Nanoflow Gradient Generator for Capillary High-Performance Liquid

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Anal. Chem. 2004, 76, 1524-1528

Nanoflow Gradient Generator for Capillary High-Performance Liquid Chromatography Kisaburo Deguchi,*,† Shinya Ito,‡ Shinji Yoshioka,‡ Izumi Ogata,‡ and Akihiro Takeda‡

Hitachi High-Technologies Company, Hitachinaka 312-8504, Japan, and Division of Biological Sciences, Graduate School of Science, Hokkaido University, Sapporo 001-0021, Japan

A novel method of generating a nanoflow gradient elution for a capillary high-performance liquid chromatography (HPLC) system has been developed. An important feature of this system is that any gradient (GR) profile generated by a conventional microflow GR pump can be asymptotically traced and converted as a corresponding nanoflow GR profile simply by using a 10-port switching valve with two injection loops installed. Consequently, it has been called an “asymptotic trace 10-port valve” (AT10PV) nanoflow GR generator. Performance of the AT10PV nanoflow GR generator was tested in the range of flow rates from 50 to 500 nL/min. The test demonstrated that the AT10PV nanoflow GR generator can asymptotically trace the original gradient profile with good reproducibility. A capillary HPLC system using the AT10PV nanoflow GR generator provides reasonably good repeatability of peak retention times on the chromatogram of the tryptic digest of a BSA sample, RSD of less than 0.3% at a flow rate of 200 nL/min. It also enables sequential running of a series of sample injections in the same manner as conventional analysis at microflow rates. The recent rapid growth of proteomics and glycoproteomics for analyzing very small sample amounts has established a strong demand for a stable and reliable microflow or nanoflow gradient elution system (hereafter, called a microflow GR or nanoflow GR generator) for a capillary high-performance liquid chromatography (HPLC). This is especially true for use in capillary liquid chromatography-mass spectrometry (LC-MS) and capillary electrochromatography-mass spectrometry (CEC-MS).1-3 Capillary HPLC systems using a small-bore column of less than 1-mm internal diameter (i.d.) have a relatively long history. Such systems were initiated by several research groups during the late 1970s. Soon, packed or open-tubular microcapillary columns with more than 100 000 theoretical plate numbers were developed.4-12 * Corresponding author. Telephone: +81-11-706-9030. Fax: +81-11-706-9032. E-mail: [email protected]. † Hokkaido University. ‡ Hitachi High-Technologies Co. (1) Aebersold, R.; Mann, M. Nature 2003, 422, 198-207. (2) Mechref, Y.; Novotny, M. V. Chem. Rev. 2002, 102, 321-369. (3) Abian, J.; Oosterkamp, A. J.; Gelpi, E. J. Mass Spectrom. 1999, 34, 244254. (4) Scott, R. P. W.; Kucera, P. J. Chromatogr. 1976, 125, 251-264. (5) Scott, R. P. W.; Kucera, P. J. Chromatogr. 1978, 169, 51-72. (6) Ishii, D.; Asai, K.; Hibi, K.; Jonokuchi, T.; Nagaya, M. J. Chromatogr. 1977, 144, 157-68.

