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Anal. Chem. 1988, 60, 1521-1524
Pneumatic Microsyringe for Use as an Injector in Open Tubular Liquid Chromatography and as a Dispenser in Microanalysis Robert T. Kennedy and James W. Jorgenson* Department of Chemistry, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599-3290 A pneumatic microsyrlnge Is described and characterized for use as a micrdnjector for open tubular ilquid chromatography and for use as a microdispenser. As a microinjector, the syringe had a relative standard devlation of 2.7% in volume delivered for 10-nL Injections on a 15 pm Inner diameter column and showed the same contribution to peak broadening as other methods of injectlon. The volume injected is easily changed by simply changing the length of tlme the Injection is made. Thls device Is useful for Injecting samples of ihnited volume. As a microdispenser, the device had a relatlve standard deviation of 3.38% In volume delivered for dispensing 0.248 nL. I n this fashlon the syringe can be used to add Internal standards or reagents to small samples. The syringe can be cailbrated for mlcrodispenslng by measuring the size of a droplet fomed from injecting an aqueous solution into mineral oil.
Open tubular liquid chromatography (OTLC) is of interest as a separation method for several reasons. These reasons are generally seen as high efficiencies, high permeabilities, low flow rates, and improved mass detection for concentrationsensitive detectors. One aspect of OTLC that is rarely mentioned and has only recently been taken advantage of ( I ) is that OTLC, because of the small column dimensions, is useful for the analysis of small samples. Columns with inner diameters of 15 pm typically have volumes of hundreds of nanoliters, and injection volumes are normally 5 nL or less. The fact that OTLC can be used for small samples offers interesting possibilities for ultramicroanalysis such as the analysis of individual cells (1). T o perform such analyses requires, in addition to columns and detectors, appropriate methods of sample introduction, and sample and reagent handling. Most current methods of sample injection for OTLC utilize a splitting arrangement (2-4), which wastes a majority of sample and so is not useful for microanalysis. One injector has been described for OTLC that is potentially useful for microsamples. This injector, which was based on connecting sample tubes to the column, was used to inject 40-nL sample onto 24 pm i.d. columns and had a reproducibility of 5-8% under limited conditions. It is not clear if this injector would be useful for smaller samples and smaller inner diameter columns, and its effect on column performance was not well-defined. This injector was also difficult to use for varying sample sizes (5). We recently described a microinjector that allowed the introduction of samples in the nanoliter range with no waste. This microinjector consisted of a syringe driven by a micrometer and connected by tubing to a micropipet. The entire system was fiied with mercury so that samples could be filled or expelled from the micropipet tip. Chromatographic injections were performed by inserting the micropipet, which contained the sample, into the inlet end of the OTLC column. The sample was forced into the column by adjusting the
* Author to whom correspondence should be addressed. 0003-2700/88/0360-1521$01.50/0
micrometer ( I ) . This microinjector was limited by the fact that it was not reproducible because of leaks and compressibility in the system. In this report we describe a new microinjector that injects volumes as small as 3 nL into columns with inner diameters at least as small as 15 Fm. Another important aspect of ultramicroanalysis is the ability to dispense samples and reagents on the subnanoliter scale. Sample and reagent handling on this scale has been done for many years, and a number of devices have been described to perform such tasks. One of the primary reasons for the development of microdispensing was for the introduction of substances into individual cells (6). Others have since used microdispensers in the analysis of nanoliter samples (7, 8). Microdispensers are of two main types. The earliest type was based on micropipets fiied with reagent to which pressure is applied to force the reagent out in a controlled manner. Pressure can be applied by either a hydraulic or pneumatic system. The other type of microdispenser is based on forming droplets, which are then allowed to fall to where they are needed (7). This type of dispenser can accurately add volumes as small as 4 nL. In this report we describe how the microsyringe can be used to dispense solutions for use as internal standards (or other reagents) in the nanoliter and subnanoliter range.
