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only four to five measurements of this type can be made. Figure 5 illustrates the raw data obtained during typical CAD experimental examination of various samples and extracts. It can be clearly observed that, in all cases, only five to six measurements for quantification purposes are obtained for each ion under the experimental protocol. In the case of the total ion chromatogram (TIC) detected through Q3, the number of data points is doubled since each point represents only half of the total experiment. In the case of reference standards (Figure 5A,B), the isotopically labeled ethyl carbamate was spiked at a slightly higher concentration than the unlabeled compound and the TIC reflected that increased level via higher alternate scan intensities that represented the total intensity observed for m / z 92 and 64. For the incurred residues (Figure 5C,D) the stable isotope was spiked at the same concentration level detected by gas chromatography with HECD-N and hence the TIC reflected an equality between the intensity of combined m / z 90 + 62 and combined m / z 92 64. Closer examination of the raw data illustrated in Figure 5A strongly suggests that the daughter ion ratio ( m / z 62:mlz 64) can be effectively used to a concentration level as low as 50 ppb of ethyl carbamate. However, the individual ratio measurements derived from each single experiment conducted within the 0.8-s-scan time frame have indicated a deviation from the expected results. It could be argued that what happens to m / z 90 should also happen to m / z 92 from the stable isotope. It should be remembered, however, that under CAD the measurement of the intensity of m / z 62 takes place about 0.5 s prior to measurement of the corresponding m / z 64. Since CAD experiments are concentration dependent, the possibility exists of a flaw in reliable quantification via isotope dilution techniques at low concentrations. While the observed
+
raw data (Figure 5) clearly indicated an elution profile under CAD, there are data points within those profiles that are of less intensity than those predicted for a smooth Gaussian shape. These discrepancies are evidence to support the notion that optimization of CAD experimental conditions (collision gas pressure and collision energy) plays an important role in ensuring precision and accuracy in quantitative results at low trace levels. In the present study, however, the elution profiles obtained by use of the megabore column were necessary to ensure sensitivity of detection to below the 30 ppb level. The exploration of daughter ions for quantitative analysis has revealed several interesting experimental factors that need to be closely monitored to ensure acceptable results. This abiality to rely on daughter ions (derived from protonated molecular ions) for quantitative purposes is an advancement in trace analysis via elimination of interfering ions observed in single-stage instruments. Registry No. Ethyl carbamate, 51-79-6.
LITERATURE CITED (1) Fed. Regist. 1972, 37(149), 15426. (2) Walker, G.; Winterlin, W.; Fouda, H.; Seiber, J. J. Agric. FoodChem. 1974, 22, 944. (3) Ough, C. S.J . Agric. FoodChem. 1976, 24, 323. (4) Ough, C. S. J. Agric. FoodChem. 1976, 2 4 , 328. (5) Ough, C. S. Chem. Eng. News 1987, 6 5 , 19. (6) Joe, F. L.; Kline, D. A.; Miletta, E. L.; Roach, J. A,; Roseboro, E. L.; Fario, T. J. Assoc. Off. Anal. Chem. 1977, 60, 509. (7) Bailey, R.; North, D.; Myatt, D. J. Chromatogr. 1986, 369, 193. (8) Dennis, M. J.; Howarth, Massey, R. C.; Parker, I. J. Chromatogr. 1986, 369, 199. (9) Bertrand, A,; Triquet-Pissard, R. Conn. Vigne V;n. 1986, 2 0 , 131. (10) Luke, M. A.; Froberg, J. E.; Doose, G. M.; Masumoto, H. T. J. Assoc. Off. Anal. Chem. 1981, 64, 1107.
RECEIVED for review October 7,1986. Accepted April 2,1987.
Spin-Deposited Submicrometer Films of Organic Molecules for Secondary Ion Mass Spectrometry Studies G . Siive,* P. Hfikansson, and B. U. R. Sundqvist Department of Radiation Sciences, Uppsala University, Box 535, S-751 21 Uppsala, Sweden U. Jiinsson' and G . Olofsson Laboratory of Applied Physics, National Defence Research Institute, Department 4, S-901 82 U m e i , Sweden
M. Malmquist' Division of Radiation Biology, National Defence Research Institute, Department 4, S-901 82 U m e i , Sweden Spin deposition is presented as a new sample preparation technique for producing submicrometer films of labile moiecules for mechanism studies of plasma desorption mass spectrometry (PDMS). Films of valine, Giy-Ala-Leu, and porcine insulin 1-400 nm thick were spindeposlted on silicon substrates with hydrophilic surfaces. PDYS, ellipsometry, and scanning electron microscopy showed that for film thicknesses greater than about 30 nm the films had a constant refractive index and were of fiat topography with a loose organic molecule air solvent structure that decomposes upon exposure to high humidity. The new technique has made it possible to study the film thickness dependence of secondary ion yields. Present address: Pharmacia Bio-sensor Sweden.
