITED (1) Knecht, L. A.; Guthrle, E. J.; Jorgenson, J. W. Anal. Chem. 1984, 56,
479-482. (2) White. J. G.;Jorgenson, J. W. Anal. Chem. 1986, 58, 2992-2995. (3) Knox, J. H.; Laird, G. R.; Raven, P. A. J . Chromatog. 1976, 122, 129-145. (4)Eon, C. H. J . Chromatogr. 1978, 149, 29-42. (5) Krlstensen, E. W.; Wilson, R. L.; Wightman, R. M. Anal. Chem. 1988, 58,986-988. (6) Kucera, E. J . Chromatogr. 1965, 19, 237-248. (7) Sternberg, J. C. I n Advances ln Chromatography; GkMings. J. C., Keller, R. A., Eds.; Dekker: New York, 1966: Vol. 2, pp 205-270.
(8) Grubner, 0. I n Advances in Chromatography; Giddlngs, J. C., Keller, R. A., Eds.; Dekker: New York, 1968;Vol. 6,pp 173-209. (9) Grushka, E.; Myers, M. N.; Schemer, P. D.; W i n g s , J. C. Anal. Chem. 1969, 4 1 , 889-892. (IO) Kirkland, J. J.; Yau, W. W.; Stoklosa, H. J.; Dilks, C. H., Jr. J . Chromatogr. Sci. 1977, 15, 303-316. (11) Foley, J. P.; Dorsey, J. G. Anal. Chem. 1983, 55, 730-737. (12)Dayton, M. A.; Brown, J. C.; Stutts. K. J.; Wlghtman, R. M. Anal. Chem. 1980. 5 2 , 946-950. (13) Wlghtman, R. M.;Wlpf. D. 0. I n Electroenalyticel Chemkhy; Bard, A. J., Ed.; Dekker: New York, 1988;Vol. 16. (14) Knox, J. H. J . Chromatogr. Sci. 1980, 18, 453-472. (15)Caudill, W. L.; Howell, J. 0.;Wlghtman. R. M. Anal. Chem. 1982, 5 4 ,
2532-2535.
RECEIVED for review June 14,1988. Accepted August 1,1988. This research was supported by the National Institutes of Health (R01-NS-15841).
Factors Affecting the Quantitation of Organic Compounds in Laser Mass Spectrometry Zbigniew A. Wilk, Somayajula Kasi Viswanadham, Andrew G . Sharkey, and David M. Hercules* Department of Chemistry, University of Pittsburgh, Pittsburgh, Pennsylvania 15260
Factors aff ectlng the reproduclbilHy of organic laser mass spectrometry have been studied. Laser focus, laser power denslty, sample preparatlon method, and Chemical effects contrlbute significantly to the variabtlity of the mass spectra. Defocuslngthe laser enhances production of structurally slgniflcant organlc ions. The yield of organic molecular Ions Is a nonflnear function of power density. The use of a polymer matrlx (5 % nitrocellulose) for dissolving an analyte(s) was determlned to be the optimum method of sample preparatlon. Using such a polymer matrlx, along wlth Internal standards, permitted relatlve standard deviations of f15% to be obtained.
Mass spectrometry of solid materials has, in recent years, been performed by using ion and atom beams as sputtering/ionization sources, providing an alternative approach for the analysis of solid samples (1). The two major advantages of a laser as a source for mass spectrometry are that it can volatilize/ionize solid samples directly and the beam can be focused to a micrometer-size spot permitting the analysis of microvolumes. Early work with the laser as an ionization source for mass spectrometry dealt with the analysis of inorganics, metals, and semiconductors. These early experiments were performed by using varied instrumental designs (e.g., different laser types and mass analyzers). An excellent review on the progress of laser mass spectrometry to 1979 is given by Conzemius et al. (2). In 1982 the laser was reviewed as an ionization source for the mass spectrometric analysis of involatile and thermally unstable organic molecules ( 3 , 4 ) .Additionally, laser ionization has seen application in diverse areas ranging from mycobacteria fingerprinting (5) to the biogenesis of coal macerals (6). Laser mass spectrometry (LMS) has been used primarily as a qualitative technique. Quantitative analysis is not a trivial problem but has been demonstrated. Schroeder deposited
standard materials on samples via a mask so that a regular pattern of coated and uncoated areas was formed on the surface. This was done to introduce internal standards (7). He also demonstrated that isotopic labeling can be an effective method for element quantitation. The local thermodynamic equilibrium (LTE) model has been used to rationalize relative peak intensities for various elements observed in the LMS of NBS glass fiber standards (8). It was suggested that the LTE model can be used as a good first approximation for the quantitative analysis of particles. Verbueken et al. studied ion exchange resins doped with successively increasing concentrations of metal complexes (9). A linear relationship between the concentrationof the metal and the metal ion peak intensity was observed, clearly demonstrating that laser mass spectrometry is capable of quantifying elements. Quantificationof a benzalkonium chloride mixture by LMS was performed; the results compared favorably with those obtained by high-performance liquid chromatography (10). Mattern et al. have applied LMS to quantify oligomer distributions for polytethyleneglycol) and poly(propy1ene glycol) (11);results compared favorably with those from other techniques. Similarly, it was possible to measure the percentage of backward addition in poly(viny1idine) fluoride; results correlated well with NMR measurements (12). The present paper addresses some factors that affect the quantitation of organics obtained by using a laser microprobe mass spectrometer (such as the LAMMA-lOOO),which operates in the reflection mode. In this mode the laser beam is focused onto the sample surface at an angle of 45" from the normal. Ions are extracted normal to the sample surface. Use of the reflection mode permits routine analysis of bulk samples; microvolume analysis is possible (13). The factors to be discussed are sample preparation, laser energy, laser focus, and chemical effects. Other factors that are important for quantitation but are not considered here include the stability of the laser, nature of the laser-solid interaction, the rate of analog-to-digital (A/D) conversion of the signal, and detector efficiency. These latter factors were
0003-2700/86/0360-2338$01.50/00 1986 American Chemical Society
ANALYTICAL CHEMISTRY, VOL. 60. NO. 21. NOVEMBER 1. 1988
2339
CONSTANT LASER ENERGV = 7.34 m i c r o - J o u l c r
0
200
IS0
100
50
OISTANCE BEHIND FOCIL P O I N f ( 1 n MICRONS)
3 . 3 3 I 108
1.19 X IO8
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x
108
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Scanning ekchon microscope pictures of ihe crator formed on bombarding polished brass sample. Field of view is 44 pm X 38 pm. w"e w ' sitioned at Um focal mint of the laser. Each successive crater lo the r5M represents mwement of the sample 50 hm behind the focal poini. Flgure 1.