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Recently, MacNair et al.13-15 reported that ultra-high-pressure capillary columns packed with 1.5-µm-i.d. nonporous silica particles provide more than 200 000 theoretical plate numbers, enabling a high-speed analysis. As for the detection unit, MS, especially, incorporating an electrospray ionization source (ESI) has become a very powerful and promising solution in micro- and nano-HPLC systems because of the ease of connection and compatibility.3 Progress in the solvent delivery unit with gradient elution capability seems to be slow, however. A simple, stable, and reliable gradient elution method at lower microliter per minute and nanoliter per minute rates is still being sought. Several solutions and methods for microflow gradient elution were proposed and developed at the early stages of micro-HPLC development. They may be classified as follows: (1) flow splitting,16,17 (2) miniaturization of high-pressure gradient pumps,16,18 (3) exponential (sigmoidal) gradient formation,19,20 and (4) offline formation and storage of gradient solvents in injection loop(s) or preformed gradient loop(s).5,16,21 The flow-splitting method is quite simple and, therefore, is most often used. Chervet et al.22 reported very good repeatability of retention times (RSD ( 0.1% for isocratic elution and (0.2% for GR elution) even on a nanoHPLC system using a UV detector. More recently, Shen et al.23 also reported peak retention times with RSD less than 1% in proteomic analysis using a nano-ESI/MS detector. However, a problem of the splitter is that it always has some potential for (7) Tsuda, T.; Hibi, K.; Nakanishi, T.; Takeuchi, T.; Ishii, D. J. Chromatogr. 1978, 158, 227-32. (8) Ishii, D.; Tsuda, T.; Takeuchi, T. J. Chromatogr. 1979, 185, 73-78. (9) Tsuda, T.; Novotny, M. Anal. Chem. 1978, 50, 271-275. (10) Novotny, M. Anal. Chem. 1981, 53, 1294A-1301A. (11) Novotny, M. Anal. Chem. 1988, 60, 500A-509A. (12) Kennedy, R. T.; Jorgensen, J. W. Anal. Chem. 1989, 61, 1128-1135. (13) MacNair, J.; Lewis, K. C.; Jorgensen, J. W. Anal. Chem. 1997, 69, 983989. (14) MacNair, J.; Opiteck, G. J.; Jorgensen, J. W.; Moseley, M. A., III. Rapid. Commun. Mass Spectrom. 1997, 11, 1279-1285. (15) MacNair, J.; Patel, K. D.; Jorgensen, J. W. Anal. Chem. 1999, 71, 700708. (16) Hirata, Y.; Novotny, M. J. Chromatogr. 1979, 186, 521-528. (17) Van der Wal, S.; Yang, F. J. J. High Resolut. Chromatogr. Chromatogr. Commun. 1983, 6, 216-217. (18) Takeuchi, T.; Ishii, D. J. Chromatogr. 1982, 239, 633-641. (19) Takeuchi, T.; Ishii, D. J. Chromatogr. 1982, 253, 41-47. (20) Ishii, D.; Hashimoto, Y.; Asai, H.; Watanabe, K.; Takeuchi, T. J. High Resolut. Chromatogr. 1985, 8, 543-546. (21) Takeuchi, T.; Niwa, T.; Ishii, D. J. Chromatogr. 1987, 405, 117-124. (22) Chervet, J. P.; Ursem, M.; Salzmann, J. P. Anal. Chem. 1996, 68, 15071512. (23) Shen, Y.; Zhao, R.; Berger, S. J.; Anderson, G. A.; Rodriquez, N.; Smith, R. D. Anal. Chem. 2002, 74, 4235-4249. 10.1021/ac0350312 CCC: $27.50

© 2004 American Chemical Society Published on Web 02/03/2004

Figure 1. Schematic diagram of the capillary HPLC system based on the AT10PV nanoflow GR generator. The section enclosed in the dotted lines is the AT10PV nanoflow GR generator, which consists of a conventional microflow GR pump with low-pressure GR capability, a nanoflow isocratic pump, a 10-port switching valve with two injection loops (A and B), and a back-pressure coil or column.