AB, S-75182 Uppsala,
The preparation of submicrometer solid films of involatile and thermally labile organic molecules is an important step in secondary ion mass spectrometry (SIMS) (1) and the high-energy primary ion version of SIMS, e.g. plasma desorption mass spectrometry (PDMS) (2,3).One question of fundamental interest in the study of these techniques is how the organic molecule desorption yields are correlated with sample film thickness. Samples of biomolecules in PDMS studies are usually prepared for analysis by using the electrospray technique (4).This method, however, gives clusterlike deposits of micrometer dimensions of the molecules on the surface, thereby making film thickness determinations unreliable. We present here a spin-deposition technique whereby it is possible to obtain homogeneous films of organic molecules in the submicrometer thickness range. Spin deposition is a well-known technique for processing of semiconductors where
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measured on the clean surface can be used to calculate the refractive index for the surface. Due to the presence of 1-2 nm thick spontaneously formed hydrated oxide on the silicon, this index will be a pseudo refractive index describing a composite first layer. According to Neal (12),for very thin films (10 nm), i.e. the hydrated oxide, the change in @ and A due to one overlaying film, i.e. the spin-deposited film, is the same as that which would occur if the film was present on a clean surface. We have therefore used a three-layer model, viz.,the ambient air, the spin-deposited film, and the silica/silicon substrate characterized by a pseudo refractive index, in our calculations of n and d of the film. Ellipsometry was performed with a Rudolph Research Ellipsometer (Model 43603) modified so that the polarizer and analyzer are controlled by a microcomputer via stepping motor drives. The wavelength of light was 632.8 nm and the measured area 0.5 cm2. The calculated n and d values are averages of measurements made at five different positions, which sample a 0.5 cm2 area in the center of each slice. Scanning Electron Microscopy. The surface topography of samples prepared by spin deposition was compared to that of samples prepared by the electrospray technique (4) in a scanning electron microscope (SEM) (JEOL JEM-100CX). Prior to examination the slices were coated with a 7 nm gold film by vacuum evaporation. PDMS. These experiments were performed as described in ref 13. The films were irradiated with a beam of 90 MeV InI ions from the Uppsala En-tandem accelerator and the masses of desorbed ions were determined by the time-of-flight method.
IPPETTE
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Figure 1. spin-deposition apparatus.
it is used to apply dopants or polymers on semiconductor surfaces. It is also used for electrode modification in eletrochemistry (5). The thickness (d) and refractive index (n)of films of organic molecules may be determined by ellipsometry or related techniques. Such studies have been performed for organic films in the monolayer range prepared by direct adsorption or Langmuir-Blodgett transfer (6-8). In this study the spin-deposition technique has been applied to three different small biomolecules. The film thickness and refractive index were determined by ellipsometry. Scanning electron microscopy was used to study surface topography. Successful tests have also been made in using the films as samples in PDMS investigations.
EXPERIMENTAL SECTION Substrates. Single crystalline silicone (1.1.1)n-phosphorus doped wafers with a resistivity of 6-8 Q cm and 330 pm thickness was purchased from Wacker-Chemitronic, FRG. The wafers were cut in 10 X 10 mm slices and these were etched for 2 min in 10% (v/v) hydrofluoric acid (Merck, FRG) and thereafter rinsed in distilled water. The slides were then rinsed as desribed in an earlier paper (9). This treatment rendered the surfaces hydrophilic. The slices were kept in ethanol until use. Before spin deposition the slices were blown dry in nitrogen gas. Organic Molecules and Solvent. DL-Valine and the tripeptide Gly-Ala-Leu were purchased from Sigma Chemical Co., St. Louis, MO, and Bachem, Switzerland, respectively. Porcine insulin was obtained from Novo, Denmark. Trifluoroacetic acid (Spectroscopicgrade, Merck, FRG) was used as solvent to prepare 5-15 mg/mL stock solutions of the organic molecules in glasa vials. All glassware was cleaned before use for 12 h in Hellmanex (Hellma, GDR), rinsed in tap water followed by distilled water, and blown dry in nitrogen gas. Spin Deposition. A spin-deposition apparatus was constructed from a dc motor (6000-12000 rpm) and a Teflon holder (Figure 1). The apparatus was placed vertically in a fume hood and the siilicon slices were fixed to the holder by double-sides adhesive tape. For spin deposition the motor was started and one drop (approximately20 pL) of the organic molecule solution was applied at the center of the spinning slice (Figure 1). The spin deposition was performed by applying 1 drop of solution containing the organic molecule to a spinning slice. After 5-10 s the motor was shut off and the slice transferred to a desiccator. The spin deposition was performed at 20 O C and at a relative humidity of 40-45 % . Ellipsometry. Ellipsometry is an optical technique in which the change in the state of polarization of light upon reflection from a surface is used to characterize the surface. For further information about theory, instrumentation, and calculations,see Azzam and Bashara (IO)McCrackin (11). Ordinary null-ellipsometry gives two parameters: change of amplitude, tan a, and phase, A, of the light upon reflection. The values of @ and A
