2070
Anal. Chem. 1888,60,2070-2075
of the mobile phase used in the gradient elution. Fluorescence intensity of PDAM derivatives was found to decrease with the increase in the content of organic solvents such as acetonitrile or methanol in the solution. Figure 9 shows the chromatogram of each 100 fmol of prostaglandins (PGs) El, E2, Fla, and F2a. All the PGs tested were excellently separated and sensitively detected. The detection limits for the PGs were about 15-20 fmol ( S I N = 3). Since the degradation products of PDAM may interfere with the determination of hydrophilic PGs such as 6-keto PGFla, they were removed by the pretreatment described previously (16, 21). The above results suggests that PDAM is suitable for the liquid chromatographic determination of biologically important carboxylic acids.
LITERATURE CITED (1) Grushka, E.; Lam, S.; Chassin, J. Anal. Chem. 1978,50, 1398-1399. (2) Lam, S.; Grushka, E. J. Chfomatogr. 1978, 158, 207-214. (3) Tsuchiya, H.; Hayashi, T.; Naruse, H.; Takagi, N. J. Chromatogr. 1982,234, 121-130. (4) Korte, W. D. J. Chromatogr. 1982,243, 153-157. (5) Yamaguchi. M.; Hara. S.; Matsunaga, R.; Makanura, M.; Ohkura, Y. J. Chromatogr. 1985,346, 227-236. (6) Kamada, T.; Maeda, A.; TsuJl, A. J. Chromatogr. 1983,272, 29-41. (7)Roach, M. C.;Unfar, L. W.; Zare, R. N.; Reimer, L. M.; Pompiiano, D. L. Anal. Chem. 1987,59, 1059-1061. (8) Matthees, D. P.; Purdy. W. C.; Anal. Chim. Acta 1979, 109, 61-66.
(9) Nimura, N.; Kinoshita, T. Anal. Lett. W80, 13, 191-202. (IO) Takadate, A.; Tahara, T.; Fujino, H.; Goya, S. Chem. Pharm. Bull. 1982,3 0 , 4120-4125. (1 1) Goya, S.; Takadate, A.; Fujino, H.; Tanaka, T. Yakugaku Zasshi 1980. 100, 744-748. (12)Lloyd, J. 8. F. J. Chrometogr. 1080. 189, 359-373. (13)Lingeman, H.; Hulshoff. A,; Underberg, W. J. M.; Offermann, F. B. J. M. J. Chromatogr. lS84,290, 215-222. (14)Goto, J.: Ito, M.; Katsuki, S.; Saito, N.; Nambara, T. J. Liq. Chromatogr . 1986,9,683-694. (15) Barker, s. A.; Monti, J. A.; Christian. S. T.; Benington, F.; Morin, R. D. Anal. Biochem. 1880, 107, 116-123. (16)Hatsumi, M.; Kimata, S.; Hirosawa, K. J . Chromatogr. 1982, 253, 271-275. (17) Shimomura, Y.; Sugiyama, S.; Takamura, T.; Kondo, T.; Ozawa, T. Ciin. Chlm. Acta 1984, 143, 361-366. (18) Ichinose, N.; Nakamura, K.; Shimiru, C.; Kurokura, H.; Okamoto, K. J. Chromatogr. 1984,295, 463-469. (19) Martinez, E. E.; Shimoda, W. J. Assoc. Off. Anal. Chem. 1985,68. 1149-1153. (20)Yoshida, T.; Uetake, A.; Murayama, H.; Nimura, N.; Kinoshita, T. J. Chromatogr. 1985,348, 425-429. (21) Yamauchi, Y.; Tomita, T.; Senda, M.; Hirai, A.; Terano, T.; Tamura, Y.; Yoshkia, S. J. Chromatcgr. 1986, 357, 199-205. (22) Hatsumi, M.; Kimata, S.; Hlrosawa, K. J. Chromatogr. 1986, 380, 247-255. (23)Shimomura, Y.; Sugiyama. S.; Takamura, T.; Kondo, T.; Ozawa, T. J. Chromatogr. 1986. 383, 9-17. (24)Kiyomiya, K.; Yamaki, K.; Nimura, N.; Kinoshita, T.; Oh-ishi, S . Prostaglandins, 1988. 3 1 , 71-82. (25)Carpino, L. A. J. Org. Chem. 1970,3 5 , 3971-3972.