susceptible and unpredictable changes in flow rate caused by unexpected changes in the pressure of the flow system by clogging. Miniaturization of high-pressure gradient pumps also has difficulties in the nanoflow rate range. For example, if a solvent composition ratio is A:B ) 99:1 at a flow rate of 50 nL/min, the pump delivering solvent B has to precisely control the flow rate to 0.5 nL/min. A combination of two electroosmotic flow (EOF)driven pumps and a combination of an EOF pump with a HPLC gradient system have been used for gradient elution in CEC.24,25 EOF-driven pumps seem to be suitable for truly miniaturized separation systems because no mechanical parts are required. However, these systems do require a special flow-feedback control to create gradient profiles and to maintain constant electroosmotic flow. In addition, the solvent composition suitable for the EOF pumps might not be suitable for column separation. Interest in the exponential (sigmoidal) gradient formation method, which was used by Ishii et al. for their micro-HPLC,19,20 has recently been revived because of its simplicity 13-15,26,27 even though a flexible gradient profile is inherently difficult to produce. MacNair et al.13-15 made their own solvent delivery system by combining this method with a splitter for their ultra-high-pressure capillary HPLC and LC-MS systems. The preformed-gradient loop method was also used by Ishii et al.6 for their first micro-HPLC and then for stepwise gradient elution.21 Davis et al.28 applied this method to micro-HPLC-MS. This idea was extended to multiple-step gradient elution by Takeuchi and Lim29 as well as Natsume et al.30 using the same number of injection loops as the number of multiple (24) Yan, C.; Dadoo, R.; Zare, R. N.; Rakestraw, D. J.; Anex, D. S. Anal. Chem. 1996, 68, 2726-2730. (25) Huber, C. G.; Choudhary, G.; Horvath, C. Anal. Chem. 1997, 69, 44294436. (26) Ducret. A.; Bartone, N.; Haynes, P. A.; Blanchard, A.; Aebersold, R. Anal. Biochem. 1998, 265, 129-138. (27) Bihan, T. L.; Pinto, D.; Figeys, D. Anal. Chem. 2001, 73, 1307-1315. (28) Davis, M. T.; Stahl, D. C.; Lee, T. D. J. Am. Soc. Mass Spectrom. 1995, 6, 571-577. (29) Takeuchi, T.; Lim, L. W. Bunseki Kagaku 2001, 50, 825-828. (30) Natsume, T.; Yamauchi, Y.; Nakayama, H.; Shinkawa, T.; Yanagida, M.: Takahashi, N.; Isobe, T. Anal. Chem. 2002, 74, 4725-4733.

gradient steps and a multiple-port selection valve to select the injection loops. Compared to the others, this apparatus is necessarily complex. Here, we propose a new nanoflow GR generator consisting of a conventional or semimicro gradient delivery system and a 10port switching valve with two injection loops, both of which are commercially available. In principle, this idea is similar to the offline formation and storage of gradient solvents in injection loops or the preformed-gradient loop method mentioned above. The similarity is that the gradient solvents are created by off-line pump(s) with low-pressure (or high-pressure) gradient capability and are alternately stored in the two injection loops installed on the 10-port switching valve. However, a major difference is that any nanoflow GR profile can be easily and simply created by shortening the switching period of the 10-port switching valve. Although most gradient solvents delivered by off-line pump(s) are wasted, they are still on the order of microliters per minute. In this paper, we investigate and discuss this method, which we call the “asymptotic trace 10-port valve (AT10PV)” nanoflow GR generator, in nanoflow rates of 50-500 nL/min. The result is reasonably good repeatability of gradient profiles and peak retention times in nanoflow GR elution chromatography. EXPERIMENTAL SECTION Figure 1 is a schematic diagram of a nano-HPLC system using the AT10PV nanoflow GR generator, denoted by the broken line. The major instrument components of the system and experimental conditions are described below. Instrumentation. The AT10PV nanoflow GR generator is composed of a conventional gradient pump with low-pressure gradient capability at a microflow rate (µL/min) (i.e., microflow GR pump), an isocratic pump capable of delivering one solvent at a nanoflow rate (nL/min) (i.e., nanoflow isocratic pump), a 10port valve with two injection loops, a back-pressure coil or column after the 10-port switching valve, and a controller to control the pumps and the 10-port valve. We used a Hitachi L-2100 LaChrom Elite pump (Hitachi-High Technologies, Tokyo, Japan) with lowAnalytical Chemistry, Vol. 76, No. 5, March 1, 2004

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Figure 2. Nanoflow gradient profile tracing the original microflow gradient profile. The solid line indicates the original microflow gradient profile, and the dotted line indicates the nanoflow gradient profile created by the AT10PV nanoflow GR generator.

Figure 3. Comparison between the original microflow gradient profile at flow rate 100 µL/min (a) and the created nanoflow gradient profiles at flow rates of 500 (b) and 200 nL/min (c). Solvent A, water; solvent B, 0.2% acetone solution. Gradient program of solvent B: 0 (0.0 min) f 100 (40 min) f 100 (80 min) f 0 (80.1 min) f 0% (105 min). UV detector wavelength, 250 nm; 10-port valve switching period, 1 min.

