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 ace- tonitrile 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 IN = 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 impor- tant 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.

(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.

1982, 234, 121-130.

(9) Nimura, N.; Kinoshita, T. Anal. Lett. W80, 13, 191-202. (IO) Takadate, A.; Tahara, T.; Fujino, H.; Goya, S. Chem. Pharm. Bull.

1982, 30, 4120-4125. (1 1) Goya, S.; Takadate, A.; Fujino, H.; Tanaka, T. Yakugaku Zasshi 1980.

(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. Chroma-

togr . 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,

(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. 1149-1 153.

(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. Prosta- glandins, 1988. 31, 71-82.

(25) Carpino, L. A. J. Org. Chem. 1970, 35, 3971-3972.

100, 744-748.

271-275.

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 spec- trometry (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 so- lutions 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, corre- spondlng to molar detectlvltles ranging between 10 I.~M and 1.0 nM. Stable Isatope dilution analysis of samples contalnlng enriched loePb demonstrated quantitative measurement ac- curacy withln 3 % of the true values for samples contalnlng 4.0 mM Pb. SIMS analyses of the NBS bovlne serum refer- ence 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 tech- nique at sample volumes in the 100-pL range, and these de- tection limits are not reduced as the sample volume increases (2). Thus, the need exists for a trace element analysis tech- nique having sub-ppm detection limits for sample volumes in the 100 pL to 100 nL range. The development of a tech- nique 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 mi- croanalysers 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 semi- conductor (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, OCTOBER 1, 1988 2071

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 1 and 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 ele- mental 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 mon- itored 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 tech- nique 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 hy- drochloric acid. Since the typical analytical field of view for the IMS-3f is 150 pm 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 interferences produced 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) stereo- microscope. I t 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 com- pensate 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. D.

(Mn : Y) MMn-12.5ng.My- 5.0Pg

Flgure 1. Scanning electron micrograph (SEM) images of 25-nL mi- crodroplet samples of X Y mlxtures containing various mass loadings.

however, minimizes the influence of sample loss on the quanti- tative accuracy. The tipa of the capillaries were 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 25nL 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 IC is 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 IC shows 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 di- mension 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 ca- pabilities. 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

i to5 h 40Ca

I I 1 I , I , 0 5 1 0 f 5 2 0 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 con- centrated 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 spec- trometer 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.

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 con- taminant 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 inten- sities 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

Table I. Relative Ion Yield and Minimum Detectable Quantity (MDQ) in SIMS Analysis of 25-nL (X:Y) Samples

X no. of measmts y(X/Y)" re1 std dev, % MDQ, pg

A1 Ca Mn Fe c o c u Zn Se(+) Se(-) Pb

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

0.084 108.3

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+ bombard- ment 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-

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 re- spective addend elements in the microdroplet. An estimate of the minimum detectable quantity (MDQ) or gram de- tectability of element X in a microdroplet can be defined as in equation 2, where 3('X+)bb is simply 3 times the background

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,/& - 1 where 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 de- viations observed for the 42Ca/40Ca and 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 ob- tained 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 trans- mission 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 inten- sities can be achieved by using larger diameter contrast dia- phragms. 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 2073

Table 11. Relative Ion Yield and Minimum Detectable Quantity (MDQ) in SIMS Analysis of 25-nL Samples as a Function of mx, my, and CD Setting

concn, X mx,pg my, pg CD, Mm y(X/Y) MDQ,pg mol/L

Ca 12500 1250

Mn 12500 12 500

1250 Zn 12500

12 500 1250

12 500

12 500 500

12 500 500

5.0

5.0

5.0

25 150

25 70

150 25 70

150

4.041 0.96 9.6 x 10-7 4.491 3.4 X 3.4 X 1.221 3.1 2.2 x 10- 0.499 0.30 2.2 x 10-7 0.724 0.002 1.4 X 0.107 71.9 4.5 x 10-5 0.049 6.2 3.9 x 10- 0.024 0.12 7.5 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 oc- curring 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 tech- nique 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/64Zn ratios 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 interfer- ences 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 log- arithm 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. I t is interesting to note that both the ratio and integrated

,02

10

1 .o

4 0 ~ a + - + Y 0.1

10-4

O2 SIMS ANALYSIS OF

Ca : Y MICRODROPLETS

7 10

4 0 ~ a + Counts

106

( 0 5

l o 4

lo3 0.1 1.0 10 IO* l o 3 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.