RECEIVED for review March 3,1988. Accepted June 14,1988.
Quantitative Trace Element Analysis of Microdroplet Residues by Secondary Ion Mass Spectrometry Robert W. Odom,* Gayle Lux, Ronald H. Fleming, Paul K. Chu, Ilsabe C. Niemeyer, and Richard J. Blattner
Charles Evans & Associates, 301 Chesapeake Drive, Redwood City, California 94063
This paper reports the results of secondary Ion mass spectrometry (SIMS)analyses of the elemental components contained In mlcrovoiume iiquld resldues deposited onto high-purity graphite substrates. These resldues were formed by evaporating the solvent in 25-nL volumes of standard solutions contalnlng the analyte element and a known mass of a yttrlum internal standard. The capablltty of the SIMS technique to quantitatively measure the mass of the anaiyte was determlned from these standard samples. The relative ion yields of AI, Ca, Mn, Fe, Co, Cu, Zn, Se, and Pb with respect to the Y internal standard were determined. The minimum detectable quantities (Mw)of these elements were measured along with the preclslon of the SIMS analysis. Gram detectlvlties for thls set of elements dlssolved in the 25-nL volumes ranged between 85 pg and 2.0 fg, correspondlng to molar detectlvltles ranging between 10 I.~Mand 1.0 nM. Stable Isatope dilution analysis of samples contalnlng enriched loePbdemonstrated quantitative measurement accuracy withln 3 % of the true values for samples contalnlng 4.0 mM Pb. SIMS analyses of the NBS bovlne serum reference standard lndlcate that this tedrnlque can provide useful quantitative analysls of selected elements contained In a mlcrovolume of blologlcal fluids.
Contemporary inorganic elemental analysis of fluid samples can be subdivided into two volumetric regimes: microliter (pL)
or larger aliquots analyzed by techniques such as inductively coupled plasma (ICP) atomic emission spectroscopy (AES) or mass spectrometry (MS) and picoliter (pL) volume samples analyzed by electron probe microanalpis (EPMA). Although the ICP-AES or MS techniques are capable of providing sub-part-per-million detection limits, these detection limits are necessarily degraded when the amount of available sample is less than approximately 1 mL (1). Part-per-million (ppm) detection limits are achievable with the electron probe technique at sample volumes in the 100-pL range, and these detection limits are not reduced as the sample volume increases (2). Thus, the need exists for a trace element analysis technique having sub-ppm detection limits for sample volumes in the 100 pL to 100 nL range. The development of a technique that could perform trace element analyses on these sample volumes would find extensive applications in such areas as microbiology, medical research, forensic science, and industrial quality control. In the past 10 years, secondary ion mass spectrometry (SIMS) as performed on the Cameca IMS-3f or 4f ion microanalysers has emerged as a viable analytical tool for the quantitative trace element analysis of solid samples (3). The applications of this technique have been primarily in semiconductor (4) and metallurgical (5)characterizations, although a number of biomedical researchers have applied the SIMS technique to the microanalysis of a wide range of biological materials (6, 7). The technique employs sputtering of the surface atoms from relatively small areas (typically, 250 wm
0003-2700/88/0360-2070$01.50/0 0 1988 American Chemical Society
ANALYTICAL CHEMISTRY, VOL. 60, NO. 19, X 250 run) of a solid sample by an energetic primary ion beam, which is generally either 0,’ or Cs*. Since the atomic sputtering rates are low (ranging between 1and 100 &), small maases of material are consumed per unit time. A routine bulk analysis of a semiconductor sample such as G& will consume a volume of material approximately 250 pm x 250 pm x 5 deep corresponding to a mass consumption of about 1 pg. In this type of SIMS bulk analysis, the ion signals of several elemental ions would normally be monitored, and the elemental detection limits range between 0.1 ppm to 0.1 parts per billion (ppb) (8). Thus,in this mode of analysis the SIMS technique can provide detection sensitivities ranging from 0.1 pg to 0.1 femtogram (fg). This type of bulk analysis requires about lC-20 min of analysis time. The elemental detection Sensitivities in the SIMS analysis are a function of the relative ease of ionizing the respective elements sputtered from the surface, and this ionization efficiency depends on either the elemental ionization potential (E’) for positive secondary ion analysis or the electron affinity (EA) of the sputtered species for negative ion analysis (9). SIMS could be a very useful complementary technique to ICP and EPMA analysis of microvolume residues, and the major problem in applying SIMS to this type of elemental analysis is one of sample preparation and presentation to the instrument. We have developed a micmdroplet technique for depositing IC-100-nL volumes onto high-purity, polished substrates. The solid residues from these droplets are then analyzed by conventional SIMS depth profiling techniques in which the ion signals from the various elements are monitored as a function of time until the residue is completely consumed. Quantitation of the mass of the analyte element(s) is performed by using either internal standard or isotope dilution techniques. In this paper, we describe the proeedures employed in the sample preparation and report results that demonstrate the overall capabilities and utility of this technique for microvolume elemental analysis.