The crater on me lefl was made wim ~
~~
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kept constant for the present work. Sample preparation, laser energy and focus, and chemical effects represent parameters that affect reproducibility and, therefore, are of prime importance in the quantitation of elements as well as organic molecules. The chemical effects to be described are those that affect molecular ion yields of organic compounds. EXPERIMENTAL SECTION Laser mass spectra were obtained with a LAMMA-1000laser microprobe mass analyzer. The basic instrument has been described elsewhere (14). The ionization source consists of a Qswitched (15 ns), frequency quadrupled (A = 265 nm) NdYAG laser in the 45'/90° configuration. The laser intensity can be varied continuously by a pair of twisted polmizen. The diameter of the laser beam at the focal point is typically 5 pm. The ions are accelerated (4 kV) into a time-of-flight (TOF) mass analyzer (1.8 m) which is equipped with a second-order energy focusing ion reflector to compensate for the initial spread of ion kinetic energies. The resolution of the instrument (MIAMIis approximately 800 at m / z 350. The ions produced hy laser ionization are postaccelerated (7 kV) and detected at a Cu-Be secondary electron multiplier (Thome-EM19643/4A). The signal is preamplified (Lecroy 100 BTB or 101 ATB) in up to two stages providing l X , lox, or l00X amplification. The preamplified signal is fed to two independent transient recorders operated in parallel. The available transient recorders consist of two Biomation 8100 transient recorders (100 MHz, 8 bit, 2 kbytes) and a CAMAC housed modular system consisting of two Lecroy TR 8828 B/C transient digitizers (&bit, 200 MHz) each with two Lecroy MM 8103A memory modules (31 kbytes/module), a modified (computer addressable) Lecroy 6102 two-channelamplifier/attenuator, a modified (two-channel,autodisplay, autorearm) Lecroy CD 8828 B control and display module, and a Lecroy 8901 GPIB/CAMAC interface. The digitized data are transferred and processed by a Hewlett-Packard 1000-Eseries minicomputer. All compounds studied were purchased from commercial sources (Aldrich Chemical Co., Milwaukee, WI) and were used without further purification. The sample plate was mounted on an x,y,z micromanipulator having a maximum scanning range of 70 mm X 50 mm X 50 mm, respectively. The sample can he viewed by a light microscope at a magnificationof 250X, and the area of interest for analysis can be positioned by using the sample manipulators Nitrocellulose solutions were prepared by using a commercially available 10% nitroeelldosesolution in amyl acetate (Polysciences, Inc., Washington, PA). The 10% nitrocellulose was diluted to 5% with methanol. Analytes were weighed out as solids and deposited directly into the 5 % nitrocellulose/methanol solution. The solutions were mixed thoroughly to ensure a complete and homogeneous distrihution. The analyte/nitrocellulose solution was deposited on brass as a small drop which was dispersed by a capillary tube into a large area (thin film). The solution was
allowed to dry for approximately 2 min and then inserted into the vacuum system. The thermospray apparatus used in this work was constructed locally. The apparatus consists of a Du Pont Instruments highpressure liquid chromatographic pump interfaced to a stainless steel microbore HPLC column having the dimensions 0.0625 in. 0.d. x 0.007 in. i.d. The microbore column was silver soldered into a copper block that contained two heating cartridges and a thermocouple (Chromel-Alumel). The heating cartridges and the thermocouple were connected to a temperature controller used to monitor and change the temperature of the copper block. With methanol as the solvent, a temperature of 145 'C and a flow rate of 3 mL/min was used. Under these wnditions a dry aerosol was produced that did not create any visible solvent droplets on the substrate. The target substrate used for deposition was a highly polished brass plate positioned approximately 10 cm from the exit end of the microbore column. The time used for deposition was approximately5 min. The concentration of each analyte was 1.0 x lo-' M. RESULTS AND DISCUSSION Defocusing. One of the major factors affecting the reproducibility and quantitative capability of laser mass spectrometry is the focus of the laser beam. Mass spectra obtained by using laser ionization differ markedly depending on how the laser is focused on the sample. As a sample is moved away from the focal point of the laser image plane, the spectra change, both with regard to ion intensities and the appearance of structurally important ions. T o study the qualitative effects of laser focus a polished brass plate was irradiated by the laser beam. Polished brass has a highly uniform surface that allows visual detection of laser damage. It has high reflectivity making the damaged areas clearly visible; thus, it is a matrix well-suited for photography and scanning electron microscopy. Irradiation of the polished brass was performed under several different laser focus conditions. Initially the brass sample was placed at the focal point of the beam. Next, the sample was moved 50 pm behind the focal point. This last step was repeated several times, each time moving the sample a n additional 50 pm behind the focal point. Figure 1 shows a series of scanning electron micrographs taken of the craters formed by the laser under different focus conditions. This figure demonstrates that as the sample is moved behind the focal point, the laser beam begins to diverge and interacts with a larger sample area. It is also seen that as the sample is moved further behind the plane of laser focus, there is a point at which no visual damage of the sample is apparent. As the sample is moved behind the focal point of the laser, the beam diverges and becomes effectively "defocused". Divergence of the beam allows the energy of the laser pulse
2340
ANALYTICAL CHEMISTRY, VOL. 60,NO. 21, NOVEMBER 1, 1988 RETlNOlC A C I D DEFOCUSED E=2.12rJ POC. I O N
100
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1
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100
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200
250
300
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Flgwe 2. Retinoic acid spectra taken at different hser energies. Each spectrum is an average of 20 spectra. Spectra were taken under focused conddons; beam diameter was approximately 5 Km. The ions in the spectra are due mainly to carbon clusters. The power denstties used are as follows: (A) 1.34X 10' W/cm2,(B) 2.23 X 10' W/cm2, (C) 5.36 X 10' W/cm2.