pressure gradient capability for the microflow GR pump, a modified Hitachi L-2100 LaChrom Elite pump for the nanoflow isocratic pump, and an Upchurch 10-port NanoPeak valve (Upchurch Scientific, Oak Harbor, WA) with two homemade 1-µL injection loops, i.e., fused-silica capillary tubes (0.05-mm i.d., 0.36mm o.d., 500-mm length). Because the microflow GR pump has a remote control function by relay, switching of the 10-port valve was controlled by this pump. The back-pressure coil was made by using fused-silica tubes (0.05-mm i.d., 0.36-mm o.d., 200-mm length), which were purchased from GL Science (Tokyo, Japan). The sample injector was an Upchurch M-435 microinjection valve (Upchurch Scientific). The UV detector was a Senshu SSC-5430 with a flow cell volume of 31 nL and an optical path length of 1 mm (Senshu Scientific. Tokyo, Japan). Operation of the AT10PV nanoflow GR generator is described in Results and Discussion. Chemicals, Sample, and Column. HPLC-grade methanol, acetonitrile, water, acetone, trifluoroacetic acid (TFA), formic acid, ammonium bicarbonate, and trypsin were purchased from Wako Chemical (Tokyo, Japan). Bovine serum albumin (BSA) was obtained from Tokyo Chemical Industry (Tokyo, Japan). BSA (1 mg) was dissolved in 1 mL of 100 mM ammonium bicarbonate buffer (pH 8.0) and kept at 100 °C for 10 min. Tryptic digestion was performed at 37 °C for 24 h by 0.01 mg of trypsin. The 1526 Analytical Chemistry, Vol. 76, No. 5, March 1, 2004

Figure 4. Smoothed nanoflow gradient profiles at flow rate 200 nL/ min by shortening 10-port valve switching period. Ten-port valve switching period, 0.25 min. The gradient program of solvent B and UV detector wavelength are the same as those for Figure 3.

Figure 5. Comparison between the steep (a), medium (b), and gentle (c) nanoflow gradient profiles at flow rate 200 nL/min. Solvent A, water; solvent B, 0.2% acetone solution. Gradient program of solvent B: 0 (0.0 min) f 100 (steep (a) 10 min, medium (b) 20 min, gentle (c) 40 min) f 100 (80 min) f 0 (80.1 min) f 0% (105 min). UV detector wavelength, 250 nm; 10-port valve switching period, 1 min.

separation column (0.075-mm i.d., 150-mm length) was a VC-15C18W-75 from Micro-Tech Scientific (Vista, CA). RESULTS AND DISCUSSION First, we describe how the AT10PV nanoflow GR generator, which is shown in Figure 1, operates to generate a nanoflow gradient elution. The microflow GR pump creates an original gradient profile mixing reservoir solvents A and B by controlling the corresponding solenoid valves (S). The mixed solvent is delivered at a given flow rate, e.g., 100 µL/min, into either injection loop A or B of the 10-port switching valve. The excess is drained after flowing through the back-pressure coil (or column). Here, it should be noted that when injection loop A is being filled (or loaded), the solvent loaded in injection loop B is being delivered to the capillary column at a nanoflow rate (nL/min) by the nanoflow isocratic pump. When the 10-port switching valve is activated, the role of injection loops A and B is also switched; i.e., the solvent loaded in injection loop A is delivered and injection loop B begins to be filled. This valve switching is cycled, e.g., every 1 or 2 min, by a signal (on/off or pulse) sent from the microflow GR pump. Figure 2 schematically illustrates how a nanoflow GR profile is created. The original microflow GR profile is indicated by the solid line; i.e., solvent B begins to linearly increase from 20% at 5 min to 100% at 18 min and then decrease

Figure 6. Repeatability of nanoflow gradient profiles at flow rate 50 nL/min. RSD values of X- and Y-axis directions: RSD(X) ) 2.36% and RSD(Y) ) 1.31% at 30 min and RSD(X) ) 0.38% and RSD(Y) ) 0.26% at 50 min. Solvent A, water; solvent B, 0.2% acetone in 80% acetonitrile. Gradient program of solvent B: 0 (0.0 min) f 100 (40 min) f 100 (80 min) f 0 (80.1 min) f 0% (105 min). UV detector wavelength, 250 nm; 10-port valve switching period, 1 min.