EXPERIMENTAL SECTION
Sam& Preparation. Elemental solution samples were atomic absorption (AA)standards purchased from Fischer Scientific. These standards contained 1000 ppm by weight (ppmw) of the element of interest dissolved in dilute (-0.1 M) nitric or hydrochloric acid. Since the typical analytical field of view for the IMS-3f is 150pm in diameter, the ideal m i m l u m e residue would be this size or smaller. In order to produce droplet residues of this size, a microvolume “pipet” was assembled consisting of a short segment of fused silica capillary tubing attached to a stainleas steel union. The capillary tubing was filled by capillary action to a known volume (determined by the displacement volume of the tube), and this volume could be dispensed onto a substrate by pressurizing the capillary with N2 gas. Both semiconductor wafers and high-purity polished graphite planchettes were used as substrates. The graphite proved superior in terms of the lower number of spectral interferencesproduced in the SIMS sputtering. The capillary pipet and the substrate were each mounted on an X,Y,Z microscope translation stage in order to precisely control positioning of the microvolume droplet on the substrate. The deposition process was viewed under a low-power (50X) stereomicroscope. It was determined that 25-nL volumes of the more concentrated standard samples typically formed droplet residues leas than 150 wn in diameter. These 25-nL volumes were produced from a 25 pm i.d. capillary 5 cm in length. To facilitate quantitation, an internal standard technique was employed in the analysis of these standard droplet residues. The ”unknown”sample solution was doped with a known volume of a yttrium solution of known Y concentration. The analyte ion intensities were normalized to the Yf intensity in order to compensate for variations in sampling losses, ion yield variations, and other instrumental effects. The quantitative accuracy of the microvolume pipet is limited hy solvent evaporation as the solution is dispensed and by sample loss due to sample adsorption onto the interior or exterior tube surfaces. Use of an internal standard,
a.
OCTOBER 1,
1988
2071
D.
(Mn : Y) MMn-12.5ng.My-
5.0Pg
Flgure 1. Scanning electron micrograph (SEM) images of 25-nL microdroplet samples of X Y mlxtures containing various mass loadings.