to be deposited over a larger sample area. The power density (watts per square centimeter) therefore decreases, and cratering effects on the sample are reduced. When the sample is moved far enough away from the laser focal plane, the power density on the sample is not high enough to damage the sample. It is at this point that no laser ablation of the sample is observed. Mass spectra can still be obtained even though the sample is not visibly damaged. Defocusing of the beam therefore affects sampling depth. As the laser beam impinging on the sample is made larger, the power density decreases, and the analysis depth decreases. Two distinct regions can be observed in the crater formed by a focused laser pulse. The scanning electron micrographs in Figure 1clearly distinguish between these two regions. The first region is a t the very center of the crater where severe damage is apparent. The second region lies at the outer fringes of the crater and appears to have suffered less damage than the center region. The existence of these two regions can be explained by considering the spatial distribution of energy in the laser beam to be Gaussian. It is probable that there are several different modes of ionization occurring during a single laser pulse. These modes may involve a combination of thermal, photo, and field ionization (15) in varying proportions at different locations in the sample crater. The nonuniformity of the laser-induced damage shown in Figure 1 can be explained by considering both the energy profile of the laser pulse and the geometry of the laser-solid interaction. The energy distribution as a function of position within the beam is approximately triangular. When the sample is moved behind the focal point ("defocusing"), the spatial energy distribution impinging on the sample may become skewed. The region of the sample being closest to the laser focal point will be defocused to a lesser degree than the region that is farthest from the focal point. The qualitative description accounts for the high intensity crater formed in
Spectrum of retinoic acid taken under defocused mndltions. Spectrum is an average of 20 individual spectra. Molecular b n at mlz 300 is clearly visible. Laser energy Is 2.12 pJ. Beam diameter is approximately 10 pm. Flgure 3.
the upper region of the defocused damage areas shown in Figure 1. To illustrate the effect of laser focus on the spectra of organic compounds, we selected retinoic acid as an example. The LMS of retinoic acid has been studied previously and did not show an (M H)+quasi-molecular ion at m / z 301 or a molecular ion (M+') at m / z 300 (16). Figure 2 shows three positive ion spectra of retinoic acid taken at different laser powers with the laser beam focused to minimum diameter. This corresponds to the focusing conditions used in Figure 1 for the "0-focal point". The threshold laser energy for retinoic acid was approximately 0.06 pJ; only low-intensity, low m / z carbon clusters were visible. As the laser energy was increased, higher mass carbon clusters were produced. Energies greater than 1.3 pJ produced spectra that were the same as those taken using 1.3 pJ but with an increase in the intensities of all carbon cluster ions. No mass spectra were collected above 2.0 pJ due to saturation of the secondary electron multiplier. It is clear from Figure 2 that under focused conditions no molecular or quasi-molecular ion (QMI) was observed at any laser power density, even at threshold. Mass spectra also were obtained for retinoic acid under defocused conditions. The average spectrum for the defocused LMS of retinoic acid is shown in Figure 3. The molecular ion (M+') at m / z 300 is clearly visible and has an intensity of approximately50% of the base peak. The peak at mlz 285 corresponds to loss of a methyl group from the molecular ion. Observation of a molecular ion by defocusing contrasts with the results obtained under focused conditions where no molecular ion could be observed. Another major difference between the spectra collected under focused and defocused conditions is in the total number of ions produced. The defocused spectra show a smaller total ion current than the focused spectra. The ions observed, although fewer in number, are structurally significant; under focused conditions, the ions show little structural significance. Such a dramatic difference in the spectra obtained on defocusing is typical of most organics studied, with the exception of high molecular weight polymers that require high threshold laser energies to produce spectra. There are both advantages and disadvantages to operating a laser mass spectrometer in the "defocused" mode. An advantage of the defocused mode is that structurally significant ions are obtained more frequently. There appear to be two factors that contribute to this effect. First, defocusing permits the use of very low power densities, thereby producing "softer" ionization conditions, avoiding severe fragmentation. Second, a larger area is sampled in the defocused mode. A larger
+
ANALYTICAL CHEMISTRY, VOL. 60, NO. 21, NOVEMBER 1, 1988 LASER INTENSITY DISTRIBUTION
X-
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DEFOCUSED
THRESHOLD LEVEL
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-
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-
Figure 4. Schematlc diagram of (A) the spatial power density distribution of the laser beam and (B) the spatlal intensity distribution for focused and defocused modes.