EXPERIMENTAL SECTION The pneumatic microsyringe that is used in this report is a modified version of an intracellular injector described by McCaman et al. (9) and Corson and Fein (10). A schematic of our design is shown in Figure 1. A double-regulated supply of pressurized helium gas is connected to a third regulator (Clippard Instrument Co. Model MAR-1) and then to a pressure gauge (Clippard). The gas line then goes through a normally closed electropneumtic valve (Clippard Model EV-3-12) and through a pneumatic s,witch (Clippard Model FTV-3) to a borosilicate glass micropipet that has an outer diameter at the tip of less than 10 Hm. The injection micropipet was pulled from 0.5 mm i.d. X 1.0 mm 0.d. glass tubing with a commercial pipet puller (Pul-1 from World Precision Instruments, Inc.). The electrical circuit operates so that when a voltage pulse is applied to the transistor from the timing circuit, the electropneumatic valve opens for the duration of the pulse. The pneumatic switch allows the user to switch between the pressurized side and a vacuum line. The vacuum line consists of a vacuum source, in this case an aspirator, connected to an electropneumatic valve, which is also controlled by the timing circuit. The vacuum line allows the pipet to be filled from the tip. To make a chromatographic injection with this pneumatic microsyringe, the inlet end of the chromatography column is removed from the pressurized mobile phase source and mounted on a positioning stage (Oriel 16616 with 16021)so that it is visible under a stereomicroscope (Wolfe Selectra 11). The micropipet containing the sample is mounted in a micromanipulator (Narishiga MM-333), and the pipet is inserted into the end of the column far enough to press against the column. When the pipet is inserted in this manner, it forms a seal that is tight enough to allow the sample to be forced into the column as previously described ( I ) . A pressure pulse is then applied to the pipet t o force the sample into the column. For the injections, pressures of 210 kPa (30 psi) were used, and times were as noted. Once the pressure pulse had been applied, the pipet was removed and the column reconnected to the mobile phase source to begin the chromatography. 0 1988 American Chemical Society
ANALYTICAL CHEMISTRY, VOL. 60,
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NO. 15, AUGUST 1, 1988
‘c
TIMING
6 Flgure 1. Schematic diagram of pneumatic microsyringe: A, third pressure regulator; B, pressure gauge; C, sdlendd valves; D, pneumatic switch; E, micropipet: F, double-regulated pressure supply; 0, vacuum
source. Our usual method of injection, to which the new injector is compared in this study, is a static split injection and has been described before (4). The column that was used in this study was a 15.1 pm i.d. column, with dimethyloctadecylsilane stationary phase bound to the inner wall, fabricated in our laboratory (11). The dimensions of the column were determined by using a previously described method (12). The mobile phase was 0.1 M phosphate buffer adjusted to pH 3.1 with sodium hydroxide. The detection system used was an on-column electrochemical detector, which has been previously described (13). The detector consisted of a carbon-fiber microelectrode inserted into the outlet end of the column and held at a fixed potential of +0.5 V versus Ag/AgCl. The current generated was amplified by using a Kiethley 427 set to have a time constant (7)equal to 150 ms. The detection system was calibrated by injecting known volumes, with the static split injector, of a series of five to six concentrations of the compound of interest. The area of the resulting chromatographic peaks was measured in coulombs and then converted to moles by using Faraday’s constant. From these data the conversion efficiency of the electrode, that is, the ratio of moles detected t o moles injected, was determined. By use of the conversion efficiency, the injected volume of standards could be determined from the area of the chromatographic peak. All chemicals were from Sigma Chemical Co. All solutions that were injected into the column were dissolved in the mobile phase. The separation efficiencies, areas, and variances of the peaks were measured by using a statistical moments computer program developed in-house.