however, minimizes the influence of sample loss on the quantitative accuracy. The tipa of the capillarieswere shaped into points in order to minimize wetting of the exterior tube walls and to provide an nnohsixucted view of the liquid/substrate contact point under the microscope. Yttrium/bovine serum solutions often plugged the capillaries. S t e p to reduce the viscosity and amount of suspended proteinaceous solids in the serum corrected the problem. A centrifuged mixture containing equal parts of bovine serum, Y standard, and methanol was relatively easy to dispense. The general morphology of several representative 2 5 n L droplet residues deposited on a carbon planchette is illustrated in the scanning electron microscope (SEM) images in Figure 1. Figure l a was produced from a binary mixture of 125 pg of Ca and 12.5 ng of Y; Figure l b is a mixture of 12.5 ng of Mn and 500 pg of Y; and Figure ICis a mixture of 12.5 ng of Mn and 5 pg of Y. The largest linear dimensions of these residues are approximately 230, 130, and 120 pm, respectively. The residue in Figure ICshows definite wetting of the carbon plancbette outside the central droplet region, which extends over a relatively long distance (-280 Fm). Spreading of the solution outside the optimal 150-Nm dimension could affect the precision and accuracy of the internal standard solution analysis if segregation occu~s.Sample transfer and spreading lows can affect the measured gram detectabilities. Analytical Conditions. A suite of binary mixtures using Y as the internal standard was prepared from the atomic absorption standards. The first set of binary mixtures contained 500 ppmw of each element. Mixtures of AI, Ca, Fe, Co, Cu, Se, and Pb with Y were prepared for SIMS analysis. Several 25-nL volumes of each mixture were deposited onto distinct locations within small squares scribed on the carbon planchette. All these binary mixtures were analyzed by using 0,’primary ion bombardment and positive secondary ion spectroscopy, while the Se:Y mixture was also analyzed with a Cs+ primary ion beam and negative secondary ion spectroscopy. Each of the SIMS analyses was performed with the electron multiplier detector in order to provide single-ion detection capabilities. Since the electron multiplier output becomes nonlinear at instantaneous count rates >5OOooO counts s-’, care was taken to perform the analyses below this signal level. The initial set of 500-ppmw droplets contained a large mass (-12.5 ng) of each
2072
ANALYTICAL CHEMISTRY, VOL. 60, NO. 19, OCTOBER 1, 1988
Table I. Relative Ion Yield and Minimum Detectable Quantity (MDQ) in SIMS Analysis of 25-nL (X:Y) Samples
i to5
h
X
A1
40Ca
Ca Mn
Fe co
cu Zn
Se(+) Se(-) Pb
no. of measmts
y(X/Y)"
re1 std dev, %
MDQ, pg
3 3 4 3 2 3 3 3 4 3
3.17 4.04 1.22 0.539 0.245 0.247 0.10 0.003 108.3 0.084
15 23 15 5 50 8 5 17 17 6
1.2 0.96 3.1 7.6 15.3 22.0 71.9 2680 0.07 84.9
All values for Oz+bombardment and positive secondary ion analysis except the Se(-) listing, which employed Cs+ bombardment and negative ion spectroscopy.
the X+/Y+ ratio observed for the 500-ppmw mixtures. Relative ion yields observed in the SIMS analyses of these mixtures are listed in Table I. The relative ion yield, y(X/Y), is defined in eq 1, where iX+and Y+ are the integrated in-
I
I 1 I , 0 5
10
I , f5
20
2 5
3EFTb imicr.rj
Figure 2. Depth proflle analysis of 25-nL samples of a Ca:Y mixture containing 12.5 ng of each element. element. Thus, it was necessary to severely reduce the ion transmission of the mass spectrometer for these relatively concentrated samples in order to insure that the electron multiplier output remained linear, The transmission efficiency was reduced by using the smallest available contrast diaphragm (CD) on the IMS-3f and by sputtering with a primary ion beam current of 1 pA. The CD is located at the crossover plane of the ion transfer optics and serves as the entrance aperture to the mass spectrometer in these analyses. The primary ion current was rastered over an area 250 X 250 pm, and the residues from these 25-nL volumes were totally consumed in approximately 15-30 min. The IMS-3f was also operated with a 75-V energy offset that effectively eliminated the transmission and detection of interferences from cluster or molecular ion signals having the same nominal mass as the elemental ion of interest (10). The elimination of these interferences was confirmed by measuring isotope ratios for those elements having more than one stable isotope.