number of structurally significant ions will be produced in the larger analysis area; detection, therefore, is more probable. On a practical level, defocusing can be used whenever molecular ion information is sought. The differences observed between the focused and defow e d modes can be described by considering the spatial power density distribution of the laser beam over the sample area. Although single values for laser power density are quoted in LMS,a more detailed analysis reveals that there is a distribution of power densities over the irradiated area of the sample. The laser power density distribution can be modeled by using the following assumptions: (1) the intensity distribution of a laser pulse over the beam diameter is triangular, as shown in Figure 4A, and (2) the shape of the laser beam at the sample surface is circular. The exact situation is more complex in that the intensity distribution more closely resembles a Gaussian (TE& is used) and the beam shape more closely resembles an ellipse (45O laser-solid geometry is used). Calculation of the total power density of a laser pulse entails integrating the power density over the analysis area and over all angles in the following manner:
where x is the diameter of the laser beam and 6 is the angle. With the assumptions stated above, the situation can be simplified whereby the power density distribution on the sample can be represented as the volume of a cone. Assuming a conical power density distribution for the laser beam, the equation for calculating the total power of a laser pulse can be expressed as follows: where PD, is the maximum power density and r is the radius of a circular beam irradiating a sample. From the retinoic acid spectra (Figures 2 and 3) dramatic differences in the mass spectra were observed for the focused and defocused modes. At a laser energy of 1.5 J, molecular ions were observed only in the defocused mode, even though the total power (energy) was identical (100 W) for both modes of operation. The presence of molecular ions_in the defocused mode can be explained by calculating the maximum power density in both modes by using eq 2. A maximum power density of 1.5 X l@ W/cmz is calculated for the focused mode (5 pm beam diameter) and 3.8 X 108W/cm2 for the defocused mode (10 pm diameter). The maximum power densities calculated for the two modes differ by a factor of 4; this is shown schematically in Figure 4B. Also shown in Figure 4B is a line representing the threshold power density level for producing ions not characteristic of structure or molecular weight; i.e., for power densities above this line, noncharac-
2341
teristic fragment ions will be produced. Two distinct areas exist within the analysis area in the focused mode. Region A is the largest and corresponds to an area of the sample in which the laser power densities exceed threshold level. This region will produce noncharacteristic fragment ions. Region B is much smaller and corresponds to an area that is irradiated by power densities that produce structurally significant ions. In the defocused mode (Figure 4B) the entire analysis area is irradiated by power densities that produce structurally significant ions. Approximate calculations reveal that in the focused mode molecular ions are produced from only 10% of the area to that of defocused mode. The observation of molecular ions of retinoic acid in the defocused mode can be explained by the combined effects of lower power densities over the analysis area and the dramatic increase of analysis area relative to focused conditions. It has been reported (17) that sample position very close to the focal plane is required to maximize metal ion intensity. Current data for organic compounds indicate that even though the total ion current is greater near the focal plane, structurally significant ions of the compounds are produced only under defocused conditions. Another factor that must be considered when defocusing the laser beam using the LAMMA-lo00 geometry is a possible shift of the ionization region. It can be seen that as the sample is moved behind the focal plane of the laser, the center of the ionization region moves away from the ion-optic axis. The geometric arrangement of the system is such that a right triangle is formed by the center of the new sample position, the ion-optic axis, and the angle for the incident laser beam. Because the laser in the LAMMA-1000 is incident 45” to the sample, movement of the sample behind the focal point shifts the ionization region away from the optic axis by an equivalent distance. This factor becomes important if the sample is moved over large distances (several millimeters), because the location of the ionization region would limit the collection efficiency of the einzel lens (i.e., the sample is removed from the field of view of the lens). If the sample is moved far enough, no ions would be collected, and therefore no mass spectrum would be produced. We have observed, however, that only small changes in the sample distance (approximately 50 pm) are required to effect significant changes in the mass spectra. Distances up to 200 bm between the sample and the focal point correspond to similar changes in the position of the ionization region with respect to the center of the ion optical axis. These changes are within the ion acceptance limits of the einzel lens. Therefore, the peak positions and shapes remain unchangect indicating that there is little change in the flight path of the ions under defocused conditions. The effect of laser focus is the most critical factor influencing reproducibility, and therefore quantitation, of organics in laser mass spectrometry. Maintaining constant laser focus from one analysis point to the next is key to obtaining reproducible spectra; obtaining constant laser focus is the major limitation in obtaining good reproducibility. This is especially true when operating in the “defocused mode”, which is often used for organic analysis. The quantitative ability of the laser mass spectrometer therefore hinges on the ability to control the laser focus from one analysis point to the next. Sample Preparation. Proper sample preparation is critical for obtaining reproduciblespectra in laser mass spectrometry. Due to the small areas that are sampled by the laser, it is imperative that components in the sample matrix be distributed homogeneously if an analysis is to be representative of the sample as a whole. Special methods must often be used to prepare samples to obtain maximum reproducibility. The three major methods of sample preparation employed in our laboratory are casting from solution, electrospraying,
2342