RESULTS AND DISCUSSION Microinjecting. An important feature of an injector is ita ability to reproducibly inject a given volume. This was tested with the new microinjector by making a series of seven injections of lo4 M hydroquinone (HQ) at 210 W a for 60 s each. The average area of the peaks was 195 nC, representing a volume of 10.1 nL, with a relative standard deviation (RSD) of 2.27%. This compares to an RSD of 1.45% by the static splitting method of injection under similar conditions. Variation in the amount injected by the microinjector may come from two sources. The first is the result of diffusion of analyk out of the tip of the pipet into the mobile phase in the column before the pressure pulse is applied. Since there is a variable amount of time that the pipet is in contact with the mobile phase before the pressure pulse is applied, variable amounts of analyte will diffuse into the column. This could be limited by using pipets of smaller tip inner diameters, although this would increase the time required to fill the pipet and increase
the possibility of clogging the pipet tip. Another possible source of error is that leaks can occur at the point of contact between the pipet and the column. Such leaks have been observed to occur no matter how carefully the pipet is placed inside the column. In spite of this problem, it is apparent that the injector exhibits acceptable reproducibility for many applications and represents an improvement over currently available injectors for samples of limited size. It should be possible to vary the amount injected by changing the applied pressure or the pulse duration. However, it is considerably easier to accurately set the time of injection rather than the pulse pressure; therefore, only the time was thoroughly studied. The pressure of injection used for this study, 210 kPa, appeared to be the most useful for this type of injection. Pressures above 275 kPa (40 psi) resulted in the pipet being pushed away from the column, allowing the majority of the sample to escape. Lower pressures could be used; however, they would result in proportionally longer injection times. Thus, 210 kPa was used for all injections. It was of interest to determine the response of the injector to different pulse duration times. This was investigated by making a series of five injections of M hydroquinone at 210 kPa for pulse durations between 30 and 200 s. The volume that was injected was determined from the peak area and the conversion efficiency of the electrode as described in the Experimental Section. There is a linear relationship between the volume injected and the pulse duration for the entire range of volumes (3.4-23 nL) tested, as the data had a linear correlation coefficient of 0.9994. The slope of the linear data plot is 0.117 nL/s and represents the rate of delivery of sample into the column during the injection. It is interesting to note that the Poiseuille equation for volumetric flow in open tubes predicts that the flow in the chromatographic column (15.1 pm i.d. X 217 cm long) of a solution with the viscosity of the mobile phase (TJ = 0.00104 N s/m2) (14) under 210 kPa would be 0.117 nL/s. This agreement with the observed flow rate leads to some important conclusions. First, it means that the majority of the resistance to flow during the injection is due to the column itself and not to the pipet tip. Therefore, all pipets of similar size and shape should give the same injection volumes, and once an injection volume calibration plot for a given column and pressure has been made, it will be useful for all pipets used with the column. Our experience with various pipets has found this to be true. The fact that the Poiseuille equation predicts the rate of sample injection also means that the volume injected can be approximated from the knowledge of column dimensions, mobile phase viscosity, and applied pressure, without a calibration curve. For accurate work, however, it is desirable to use internal standards or a calibration curve to determine the exact volume injected. The calibration plots indicate that there is a small negative intercept; the intercept for the line discussed above was -0.341 nL. It is not clear what causes this nonzero intercept, but it has occurred with all calibration plots and suggests some loss of sample that is independent of the volume injected. It may be due to loss of samples as the pipet is removed from the column. The nonzero intercept causes the injector to be more accurate the larger the volume injected. Another important characteristic of a chromatographic injector is its injection profile. The injection profiles for both the pneumatic microsyringe and the static split injector were investigated by using the method outlined by Sternberg (15) and Karger et al. (16). If the only sources of variance in the chromatographic system are the column (col) and the injector (inj) then the total variance of the system (u?) can be expressed as follows: where u,?