tensities of the ith isotope of elemental ion X+ and yttrium, respectively; if is the isotopic abundance of the ith isotope (in atomic fraction), and my and mx are the masses of the respective addend elements in the microdroplet. An estimate of the minimum detectable quantity (MDQ) or gram detectability of element X in a microdroplet can be defined as in equation 2, where 3('X+)bb is simply 3 times the background
-
RESULTS AND DISCUSSION Analysis of Binary Mixtures. A typical SIMS analysis of a 500-ppmw droplet mixture is illustrated in the depth profile plot in Figure 2. The depth profile was obtained from a sample of 25 nL of a Ca:Y mixture. The ion intensity of various isotope signals is displayed as a function of the sputtered depth into the surrounding carbon planchette. The analysis was continued until the Ca and Y ion signals reached a background level of -10 cps. Although this sample was composed primarily of Ca and Y, there are readily detectable signals for Al, Cr, Fe, Cu, and Zn in the profile. These contaminant signals are most likely in the atomic absorption standards themselves, since each fused silica capillary tube was rinsed several times in high-purity MeOH and baked in an oven. Individual tubes were employed with each solution and discarded after use. The contaminant ion signal intensities were typically 3-4 orders of magnitude below those of the intentionally added elements and therefore represent a negligible fraction of the total ion signal. The X+/Y+ signal intensity ratios (where X is one of the binary mixture elements and Y is the yttrium) in background analyses of H20:Y mixtures were also typically 3-4 orders of magnitude below
intensity of rX*. Table I summarizes the relative ion yields and calculated minimum detectable quantities obtained in the SIMS analysis of the 12.5-ng (X:Y) binary mixtures. The voltage offset technique has effectively reduced cluster or molecular ion interferences for most of these analyses. The average deviation of the various isotope ratios, defined as A = I,/& - 1where I , and It are the measured and true isotope ratios, respectively, were the following: 42Ca/40Ca= +0.274, j7Fe/j6Fe = +0.188, 65Cu/63Cu= -0.070, 66Zn/64Zn= -0.076, 78Se/80Se(positive ion) = +0.138, 78Se/BOSe(negative ion) = +0.022, and 207Pb/208Pb = +0.070. The relatively large deviations observed for the 42Ca/40Caand 57Fe/56Fe ratios suggest that significant ion interferences are present a t m / z 42 and 57. However, the true values for these two ratios are 0.0066 and 0.024 58, respectively, and hence, relatively low intensity interference signals at these two masses could cause substantial deviations in the measured isotope ratios. The initial set of SIMS analyses given in Table I was obtained under instrumental conditions in which the secondary ion signals were significantly attenuated. Attenuation was necessary so that the electron multiplier detector operated below its saturation level. Microvolume samples containing lower masses of the analyte element and the internal standard could be analyzed by using higher instrumental ion transmission efficiencies, which should provide lower MDQ's. Since the ion transmission efficiency in these SIMS analyses was controlled primarily by the size of the CD, higher ion intensities can be achieved by using larger diameter contrast diaphragms. SIMS analyses of 25-nL residues having lower mass loadings of Ca, Mn, and Zn with the Y internal standard were performed with larger CD sizes. These results are given in Table 11. The data demonstrate that lower sample loadings
ANALYTICAL CHEMISTRY, VOL. 60, NO. 19, OCTOBER 1, 1988
Table 11. Relative Ion Yield and Minimum Detectable Quantity (MDQ) in SIMS Analysis of 25-nL Samples as a Function of mx, m y ,and CD Setting
,02
O 2 SIMS ANALYSIS OF 10
Ca : Y MICRODROPLETS
concn, X Ca
Mn
Zn
mx,pg
my,
pg
12500 12 500 5.0 1250 12500 12 500 500 12 500 5.0 1250 12500 12 500 12 500 500 5.0 1250
CD, Mm y(X/Y) 25 150 25 70 150 25 70 150