ANALYTICAL CHEMISTRY, VOL. 60, NO. 21, NOVEMBER 1, 1988
Table I. Reproducibility as a Function of Sample Preparation
intensitpd std dev powdered solid" evaporating from solutionb thermospray electrospray* nitrocellulose solutionb
215 96 156 103 90
220 83 66 31 19
re1 std dev, % 102 86 64 25 21
'Data obtained from an average of 20 spectra. boPhenanthrolinewas used for all preparations except thermospray. Thermospray experiments used a mixture of quaternary amines. dPowerdensity used was approximately 1 X lo' W/cm2. and casting from a 5% nitrocellulose solution. These and other methods have been studied to determine which sample preparation technique provides the most reproducible laser mass spectra. Table I lists the various sample preparation methods studied, along with the average intensity and standard deviation observed for each. The method used most frequently is casting from solution. The soluble analyte(s) of interest is dissolved in an appropriate solvent and deposited on a sample support. Conductive metal supports such as zinc are often used to eliminate sample charging. The solvent is allowed to evaporate, leaving the analyte attached to the support usually as small crystallites and sometimes as thin films. These crystallites and films are easily observed by using an optical microscope. Casting from solution is valid only for qualitative characterization of an analyte. This is due to the poor reproducibility (Table I) of the spectra obtained because of nonuniform crystallite deposits on the support. Mixture analysis is virtually impossible with the solution casting method because of segregation effects. Spectra obtained from mixtures display high variability from one analysis point to the next. Sometimes there is an absence of one component in the mass spectrum obtained from a given sample area. To circumvent some problems of obtaining sample uniformity, the electrospray technique (18)has been employed. This method of sample Preparation utilizes a high electric field to deposit analytes from a solution. The analyte is deposited as either crystallites of uniform size or a film of uniform thickness depending on the compound. Samples prepared by electrospraying are homogeneous and superior in reproducibility to those prepared by solution casting. The electrospray technique does, however, suffer from solvent restrictions that limit the types of compounds for which the method is useful. The segregation of individual components in a mixture is also minimized because the majority of the carrier solvent is evaporated before contact with the substrate. A third method is the use of a polymer solution. In this method an analyte or mixture of analytes is dissolved in a 5% nitrocellulose/amyl acetate-methanol solution. The analyte is completely dissolved in the polymer solution. A polymer film of the mixture is then cast onto a support by a capillary tube and allowed to dry. No crystallites can be observed with an optical microscope (300x) in the polymer film after solvent evaporation. The higher viscosity of the polymer solution has the advantage of maintaining analyte homogeneity by reducing component diffusion during evaporation. Reproducibility in mixture analysis is improved due to reduction of segregation effects by the viscous polymer matrix. The mass spectrum of nitrocellulose consists primarily of carbon clusters that do not interfere with the measurement of analyte ions above m / z 60 at low laser powers and above m/z 120 at high laser powers. The polymer matrix has proven to be suitable for obtaining laser mass spectra with good reproducibility for many organic compounds.
I I I I I
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Schematic of laser-sample interaction. The sample with the irregular surface effectively defocuses the laser beam.
Flgure 5.
Additionally, we have studied thermcspray (19)as a possible sample preparation method for use in LMS. Thermospray produces a fine mist of solute from a solution that is pumped under high pressures through a heated microbore high-pressure liquid chromatography column. The nebulized solution was placed in the line of sight of a metal target used as a substrate for deposition. Examination of the metal target after deposition of quaternary amines showed the formation of islands of small crystallites. Nonuniform distribution of the material was observed to be the reason for the poor reproducibility, as shown in Table I. Thennospray, therefore, does not appear to be a highly reproducible sample preparation method for LMS. An additional concern in the preparation of samples for quantitative analysis is the topography of the sample surface. When the laser beam is focused on the sample surface, it is necessary that the surface be smooth so the laser power density is the same from one analysis point to the next. Relatively small variations in surface structure (tens of micrometers) can significantly affect mass spectra due to a defocusing effect. Figure 5 illustrates how variation in sample topography can defocus the laser beam and affect the power density a t different positions on the sample. Portions of the nonuniform surface are a t varying distances from the focal plane. The beam at location A strikes a relatively flat part of the sample. However, the analysis area and the topography of region B are significantly different from those of region A; region B is much larger and has portions of the sample lying out of the plane of the laser focus. Different locations of the sample, such as regions A and B, will be irradiated under different conditions of focus and, therefore, different effective power densities. Reproducing exact focusing conditions on an irregular surface at different locations is very difficult, if not impossible. The inability to reproduce focusing conditions accounts for, in part, the variability among laser mass spectra. The goal of sample preparation is then to reduce the variation of mass spectra by obtaining a uniform sample distribution and by generating a topographically smooth surface. Table I illustrates the reproducibilities that one can expect by using the sample preparation methods described. The data compiled in the table were obtained by averaging the absolute intensities of (M + H)+ions from 20 spectra obtained from o-phenanthroline. The thermospray data were obtained by using tetraethylammonium iodide (TEAI), because TEAI did not decompose a t the high capillary temperatures necessary, as did o-phenanthroline. The data from Table I demonstrate that the polymer matrix and electrospray methods provide the best reproducibility among the methods evaluated. The
ANALYTICAL CHEMISTRY, VOL. 60, NO. 21, NOVEMBER 1, 1988 2343