3.0

0.1

64 Zn+

y -3

- + 10

-4 10

- 5 10

+ O 2 SIMS ANALYSIS

OF Zn : Y MICRODROPLETS

64 Zn+

10’

106

l o 5

“ ~ n - Counts l o 4

l o 3

2 I O

0 1 1 0 I O l o +

Mass Zn (pg)

Figure 4. ‘‘Zn+/Y+ ratio and integrated e4Zn+ intensity as a function of Zn loading for 25-nL samples containing 500 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/40Ca isotope 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 il- lustrated 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+ in- tensity 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/64Zn isotope 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

2074 ANALYTICAL CHEMISTRY, VOL. 60, NO. 19, OCTOBER 1, 19 188

O2 SIMS ANALYSIS OF

M, TV 5.0 pg 1__

i 1 0 4

I ,L I 1303

0.1 1 .0 10 i o 2 l o 3 i o 4

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 ob- served 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 con- taminants 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, non- wetting polished substrates. We are at present evaluating the use of several metal substrates, such as high-purity W or Ta 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 bio- logical 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 pico- grams 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.

Table 111. Recommended Values and Estimated Uncertainties of Inorganic Constituents in Bovine Serum Reference Material 8419

recommended value' (f estimate of uncertainty)

element (mM1 pg18.33 nL

Na 141 f 2 27000 f 383 K 5.1 f 0.2 1660 f 65 Ca 2.5 f 0.1 833 f 33

172 f 20 16.6 f 4.1 Fe 0.036 f 0.009

c u 0.012 f 0.002 6.2 f 1.0 9.2 f 1.1 Zn 0.017 f 0.002

A1 [(4.8 f 1.8) X lo4] 0.11 f 0.04

0.85 f 0.1 Mg

c o ~ (2 .0 f 0.5) x 10-51 0.01 f 0.002 Cr [(5.8 f 1.0) X IO"] ~(2.5 0.4) x 10-31 Mn [(4.7 f 0.9) X lo"] 0.022 f 0.004 Mo [(1.7 f 0.4) X lo-'] 0.13 f 0.03 Ni [(3.i f 1.0) x 10-51 0.015 f 0.005 Se [(2.0 f 0.2) x 10-41 0.13 f 0.016 V <3.9 x 10-5 <0.017

Values in brackets are not certified.

Table IV. Measured Mass of Selected Elements in Bovine Serum Microdroplets

mass, ~g

element SIMS NBS

A1 10.2 f 5.5 0.11 f 0.04 Ca 1270 f 340 833 f 33 Mn 0.038 f 0.026 0.022 f 0.004 Fe 15.2 f 2.1 16.7 f 4.1 c u 190 f 86 6.2 f 1.0 Zn 4.4 f 2.0 9.2 i 1.1

capillaries, as discussed above. Consequently, equal volume mixtures of serum, spectroscopic grade methanol, and the 0.4-ppmw Y standard were prepared and centrifuged for ap- proximately 15 min. This centrifugation produced a very slight settling of solids that were assumed to be proteinaceous material. Twenty-five-nanoliter volmes of the supernatant solution were easily dispensed onto the carbon planchettes.

Low Y loadings (3.3 pg) were chosen for these samples in order that the SIMS instrument could be operated at maxi- mum detection sensitivity. The bovine serum mixtures were analyzed for Al, Ca, Mn, Fe, Co, Cu, and Zn by using 02+ SIMS. The measured 'X+/ Y+ intensities were converted to X mass values by using eq 1. The average and standard deviations of these calculated mass values are listed in Table IV along with the certified values for an 8.33-nL volume of bovine serum.

The measured Ca, Mn, and Zn masses agree approximately within a factor of 2 with the certified values, while the A1 and Cu values are significantly higher than the true values. The elemental masses in these serum residues were calculated by using relative ion yields determined from simple inorganic matrices containing 12.5-ng Y loadings, and the complex composition of the bovine serum matrix undoubtedly influ- ences the relative ion yields. The relatively good agreement between the measured and certified values for Ca, Mn, Fe, and Zn strongly suggests that the total sample consumption technique employed for these analyses minimizes SIMS matrix effects and provides reasonably constant relative ion yields. More accurate measurements of the elemental masses in these serum analyses could possibly be obtained by removing the proteinaceous components in the serum residues via a residue ashing procedure (12). We are currently investigating various methods of ashing these microvolume residues. Isotope ratios were measured for the 42Ca/40Ca, 54Fe/56Fe, and 66Zn/64Zn