4.041 4.491 1.221 0.499 0.724 0.107 0.049 0.024
MDQ,pg 0.96 3.4 X 3.1 0.30 0.002 71.9 6.2 0.12
2073
mol/L 9.6 x 10-7 3.4 X 2.2 x 102.2 x 10-7 1.4 X 4.5 x 10-5 3.9 x 107.5 x 10-8
and larger ion transmission efficiencies do, in fact, yield lower MDQs. The relative Mn and Zn ion yields (y(X/Y)) also tend to decrease with lower mass loadings. The relative standard deviations of all these ion intensities were typically less than *20% as shown. The observed decrease in relative ion yield probably reflects a differential loss of one element (e.g., Y) with respect to the other in the analytical volume at these lower concentrations, which is possibly the result of a selective segregation phenomenon. This hypothesis is corroborated by the SEM images of a lower concentration droplet (e.g., Figure IC)in which the solution appears to have wetted an area significantly larger than the droplet diameter. A rapid freezing and freeze-drying approach as is used in electron microprobe analysis of tissue may prevent this phenomenon from occurring and thereby improve the results (11). A reduction in the relative ion yields with lower sample loading suggests that the MDQ values in Table I1 are upper limits that could be reduced further by improving the confinement of the droplet residue. I t is conceivable that subpicogram detection limits in 25-nL volumes can be achieved by using the SIMS technique once improved preparation procedures are implemented. The far right column in Table I1 lists the measured minimum detectable concentration of these three elements in 25-nL volumes. These detection limits range between 45 pM for Zn and 0.3 nM for Ca. Isotope ratios were also measured in these Ca and Zn analyses. The average deviation of the 42Ca/40Ca ratio from the true value was +0.003 a t the 5-pg Y loading, while the deviation of the 66Zn/64Znratios were -0.051 and -0.001 at 500- and 5-pg Y loadings, respectively. The accuracy of these isotope ratios demonstrates that the voltage offset technique has effectively eliminated mass spectral interferences in the analysis of these samples. The variation of the X+/Y+ intensity ratios as well as the integrated X+ intensity in the SIMS analyses were evaluated as a function of the X mass loading (weight concentration of solution) for several elements. Figure 3 is a plot of the logarithm of the measured @Ca+/Y+ratio and the @Ca+intensity as a function of the mass of Ca in 25-nL residues containing a Y loading of 12.5 ng. The error bars in these data are la limits based on three measurements at each mass loading. The intensity ratio varies in an approximately linear fashion with the Ca concentration in the microvolume. A least-squares fit of the data produces the line drawn through the data points. This line has a slope of 0.94 and a correlation coefficient of 0.997. The integrated 40Ca+intensity includes a correction for the Ca+ background observed in the analysis of the H20:Y sample. The Ca+ intensity varies in an approximately linear manner with the Ca concentration, suggesting that there were no significant variations in ion yields or sample deposition over this Ca concentration range. The integrated intensity does show significant deviation from “linearity” a t both high and low mass loadings. The good linearity of the ratio data coupled with the deviations observed in the Ca+ intensity data is direct evidence of the need for some form of internal standardization. It is interesting to note that both the ratio and integrated
1.o
10
40~a+ -Y+
7
40~a+ Counts 106
0.1
(05
lo4
lo3
10-4
0.1
1.0
10
IO*
lo3
lo4
Mass Ca (pg) Figure 3. ‘OCa+/Y+ ratio and integrated %a+ intensity as a function of Ca loading for 25-nL samples containing 12.5 ng of Y.
+
O2
SIMS ANALYSIS OF
3.0
10’
Zn : Y MICRODROPLETS 0.1
106
64 Zn+
64
lo5
Zn+ -
“~nCounts
+
y
-3
lo4
10
10
-4
lo3
-5 10
IO 0 1
10
IO
2
lo+
Mass Zn (pg) Figure 4. ‘‘Zn+/Y+ ratio and integrated e4Zn+ intensity as a function of Zn loading for 25-nL samples containing 5 0 0 pg of Y.