is the variance due to the column, Vhj is the volume
ANALYTICAL CHEMISTRY, VOL. 60, NO. 15, AUGUST 1, 1988
Table I. Data for Investigation of Injection Profiles, Plot of u t versus Vinf
static split
microinjector
vol range, nL corr coeff
4.3-17.2 0.9999
slope ( 1 / P ) intercept, nL2
0.0509
3.4-16.6 0.9997 0.0322 3.57
3.30
Table 111. Calibration Curves for Microsyringe Obtained with Different Volume-Measuring Techniques
slope, nL/s time axis intercept, ms linear corr coeff
~~
~
Table 11. Comparison of Efficiencies for the Mi’croinjector and the Static Split Injector
N M (k’= 0) NHQ(k’= 0.26) NCAT(k’= 0.71)
static split 3.3 nL
microinjector 3.8 nL
114000 129000 113000
127 000 126 000 112000
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injected, K is a constant characteristic of the injector, and the V,j2/p term is the total variance due to the injector. This equation assumes that the detector is not a source of variance, which is approximately true in this case because of the oncolumn detection. Given that the detector cell volume is 0.115 nL, the amplifier time constant is 150 ms, the data acquisition rate is 1 Hz,and the mobile phase flow rate is 0.88 nL/s, the corresponding volumetric variance associated with the entire detection system is 0.079 nL2, which is insignificant relative to the column variance. This treatment assumes a plug-type flow profile for analyte flow. Since the mean time for diffusion from the wall to the center of a 15-pm capillary is typically 50 ms, this assumption will introduce minimal error. It is apparent from eq 1that the larger the value of K , the less total dispersion of a peak. K can be determined from the slope of the linear plot of u t versus Vinj2.K was determined for the microinjector to be 5.56 and for the static split injector to be 4.43 by using the data in Table I. According to Sternberg’s theory, an exponential profile gives K = 1, a plug injection profiie gives K = 3.5, and a Gaussian injection profie has K = 6. The K values obtained indicate that the injection profiie for the static split injector is something between a plug and Gaussian. This is a surprising result, as it was expected that this injector would yield a plug profile. The difference may be due to the laminar flow profiles in an open tube. The K value for the microinjector indicates that its profile is approximately Gaussian. Guiochon has observed that a syringe injector for gas chromatography has a Gaussian injection profile (17),and the same principles may apply for the microinjector. It should be noted that the K values reported here are considerably higher than those that have been reported for other injectors for liquid chromatography (16,18). The intercept of eq 1 is equal to the variance of the column; therefore, no matter what injector is used, this value should be the same. It is interesting to note the agreement between the two data sets in Table I for the value of the column variance. Although the profiles give an indication of the effect that the injector has on the efficiency of the columns, it is also useful to measure the theoretical plates under high-resolution conditions. The average plate numbers for three runs of three different compounds, ascorbic acid (AA), HQ, and catechol (CAT), were measured for the static split injector and the microinjector and are given in Table 11. The mobile phase flow rate was 0.079 nL/s for the runs. The efficiencies shown are not strictly comparable because slightly larger volumes were injected with the microinjector. Nevertheless, the theoretical plates measured show that there is virtually no difference between the two injectors, which is expected because
radioactivity
optical
2.61 4.4 0.9984
2.63 60.5 0.9960