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Flgure 6. Plot of the ion intensity ratios of tetrapentylammonium ion to tetraethylammonium ion versus data set for the thermospray (B)and nitrocellulose ( 0 )methods of sample preparation. Each data set consists of an average of 10 irdMdual spectra. Spectra were collected in the focused mode with a constant laser energy of 3.0 CIJ. Error bars shown are l a values. relative standard deviations observed for o-phenanthroline prepared by electrospraying and casting from nitrocellulose solution are 25% and 21%, respectively. The methods of evaporating from solution and mounting of a powder onto a support show poor reproducibility. The poor reproducibility of the evaporation method is attributed to nonuniform deposition of the analyte during solvent evaporation. The poor reproducibility of powdered samples is attributed to defocusing effects occurring at the nonuniform sample surface. The use of internal standards has been employed in our laboratory to enhance the quantitative capability of LMS. Ratioing the analyte of interest to an internal standard serves to decrease the effect of instrumental fluctuations and sample preparation methods on ion intensities. To illustrate the increased reproducibility obtained through the use of internal standards, the absolute ion intensities of the tetrapentylammonium (TPA) ion in nitrocellulose were obtained. A total of 20 spectra were averaged, and a relative standard deviation of f20% was observed. For comparison a binary mixture of TMAI (tetramethylammonium iodide) and ETMAI (ethyltrimethylammonium iodide) was analyzed and the TPA ion ratfoed to the ETMA ion. For a set of 20 spectra the relative standard deviation was *15%. This result illustrates that the use of internal standards enhances reproducibility. It is therefore highly desirable to include internal standards when possible for quantitative LMS. Internal standards were used to assess the utility of the thermospray method as a sample preparation method for LMS. A mixture of two quaternary amines (TPAI and TEAI) was prepared and thermosprayed onto a polished brass support. The results are presented in Figure 6. It is graphically illustrated that reproducibility of the thermospray method is poor; the relative standard deviation is approximately *75% for the data set in Figure 6. The thermospray data are compared with results obtained from a similar binary solution of quaternary amines (TPAI and TEAI) using a nitrocellulose matrix; these data also are plotted in Figure 6. The reproducibility of the nitrocellulose matrix data is good, not only in the standard deviation for each data set but also in the average values between data sets. The relative standard deviation among data sets is approximately f15%. Comparison of the data for the two sample preparation techniques, thermospray and polymer matrix, clearly shows that the use of a polymer matrix is superior. Analysis of Variance of Intensity Measurements. Another parameter that has been studied is variation among relative intensity ratios for a given sample. Two data sets were
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Flgure 7. Ratios are the (M + H)' ion intensities of 2-methyl-ophenanthroline divided by (M 4- H)' of o-phenanthroline. The binary mixture was dissolved in 5 % nitrocellulose and deposited on a brass support. Each data point is an average of 20 individual spectra. All mass spectra were obtained from one sample. Data for set 1 were obtained on one day; data for set 2 were obtained a day later. obtained on different days for samples prepared from a solution containing o-phenanthroline and 2-methyl-ophenanthroline in a nitrocellulose matrix. A total of 200 spectra were collected for each data set; the data are summarized in Figure 7. Each data point in Figure 7 corresponds to an average of 20 mass spectra. The choice of averaging 20 spectra was made for two reasons: (1) 20 replicates is considered a sufficient data set for statistical purposes and (2) averaging 20 spectra can be performed by the computer in a short amount of time. Plotting the data as an average of 20 spectra illustrates the variation within and between data points. For the purposes of this discussion each data point will be considered to be an individual run within a set. A one-sided analysis of variance (ANOVA) calculation was performed by using the data from set 1 to determine if the within-run variance was significantly different from the between-run variance. An F test showed that at a confidence level of 99.5% the variances from the within-run and between-run data did not differ significantly. Relative standard deviations (RSD) were calculated for each run and ranged from 25% to 45%. Additionally, each averaged intensity ratio in set 1was taken as representing a replicate, and the RSD was calculated for all the replicates in set 1. The RSD among the ratioed intensities was calculated to be 6.7% showing significantly smaller variability between the averaged intensity ratios than within each intensity ratio. The data plotted in set 2 of Figure 7 represents an analysis of the same mixture obtained 1day after obtaining the data in set 1. An ANOVA calculation was performed to estimate the variances within-days and between-days. An F test revealed that there was no difference in the variance of the within-run and between-run data of set 2 at a confidence level of 99.5%. The RSDs were also calculated for each run and again ranged from 25% to 45%. The RSD calculated between runs was 12.2%. As was observed for set 1, the RSD calculated among the runs was lower than the RSDs for each individual run. A t test was performed to compare the average of ratios between set 1 and set 2. The test showed that the averaged ratios from the two sets can be considered identical at a confidence level of 95%. The data plotted in Figure 7 show that the averaged intensity ratios are more reproducible than the variation of individual mass spectra would indicate. It is therefore suggested that quantitative LMS data should be presented as averaged data sets consisting of at least 20 spectra. Laser Power Density. A factor that affects relative ion intensities in laser mass spectrometry is the laser power
2344
ANALYTICAL CHEMISTRY, VOL. 60, NO. 21, NOVEMBER 1, 1988
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Flgure 8. (A) Plot of the quasi-molecular ion (M H)+ intensity of 2-methyl-o-phenanthroline as a functlon of laser energy. (E) Plot of the total ion current as a function of laser energy. Spectra were collected under focused conditions. Nitrocellulose was used as the sample matrix. Absolute intensities were measured.