intensity data extrapolate to approximately 1-pg MDQ values at these Y loadings. These MDQ values are in good agreement with the MDQ value calculated from the Ca+/Y+ relative ion yield and background noise levels (Table I). The average 42Ca/40Caisotope ratio for these analyses was 0.007 19 (k0.00047), which is 9% higher than the true value. A second example of a response function evaluation is illustrated in Figure 4. Zinc is the analyte element in this study, and the Y mass loading is 500 pg. The lower internal standard concentration permitted operating the instrument a t a higher ion transmission efficiency (70-pm CD). The 64Zn+/Y+intensity ratios exhibited more scatter than the corresponding Ca data. The slope of the fitted line in this case is 0.825, while the correlation coefficient is 0.993. The integrated MZn+ counts follow a similar response function and exhibit slightly more scatter than the ion intensity ratios. The extrapolated MDQ values from either the intensity ratio or integrated intensity are on the order of 5.0 pg, which is approximately equal to the value calculated in Table 11. The average 66Zn/64Znisotope ratios for these Zn analyses were 0.5242 (f0.0106), which is 7.8% lower than the true value. Figure 5 illustrates a similar signal response evaluation performed on 25-nL residues of a Mn:Y mixture having a 5.0-pg Y loading. The slope of the fit of the intensity ratios
ANALYTICAL CHEMISTRY, VOL. 60, NO. 19, OCTOBER 1, 19188
2074
1_
O 2 SIMS ANALYSIS OF M,
TV
5.0 pg
Table 111. Recommended Values and Estimated Uncertainties of Inorganic Constituents in Bovine Serum Reference Material 8419
element Na
K
I 0.1
i104
,L 1 .0
I
10
io2
lo3
1303
io4
Mass Mn (pg) Flgure 5. Mn+/Y+ ratio and integrated Mn+ intensity as a function of Mn loading for 25-nL samples containing 5.0 pg of Y.
is 0.646, indicating that this ratio does not vary linearly with the mass of Mn in the residue. The least-squares slope of the Mn+ ion signal versus Mn mass is 1.0, although it exhibits fairly strong variability over the Mn mass range. Since there was essentially no Mn+ background signal produced in the H20:Y blank sample, the relatively high Mn+/Y+ ratios observed at the lower Mn loadings suggests either that the measured Y+ signal is lower than it should be or that there is a molecular ion interference a t m / z 55 arising from the standard (Mn is monoisotopic). If there is a Y-Mn segregation in these samples and if the volumes disperse over an area larger than the analytical field of view (150 pm), this could explain a relatively low Y+ signal intensity and the deviation of the Mn+/Y+ ratio from a linear dependence on the mass of the Mn residue. Several SEM images of the 5.0-pg Y samples (e.g. Figure IC)suggest that the solution has wetted a region significantly larger than 150 pm in diameter. As for the interference question, the voltage offset used for these measurements should be sufficient to eliminate molecular interferences (such as KO+) at m/z 55. There is the possibility, however, that the Mn stock solution contains different contaminants than those in either the Zn or Ca solutions, which did not demonstrate interference signals at m / z 55. These issues remain unresolved and are being investigated. The apparent large area dispersion of these low-concentration droplets might be eliminated by utilizing other clean, nonwetting polished substrates. We are at present evaluating the use of several metal substrates, such as high-purity W or T a for these microvolume analyses. Analysis of Bovine Serum. One of the primary aims of this research was to evaluate analytical methods for the quantitative analysis of trace elements in microvolume biological samples. We have utilized NBS bovine serum as an example of a multicomponent proteinaceous biological fluid. The concentrations of a wide range of inorganic constituents have been certified by NBS and are listed in Table 111. These certified values are given in both millimolar units and picograms per 8.33 nL. This latter specification relates to the SIMS analyses described below. It is important to emphasize that the masses of several of these elements in a nanoliter volume of serum are in the subpicogram range. Bovine serum is a typical biological fluid containing protein, fat, and glucose. Equal volume mixtures of bovine serum and Y standard were prepared, but these mixtures plugged the 25 pm i.d.
Ca Mg Fe cu Zn A1 co Cr Mn Mo Ni Se V
recommended value' (f estimate of uncertainty) (mM1 pg18.33 nL 141 f 2 5.1 f 0.2 2.5 f 0.1 0.85 f 0.1 0.036 f 0.009 0.012 f 0.002 0.017 f 0.002 [(4.8 f 1.8) X lo4] ~ ( 2 . 0f 0.5) x 10-51 [(5.8 f 1.0) X IO"] [(4.7 f 0.9) X lo"] [(1.7 f 0.4) X lo-'] [(3.i f 1.0) x 10-51 [(2.0 f 0.2) x 10-41