ANALYTICAL CHEMISTRY, VOL. 60, NO. 19, OCTOBER 1, 1988 2075

DEPTH lmicronsl

Figure 6. Depth proflle analysis of isotopically enriched Pb sample.

species in these serum analyses, and the deviations (A) of these ratios from the true values were -0.062, +0.098, and -0.222, respectively. The Ca and Fe isotope ratios are in good agreement with the true values, which again indicates that the voltage offset technique eliminates cluster ion interferences at these masses even in the analysis of a complex matrix such as bovine serum. The large deviation observed for the Zn ratios is attributable in part to the low intensity of the Zn+ signals detected (average of 64Zn+ counts was 29) as well as a possible interference at m/z 66.

Only one of the three analyses for Co produced m/z 59 counts above the background level, and the calculated Co mass for this single analysis was 3.2 pg, which is substantially in error with the certified value of 0.01 f 0.002 pg. This large error suggests the presence of a mass spectral interference at mass 59, which could be the AlOz+ ion. Relatively intense (2000-5000) counts of Al+ were observed in these analyses, and thus an AIOz+ interference is a distinct possibility.

The high measured values observed for the A1 and Cu masses suggest possible contamination of the serum samples. The deviation of the measured e5Cu/s3Cu isotope ratio from the true value is -0.106, indicating that the m f z 63 and 65 ions are composed primarily of the copper species. A further possible source of error is the differential precipitation of Y and the analyte elements in the proteinaceous residue, which could give rise to variations in the relative ion yields for the different elements.

Stable Isotope Dilution Analysis. The isotope dilution technique consists of adding a known concentration or weight of an isotopically enriched sample of known isotopic abun- dance to the analyte sample and accurately measuring isotope ratios of the mixture (13). Since a SIMS isotope dilution analysis involves measuring the intensities of different iso- topes, there is no ion yield variation between the species of interest. Thus, SIMS quantitation of single elements by isotope dilution should be both precise and accurate.

The isotope dilution technique was evaluated by using an enriched P b sample obtained from Oak Ridge National Laboratories. The isotopic abundance5 for this material were as follows: 2"Pb, 61.0 f 0.1%; 206Pb, 19.63 f 0.05%; 207Pb, 7.89 f 0.05%; mPb, 11.49 f 0.05%. A measured mass of the enriched sample was dissolved in a solution containing a known mass of natural abundance lead. Four 25-nL droplets of this mixture were deposited onto separate areas of a carbon planchette, and the integrated Pb isotope intensities were determined for each residue. Figure 6 illustrates a typical depth profile analysis of one of these microdroplet mixtures. The Pb concentrations in the samples were approximately 0.8 mg/mL of 4.0 mM. The strong 'Pb+ intensities observed in these analyses were obtained at the smallest CD setting, which suggests that isotope dilution analyses could easily be per- formed at micromolar P b concentrations. The Oz+ SIMS ionization of Pb is not particularly efficient (Table I), and we envision performing isotope dilution analyses a t even lower analyte concentrations for the more easily ionized elements such as Ca, Fe, or Se (using Cs+ SIMS).

The Pb concentration in the analyte was calculated with standard isotope dilution formulas (13). Average Pb con- centrations were calculated to be 0.856 f 0.01 mg/mL, which is within 3% of the true value of 0.837 mg/mL. The precision of the isotope dilution analysis was f l % (relative standard deviation). We want to emphasize that these results were obtained from single 25-nL droplets. Clearly, the high pre- cision and good accuracy of this method indicates its potential utility for accurate quantitative analysis of microvolume bi- ological samples.

In summary, these results demonstrate that SIMS can provide very sensitive elemental analysis of microvolume sample residues. The development of more accurate methods will require improvements in both sample preparation and analysis. The quantitative results obtained in the SIMS isotope dilution experiment are very encouraging, and further investigations are in progress. Registry No. AI, 7429-90-5; Ca, 7440-70-2; Mn, 7439-96-5; Fe,

7439-89-6; Co, 7440-48-4; Cu, 7440-50-8; Zn, 7440-66-6; Se, 7782-49-2; Pb, 7439-92-1; graphite, 7782-42-5.

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RECEIVED for review January 4,1988. Accepted May 25,1988. Support for is research was provided by NIH Grant 1R43GM37898-01 through the Small Business Innovation Research Program.