density. Figure 8a shows a plot of the intensity of the (M + H)+ions of 2-methyl-o-phenanthroline as a function of laser power. These data were collected under focused conditions and therefore at constant beam diameter; the power density can be calculated directly from the laser energy. By use of a 3-pm beam diameter the relationship between power density (PD) in W/(s cm2) and laser energy (L.E.) in microjoules is PD = (9.43 X lOI4)LE. The laser energy, as measured in these experiments, is the total energy reaching the sample. This is distinguished from the single photon energy that is calcuJ or 4.68 eV. lated to be 7.5 X It is observed that above some critical energy value the QMI intensity begins to decrease due to the breakup of the 2methyl-o-phenanthrolineat higher energies. Lower mass ions consisting of carbon clusters are formed at higher laser energies only. Figure 8b also shows a plot of total ion current as a function of laser energy. This reveals that at higher laser energies the total ion current remains relatively constant while the (M + H)' ion intensity decreases. Although not all compounds may behave precisely in this manner, results from this laboratory indicate generally that the intensity of a molecular ion is not a simple linear function of the laser energy or power density. For this reason all data collection taken for the purposes of quantitation should be performed at constant laser power density. C h e m i c a l E f f e c t s and Mixture Analysis. A difficulty with the quantitative analysis of organics by using LMS can arise from chemical interactions between the anal* and other
components in a sample matrix, including the matrix itself. A variety of chemical interactions are possible, depending on the nature of the analyte and the matrix. One such interaction that has been demonstrated for secondary ion mass spectrometry (SIMS) is the use of proton donors such as p toluenesulfonic acid (p-TSA) to aid in production of (M H)+ ions (20). Similar results for organic compounds with proton acceptor groups have been observed in our laboratory by using laser ionization. An example that dramaticallyillustrates the effect of matrix composition on organic analysis is a mixture of an organic acid (2-naphthoic acid) and an organic base (o-phenanthroline). The positive ion LMS of 2-naphthoic acid shows an M+' molecular ion when analyzed as a single component in a nitrocellulose matrix. The positive ion LMS of o-phenanthroline in nitrocellulose showed an (M + H)+ ion under the same conditions. In negative ion LMS an (M - H)- ion was observed for 2-naphthoic acid; the negative ion LMS of ophenanthroline showed only carbon clusters. The averaged laser mass spectra of a 1:l binary mixture of these compounds is shown in Figure 9a. Only the (M H)+ ion from ophenanthroline is observed in the positive ion LMS. This result is obtained by using laser energies of 0.1 &(threshold) I and higher. In the negative ion LMS (Figure 9b) only the (M - H)-ion from 2-naphthoic acid is observed. Proton NMR indicates attachment of protons to both nitrogen atoms of the o-phenanthroline molecule in the presence of the carboxylic acid. The LMS results therefore can be explained by the transfer of the acidic proton from the carboxylic acid to the basic amine in the matrix before or during laser mass spectrometric analysis. Next we present calibration curves constructed for two distinct classes of organic compounds to assess the potential of laser mass spectrometry for quantitative analysis of organic compounds. These calibration curves were obtained under conditions required for optimum reproducibility: the use of
+
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ANALYTICAL CHEMISTRY, VOL. 60, NO. 21, NOVEMBER 1, 1988 1.8 1
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tration of TMAI can be followed by measuring the intensity of the TMAI+ ion. Comparison of the two calibration curvw in Figure 10 shows that both are linear over a large portion of the concentration range studied. The curves also demonstrate that different compounds have different intensity responses as a function of concentration in LMS. Additionally, the slopes of the curves illustrate the effective range of analyte concentrations that can be studied. The simultaneous operation of two transient recorders at different input ranges can be used to increase the dynamic range of intensities studied. Obtaining different calibration curves for different organic compounds demonstrates that calibration curves must be constructed for each compound to be quantified. Calibration curves are necessary because different compounds will have different intensity responses to different concentrations. For example, at a mole ratio of 1.2, the intensity ratio of the o-phen mixture is 1.2, whereas the intensity ratio of the TMAI is 0.93.
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CONCLUSIONS The reproducibility and therefore the quantitative ability of laser mass spectrometry have been shown to be dependent on several parameters: sample preparation, laser focus, and laser energy. The sample preparation technique providing the highest reproducibility involves dissolving an analyte in a polymer matrix such as nitrocellulose. Use of the nitrocellulose matrix allows for the uniform distribution of analyte(s) and a uniform surface for analysis by the laser beam. The laser focus has also been observed to be a critical parameter in obtaining reproducible mass spectra. Focusing conditions may be altered by moving the sample away from the focal plane of the laser optics or by sample surface irregularities. The sample surface irregularities of most realworld samples make obtaining reproducible mass spectra difficult when compared to laboratory prepared polymer f i i . The use of such films is necessary, however, to define the optimum reproducibility of the laser mass spectrometric technique. To maximize reproducibility, therefore, the sample preparation must provide uniform analyte distribution and a uniform surface. The laser focus must be kept constant to provide a uniform power density over the analysis area. Changes in the laser energy affect the relative ion intensities and is, therefore, another parameter to be considered. As with laser focus, the laser energy must be held constant to minimize intensity fluctuations. Controlling the parameters described above will serve to maximize the reproducibility of laser mass spectra. In order to fully understand the quantitative aspects of LMS, additional work is required in the detailed mechanism(s) of laser interactions with organic solids.
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Flgure 10. Calibration curves constructed for (A) 2-methyl-o phenanthroline using 0-phenanthroiine as an internal standard and (B) for tetramethylammonium iodide using trimethylammonium iodide as an internal standard. The mixtures were prepared in a 5 % nitrocellulose solution using amyl acetate and methanol as solvents. Laser energy used was 0.4 pJ.