to the initial 20%. The dotted line indicates the corresponding nanoflow GR profile created when the 10-port switching valve is activated every 2 min. That is, it traces the original microflow GR profile in a stepwise manner. It should be noted here that a nanoflow GR profile could be used to asymptotically trace the original microflow GR profile simply by shortening the switching period without a problem of solvent mixing at nanoflow rates. Figure 3 compares the original microflow GR profile and a nanoflow GR profile. Solvents A and B were water and 0.1% acetone solution, respectively. The flow rate of the microflow GR pump was 100 µL/min. The gradient program of solvent B was linearly increased from 0% at 0.0 min to 100% at 40 min, kept at 100% until 80 min, and then returned to the initial condition of 0%. The original microflow GR profile (a) was monitored by the UV detector at 250 nm. The nanoflow GR profiles (b, c), which were created by activating the 10-port switching valve every 1 min and by running the nanoflow isocratic pump at flow rates of 200 and 500 nL/min, were monitored by the UV detector in the same manner. The sample injector and the capillary column shown in Figure 1 were removed in this study. Although gradient dwell times do not coincide exactly, it can be seen that these gradient profiles are very similar. “Steps” still remain (see the inset in the figure). However, they can be easily eliminated by changing the 10-port valve switching period from 1 to 0.5 or 0.25 min. Figure 4 shows the result of a 10-port valve switching period of 0.25 min at the flow rate 200 nL/min. The nanoflow GR profile is very smooth and the “steps” seen in Figure 3 disappear. In other words, the asymptotic trace under these conditions seems to be almost perfectly performed. Figure 5 compares the steep (a), medium (b), and gentle (c) slope nanoflow GR profiles. The gradient program of solvent B was linearly increased from 0% at 0.0 min to 100% at 10 (a), 20 (b), or 40 min (c), kept at 100% until 80 min, and then returned to the initial condition of 0%. The flow rate was 200 nL/min, and the other conditions were the same as those for Figure 3. The “steps”

seen in the figure can be smoothed by simply shortening the 10port valve switching time from 1 to 0.25 min, as discussed above. Figure 6 shows the repeatability of the nanoflow GR profiles obtained at a flow rate of 50 nL/min of the nanoflow isocratic pump. In this case, solvent B was 0.1% acetone in acetonitrile. The gradient program of solvent B and other conditions were the same as those for Figure 3. The nanoflow GR profile looks “sigmoidal”, slightly differing from those in Figure 3. This may be due to that the UV spectral band of acetone red-shifts while a composition of acetonitrile (solvent B) increases. The nanoflow GR profiles were taken six times and overlaid. The insets in the figure magnify the nanoflow GR profiles at 30 and 50 min. RSD values of X- and Y-axis directions at these points were calculated for quantifying a repeatability. The results were RSD(X) 2.36% and RSD(Y) 1.31% at 30 min and RSD(X) 0.38% and RSD(Y) 0.26% at 50 min. The repeatability of these profiles is good even at the flow rate of 50 nL/min. The repeatability of the gradient profile was tested at other flow rates in the range of 50-500 nL/min. They also showed repeatability similar to that shown in Figure 6 (data not shown). Figure 7 shows the chromatograms obtained by the nanoHPLC system shown in Figure 1 at a flow rate of 200 nL/min. Solvents A was 0.1% formic acid and 0.01% TFA in 5% acetonitrile solution. Solvent B was 0.1% formic acid and 0.01% TFA in 95% acetonitrile solution. The flow rate of the microflow GR pump was 100 µL/min. The composition of solvent B was linearly increased from 0% at 0.0 min to 100% at 60 min, kept at 100% until 70 min, and then returned to the initial condition of 0%. The nanoflow GR profile was created by activating the 10-port switching valve every 1 min. The sample was a mixture of BSA peptides. The manual microinjection valve was used to inject 50 nL of the sample into the capillary silica-ODS column (VC-15-C18W-75, 0.075-mm i.d., and 150-mm length). The monitoring wavelength was 214 nm. The chromatograms were taken six times and overlaid. Table 1 summarizes the retention times (RT) of the five component peaks Analytical Chemistry, Vol. 76, No. 5, March 1, 2004

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changes during the nanoflow GR elution, it would be preferable to use a dummy column for this purpose rather than a tube coil because we can easily estimate its internal diameter by calculating the linear velocity (mm/min) in the column if the same packing material and column length as the capillary column are chosen. The second point is that solvent C, which is delivered by the nanoflow isocratic pump, does not actually enter the capillary column if the 10-port switching valve is activated before the gradient solvent loaded in the injection loop runs out. This means that not only is the composition of solvent C necessarily the same as that of solvent A or B, but the type of nanoflow isocratic pump is not necessarily restricted to a mechanical one. If an EOF pump is used as the nanoflow isocratic pump, the best solvent for the EOF-driven pump can be chosen as solvent C without regard for the nanoflow gradient elution. Even a gas-driven pump (i.e., pressure cylinder) could be used for a constant-pressure nanoflow GR generation.