RESULTS AND DISCUSSION Spin Deposition. The most essential factor in order to obtain reproducible n and d of the films was found to be application of the droplet in the center of the spinning slide, Figure 1. Furthermore the solution must be free of contaminates such as dust particles. However, it was not necessary to work in a strick dust-free environment. It was found that another important parameter was the relative humidity of the air. Above a relative humidity of about 60% the valine and the tripeptide films especially showed tendencies to crack and the formation of a heavy haze on the surface was observed. Apparently the structure of the films could not be preserved above a certain degree of film hydration. The spin-deposited films were therefore stored in a desiccator and the integrity of the films could in this way be preserved for several months. Another important property of sample films to be used in PDMS is that they are not influenced by vacuum. This was checked by comparison of ellipsometric measurements of the films before and after the vacuum treatment in the plasma desorption experiments. The films were indistinguishable from the bare silicon slices when comparing the light scattering under oblique illumination in the ellipsometer indicating the absence of articulate matter in the films. Thickness dependent interference colors were seen for thickness above 60 nm. The colors could be used as an aid to develop the technique for a certain solvent/organic molecule combination. Thus,to a f i t approximination, the smoothness of the films could be estimated by the unaided eye during the spin deposition and later verified by the ellipsometer. Ellipsometry. Figure 2 shows n as a function of d for spin-deposited fiis of valine, Gly-Ala-Leu, and insulin. The variation in @ and A for the five measurements made on each slide was always within f0.1'. The n values are somewhat scattered and increase drastically to values of around 1.8-2.0 below a film thickness of around 30 nm. Above this thickness the n values converge to 1.46, 1.47, and 1.53 for valine, GlyAla-Leu, and insulin, respectively. The optical model assumed for the spin-deposited films is that of a homogenous film with discrete boundaries. This is probably not true for very thin films, where refractive index inhomogenieties normal and parallel to the surface may appear (14). Another aspect that also must be taken into account
ANALYTICAL CHEMISTRY, VOL. 59. NO. 17. SEPTEMBER 1, 1987
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REFRLICTIVE INDEX
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is the limitations of McCrackin's computational method in the case of very thin films on silicon. This has been discussed by Yoriume (15). Our situation may be similar to the study of Vedam et al. (16). Their results indicated that McCrackin's computational method was not sufficiently accurate to determine both n and d for films thinner than 25 nm. In most ellipsometry studies of Langmuir-Blodgett films the actual value of n for a close-packed monolayer is assumed to differ only slightly from the bulk material. The bulk value of n is accordingly aasigned in the calculations and the f h thickness may be calculated with good accuracy. The basic assumption is of course that a homogenous monolayer is formed in the Langmuir-Blodgett transfer. The smooth reproducible behavior of yield vs. film thickness in Figure 5 indicates that this may be true also in our case and that the problem is in the computational method. However, this conclusion should be checked in an experiment where it is known that the film
experiments are under way in our laboratory. The films were easily reproduced and the results for thickness above 30 nm, where the refractive index is independent of thickness, suggested homogenous films. In the case of insulin some experiments were performed in which the film thickness was extended to the range of 100-400 nm. Also in this case the n values were constant (1.53). The amount of material in the films expressed as pg/cm2 can be calculated from the obtained n a n d d values (8,11). These calculations require knowledge about molar refractivity and partial specific volume. These constants are not easily obtainable, especially in the case of proteins. The constants may, however, be estimated in an indirect way by a combination of radiotracer methods and ellipsometry (8).We are currently investigating the combination of these two methods in order to obtain the amount of material in the films. Scanning Electron Microscopy. Figure 3 shows a comparison of spin-deposited and electrosprayed insulin. SEM of spin-deposited filmsindicate contrary to the electmaprayed samples, no particular matter larger than 10-20 nm. The same type of result was found for spin-deposited films in the 10-100 nm range. PDMS. Figure 4 shows the results of positive ion desorption from f h of valine, Gly-Ala-Leu, and porcine insulin. No significant differenee was observed in the PDMS spectra from spin-deposited (Figure 4c) and electrosprayed samples (Figure 4d), with regard to the degree of cationization and
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