of the similar K values. Therefore, the microinjector can be used to inject samples of nanoliter volume and achieve results comparable to those obtained by using the static split injector. Microdispensing. The pneumatic microsyringe can also be used to accurately add subnanoliter amounts of liquid to small volumes of solutions. This will undoubtedly be useful in a number of applications in the area of microanalysis. One possibility, which is currently being exploited in our laboratory, is to add internal standards to small samples. Another possibility is to add reagents to samples of small volume. The operation of the microsyringe in this capacity is different from that as a microinjector. To dispense solution into a liquid, the pipet tip containing solution is lowered into the solution of interest, a pressure pulse is applied (the duration of the pulses is generally shorter for a given volume than when injecting into the column because of considerably lower resistance to flow), and the pipet is then quickly withdrawn. It is important to insert and withdraw the pipet quickly when adding subnanoliter volumes because diffusion out of the tip of the pipet can affect the amount added. In order to use the pipet it is necessary to be able to reliably calibrate it. This was done initially by making a series of five injections of a solution of radioactive iodide (lZ5I) into deionized water, using varying pulse times, and then determining the volume added from the resultant radioactivity of the water. The syringe exhibited a linear relationship, with a correlation coefficient of 0.9998, between the volume added and the pulse time for volumes ranging from 0.245 to 1.32 nL and for pulse times ranging from 103 to 517 ms. Different pipets showed different calibration curves; however, all were linear with high correlation coefficients. The calibration curves all showed intercepts on the time axis of around 4 ms. This presumably represents the difference between the valve opening and closing times at 210 kPa and is a characteristic of the valve. The syringe also showed good reproducibility, as seven injections at 103 ms were done and the average volume ejected was 0.248 nL with a RSD of 3.38%. The syringe was also calibrated by injecting aqueous solution into mineral oil and then calculating the volume of the resultant drop by using a microscope fitted with a reticle, as described by others (9, IO). This optical method is considerably more convenient than the radioactivity method; however, the calibration curves are slightly different, as shown in the example given in Table 111. In general, it was observed that the slopes obtained by using the two methods were similar but that the time axis intercepts were higher and more variable with the optical method. (The cause of this is not certain, but it is probably due to the surface tension between the water and the mineral oil.) This suggests that good calibrations can be obtained by using the optical method to calibrate the pipet and then adjusting the resultant line. The working calibration curve is a line that has a slope equal to that which is determined from the optical calibration and a time axis intercept of 4 ms, the delay time of the solenoid valve. This correction helps to account for the error in the time axis intercept associated with the optical calibration.
ACKNOWLEDGMENT We thank David Weber for assistance in performing the radioactivity measurements and Robert L. St. Claire I11 for fabricating the chromatography column used in this work.
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Anal. Chem. 1988, 60, 1524-1529
LITERATURE CITED Kennedy, R. T.; St. Claire, 111; R. L., White. J. G.: Jorgenson, J. W. Mlkrochim. Acta 1988, 1967, II, 37-45. Manz, Andreas; Simon, Wlihem J. Chromatogr. 1987, 387, 187-196. Yang, F. J. Chromatogr. 1982, 236, 265-277. Jorgenson, J. W.; Guthrie, E. J. J. Chromatogr. 1983, 255,335-348. Capacci, M. J.; Sepaniak, M. J. J. Liq. Chromatogr. 1986, 9(15), 3365-3376. Graessmann, M.; Graessmann, A. Methods Enzymol. 1983, 101, 482-493. Shabushnig, J. G.; Hieftje, G. M. Anal. Chim. Acta 1981, 126, 167-1 74. Vurek, G. G.; Bowman, R. L. Anal. Chem. 1984, 56, 391A-405A. McCaman, R. E.; McKenna, D. G.: Ono, J. K. Brain Res. 1977, 136, 141-147. Corson, D. W.; Fein, A. Biophys. J. 1983, 4 4 , 299-304. St. Claire, R. L., 111 PhD. Thesis, University of North Carolina at Chapel Hill, Dec. 1986. Guthrb, E. J.; Jorgenson, J. W.; Knecht, L. A,; Bush, S. G. HRC CC, J. Hiah Resolut. Chromatoor. Chromatoor. Common. 1985, 6, 566-567. Knecht, L. A.; Guthrie, E. J.; JOrQWIson, J. W. Anal. Chem. 1984, 56, 479-402.