Figure 7. Repeatability of chromatograms of BSA tryptic digest at flow rate 200 nL/min. Column: VC-15-C18W-75 (0.075-mm i.d., 150mm length). Sample, mixture of BSA peptides; injection volume, 50 nL. Solvents A, 0.1% formic acid and 0.01% TFA in 5% acetonitrile solution; solvent B, 0.1% formic acid and 0.01% TFA in 95% acetonitrile solution. Gradient program of solvent B: 0 (0.0 min) f 100 (60 min) f 100 (70 min) f 0% (70.1 min). UV detector wavelength, 214 nm; 10-port valve switching period, 1 min. Table 1. Repeatability of Peak Retention Times (min) of Chromatograms Shown in Figure 7 data no.

RT (Pk1)

RT (Pk2)

RT (Pk3)

RT (Pk4)

RT (Pk5)

1 2 3 4 5 6

27.19 27.20 27.39 27.15 27.23 27.25

35.84 35.89 36.00 35.89 35.92 36.03

37.49 37.52 37.63 37.49 37.52 37.63

45.52 45.52 45.65 45.49 45.57 45.60

49.15 49.17 49.33 49.20 49.23 49.28

av % RSD

27.235 0.30

35.928 0.20

37.55 0.17

45.558 0.13

49.23 0.14

on the chromatograms and their statistics. The capillary HPLC system based on the AT10PV nanoflow GR generator provides reasonably good repeatability of peak retention times, i.e., less than RSD 0.3%. Finally, we would like to point out two additional features not described above. The first feature is the role of the back-pressure coil or column. This device works to minimize the pressure difference between the microflow GR solvent in the injection loop and the nanoflow GR solvent that is to be delivered into the capillary column. Without this device, the nanoflow rate might change slightly and disturb the stability of the nanoflow whenever the 10-port switching valve is activated. Considering pressure

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CONCLUSIONS A capillary HPLC system based on the AT10PV nanoflow GR generator proposed and shown to provide reasonably good repeatability at flow rates of 50-500 nL/min was investigated in this study. An important aspect of this device is that any gradient profile created by the microflow GR pump can be asymptotically traced as a corresponding nanoflow GR profile, simply by using the 10-port switching valve incorporating two injection loops and without a problem of solvent mixing at nanoflow rates. A series of sample injections can be run continuously in the same manner as in conventional sequential analysis at a microflow rate. The low-flow electrospray techniques such as nano-ESI-MS31,32 and micro-ESI-MS33,34 require a rate lower than the nanoflow rate of 50 nL/min. This is the target of our next study. We expect to report some results from this next study in a subsequent paper. ACKNOWLEDGMENT The authors thank Mr. K. Uchida, Dr. A. Hirabayashi, and Dr. I. Waki of Hitachi Central Research Laboratory, Mr. T. Okumoto, Mr. M. Ito, and Mr. K. Tsukada of Hitachi-High Technologies, and Prof. S.-I. Nishimura and Dr. H. Nakagawa of Hokkaido University for their participation in a useful discussion about the nanoflow LC-ESI-MS system. Received for review December 7, 2003.

September

2,

2003.

Accepted

AC0350312 (31) Wilm, M. S.; Mann, M. Int. J. Mass Spectrom. Ion Processes 1994, 136, 167. (32) Wilm, M.; Mann, M. Anal. Chem. 1996, 68, 1-8. (33) Emmett, M. R.; Caprioli, R. M. J. Am. Soc. Mass Spectrom. 1994, 5, 605613. (34) Hendrikson, C. L.; Emmett, M. R. Annu. Rev. Phys. Chem. 1999, 50, 517536.