a nitrocellulose matrix to provide a uniform analyte distribution; a smooth sample surface; constant laser focus to provide a constant, reproducible sampling volume; and constant laser energy to provide the same power density for each analysis. The first calibration curve, shown in Figure loa, is for a mixture of o-phenanthroline (0-phen) and 2-methyl-ophenanthroline (2-Me-o-phen). These compounds showed no fragment ions in their positive ion LMS at the energies used for analysis. The (M H)+ ions have a mass difference of only m/z 15; corrections for mass discrimination of the secondary electron multiplier can be neglected. The calibration curve was constructed by monitoring the (M + H)+ ion intensity of o-phen ratioed to the (M + H)+ion intensity of 2-Me-o-phen; i.e., 2-Me-o-phen was used as an internal standard. Figure 10a illustrates the sensitivity of the laser microprobe mass analyzer to concentration changes over a limited concentration range. The curve in Figure 10a shows an initial nonlinear intensity response to changes in concentration. The reason for the nonlinear response in the low concentration region is unclear. Aside from the low concentration region, the intensity ratios are a linear function of the concentration ratios. Figure lob shows a plot of the calibration curve obtained for two quaternary ammonium salts, tetramethylammonium iodide (TMAI), and ethyltrimethylammonium iodide (ETMAI). Figure 10b demonstrates that changes in the concen-
+
2345
LITERATURE CITED Honig, R. E.; Woolston, J. R. Appl. Phys. Len. 1963, 2 . 138. Conzemius, R.; Capellen, J. Int. J. Mess Specfrom. Ion Phys 1980, 34, 187-271. Denoyer, E.; Van Grieken, R.; Adams, F.; Natusch, D. F. S. Anal. Chem. 1982, 54. 26A. Hercules, D. M.; Day, R. J.; Balasanmugam, K.; Dang, 1.A,; Ll, C. P. Anal. Chem. 1982, 54,280A-290A. Seydel, U.; Lindner, 6. Frezenlus' Z . Anal. Chem. 1981, 308, 253-257. Lyons, P. C.; Hercules, D. M.; Morelll, J. J.; Sellers, 0. A.; Mattern, D.; Thompson, C. L.; Millay, M. A. fnt. J . Coal W l . , In press. Schroeder. W. H. Freszenlus' Z . Anal. Chem. 1981, 308,212-217. Haas, U.; Wieser, P.; Wurster, R. Fmzenius' 2.Anal. Chem. 1981, 308,270-273. Verbuecken, A. H.; Van Grieken, R. E.: Paulus, 0. J.; de Bruijn, W. C. Anal. Chem. 1984, 56, 1362-1370. Balasanmugam, K.; Hercules, D. M. Anal. Chem. 1983, 55,145-146. Mattern, D. E.;Hercules, D. M. Anal. Chem. IB85, 57. 2041. Mattern. D. E.;Lln, F. T.; Hercules. D. M. Anal. Chem. 1984, 56, 2762-2789. Hillenkamp, F.; Feigl, P.; Schueler, 6. Microbeam Anal. 1982, llfh, 359-364. Heinen, H.; Meler, S.: Vogt, H.: Wechsung, R. Int. J. Mass Specfrom. Ion phvs. 1983, 47, 18-22. Manakov, N. L.; Ovslannlkov, V. D. Phys. Lett. 1983, 96A. 121-124.
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Anal. chem. 1988, 6 0 , 2346-2353
(16) Day, R. J.; Forbes, A. L.; Hercules, D. M. Spectrosc. Lett. 1981, 74, 703-727. (17) Conzemius. R. J.; Zhao, S.; Houk, R. S.; Svec, H. J . Int. J . Mass Spectrom. Ion Phys. 1984, 67, 277-292. (18) Brunnix, E.;Rudstam, 0. Nucl. Instrum. Methods 1981, 73,131-140. (19) Hardin, E. D.; Fan, T. P.; Blakely, c. R.; Vestal, M. L. Ana/. &em. 1984, 56, 2-7.
(20) Busch, K. L.; Unger, S. E.; Vincze, A.; Cooks, R. 0.; Keough, T. J . Am. Chem. SOC. 1982, 704, 1507-1511.
for review May 18, 1987. Resubmitted May 21, 1988. Accepted August 1, 1988.
RECEIVED
Ion-Molecule Reactions in the Negative Ion Laser Mass Spectra of Aromatic Nitro Compounds Somayajula K. Viswanadham and David M. Hercules* Department of Chemistry, University of Pittsburgh, Pittsburgh, Pennsylvania 15260 Emanuel M. Schreiber, Robert R. Weller,' and C. S. Giam Department of Industrial Environmental Health Sciences. Graduate School of Public Health, University of Pittsburgh, Pittsburgh, Pennsylvania 15261
Negative Ion laser mass spectra of many aromatk nitro compounds show formatlon of an Ion corresponding to (M 0 H)- formed by an ion-molecule reaction. I t was establlshed that Interaction of NO,- wlth neutral molecules Is responslble for formation of (M 0 H)-, The presence of substituents like -CI and -SOs- in aromatic nltro compounds leads to formation of (M 0 C1)- and (M 0 SO,Na)-, respectively. The presence of labile hydrogens (-OH, -COOH, -NH-) causes Intense (M H)- peaks, suppressing (M 0 H)- ion formatlon. Use of defocused laser condltlons (30 pm above the sample) Is essential to obtaln reproducible production of (M 0 H)-. Formation of molecular anlons (We)was observed when the laser was focused directly onto thln (-0.1 pm) flims. The Intensity of the molecular antons was highly sensitive to both laser focus posltion and sample thickness.
+ -
+ + -
+
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-
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Polycyclic aromatic nitro compounds (nitro-PAH) have been found in air particulates ( I ) , diesel exhaust particles (2), carbon black (31, and in the atmosphere (4). Most analytical methods used to identify nitro-PAHs involve extraction followed by chromatographic analysis (2,5,6). Due to low levels of nitro-PAHs in environmental samples, in situ analysis is desirable. Laser mass spectrometry (LMS) has evolved as a powerful technique (7-11) for the analysis of nonvolatile and thermally labile organic compounds directly from solids without complicated sample preparation. The microprobe capability (12) of LMS (95% of the d3 compound. Samplesfor LAMMA analysis were prepared by dissolving the appropriate compound (-0.5 mg/mL) in a solvent (methanol or acetone); 5-10 pL of the solution was placed on a metal support, and the solvent was allowed to evaporate. Compounds 1,2,5,
0003-2700/8~/0360-2346$01.50/0 0 1988 American Chemical Society