(14) Weest. R. C. Herhibook of Chemkby and Physics, 61st ed.; CRC Press: Boca Raton, FL, 1980: p 13226. (15) Sternberg, J. C. Advances In Chromtogrephy Volume 2 ; Gddings, J. C., Keiler, R. A., Eds.; Marcel Dekker: New York, 1966; pp 205-270. (16) Karger, B. L.; Martin, M.; Guiochon, G. Anal. Chem. 1974, 4 6 , 1640-1647. (17) Guiochon. G. Anal. Chem. 1983, 35, 399-400. (18) Gluckman, J. C.; Hirose, A.; McGuffin, V. L.; Novotny, M. Chromatographk, 1983, 77, 303-309.
RECEIVED for review December 7,1987. Accepted March 28, 1988. This work was supported by the donors of the Petroleum Research Fund, administered by the American Chemical Society, and the University Research Council of the University of North Carolina. R.T.K. received support from a North Carolina Governor's Board of Science and Technology Fellowship and from an American Chemical Society Analytical Fellowship sponsored by the Society for Analytical Chemistry of Pittsburgh.
Determination of Phosphorus Distribution in the Silicon Dioxide/Silicon Layer System by Secondary Ion Mass Spectrometry Gerhard Stingeder Institute of Analytical Chemistry, Laboratory for Physical Analysis, Technical University Vienna, Getreidemarkt 9/151, A-1060 Vienna, Austria
A measurement technique for quantitative distrlbutlon analysis of phosphorus in the layer system SIOz/SI was developed. Oxygen primary Ions and an increased oxygen pressure (5 X l o 4 mbar) In the sample chamber were used for ellmlnatlon of the matrix effect. Preclse adlustment of the mass spectrometer during depth profiling wRh high mass resolution was controlled by a computer routine. Charging effects were compensated by flooding of the sample wlth electrons, optimized biaslng of the accelerating potential of the secondary ions, and nonnaikatton to a reference stgnal. A detection limit of 1 X lo'@ atoms-cm-8 and an accuracy of f30% were obtained. The improved measurement technique was used for determinatlon of segregatlon coefficlents, whlch are used as Input parameters for process modeilng in metal oxlde semiconductor transistor production.
dopant elements, oxidation, and nitriding). To obtain physical data, suitable experiments have to be developed (see for example ref 1 and 2) and depth distributions of dopant elements have to be determined with high accuracy (3-6). Phosphorus is one of the major n-dopants in silicon. Its diffusion mechanism in silicon is still under intensive discussion (for a review see ref 7). Also, the redistribution in the layer system Si02/Si is of great importance for the optimization of the oxidation process in metal oxide semiconductor (MOS) production. To study the segregation, simultaneous measurements of P in Si02 and Si are necessary. Chu et al. (8) performed quantitative determination of high concentrations of P in borophosphosilicate glass without separation of the interfering SiH and obtained a detection limit of -0.1 atom %. Measurements of the distribution of P with secondary ion mass spectroscopy (SIMS) down to concentrations of 1015 a t ~ c m -are ~ difficult because high mass resolution is necessary to separate the 30Si1Hinterference. In a previous paper we have presented an analytical approach for the quantitative determination of P in silicon with an accuracy of f25% (relative) and a detection limit of 1015a t ~ c m -(9). ~ In this paper optimization of the measurement technique for quantitative distribution analysis of P in the layer system Si02/Si is presented. This approach is generally applicable to depth profiling with high mass resolution in insulating (poorly conducting) materials. For quantitative measurements the following three requirements have to be fulfilled: (1)precise peak switching of the mass spectrometer between analytical masses and the
-
In very large scale integration (VLSI) technology, tolerances of process parameters are decreasing. Process modeling (specifically the simulation of dopant profiles) has become an essential development tool. Due to rapid downscaling in the last few years, process models have to be improved to describe the dopant profiles with sufficient accuracy for integrated circuit manufacturing. Temperature steps (typical temperatures 800-1100 "C) are used in every production process. In order to obtain models with a sound physical base, understanding of the physical processes influencing the diffusion of dopants and self-diffusion is very important (e.g. mutual diffusion of different
0003-2700/88/0360-1524$01.50/00 1988 American Chemical Society
