Anal. Chem. 1996, 68, 1164-1168
Quantitative Injection from a Microloop. Reproducible Volumetric Sample Introduction in Capillary Zone Electrophoresis Purnendu K. Dasgupta* and Kazimierz Surowiec†
Department of Chemistry and Biochemistry, Texas Tech University, Lubbock, Texas 79409-1061
A wire loop deployed at the tip of a capillary electrophoresis system has been investigated as a means of quantitative injection. A thin film of a liquid is formed on the loop, the loop is transferred to a sealed chamber, and then pneumatic pressure is applied to introduce the contents of the loop into the capillary. As long as the applied pressure is below a certain threshold, no air is introduced into the capillary, even after the loop contents have been fully introduced. Sample surface tension and viscosity do not have a significant effect on the injected volume. The small loop injection technique appears to be a robust and reproducible alternative to presently practiced approaches to sample injection in CE. The reproducible introduction of a known amount of a sample into an analytical system is of paramount importance in achieving good accuracy in quantitative analysis. In liquid phase analysis methods, such as flow injection analysis or liquid chromatography, the use of a loop type injector dominates present practice. In capillary zone electrophoresis (CZE), perhaps the liquid phase separation technique of the greatest current interest; there are difficulties in pursuing such an approach, however. Commercially, the smallest internal loop injector valve available has a specified injection volume of 20 nL. Even in applications where this injection volume might be acceptably small, the channel geometry of the internal loop creates problems. Manufacturing reproducibility requires that the channel be relatively wide and short in length, rather than narrow and long; this inhibits effective electrostacking.1 Other constant volume injection techniques that have been described for capillary-based applications include the use of miniaturized sliding or rotary type injection valves,2,3 syringe injection (with either hydrodynamic or electric spliters4,5 ), and other techniques;6 all of the above rely on flow splitting in some form or other. Techniques that are more commonly in use today are (1) injection by hydrodynamic flow caused by a pressure difference across the capillary, realized by (a) gravity-induced sample siphoning by raising the sampling end of the capillary † Permanent address: Department of Chemical Physics, Maria Curie Sklodowska University, Lublin, Poland. (1) Liu, S.; Dasgupta, P. K. Anal. Chim. Acta 1993, 283, 739-745. (2) Mikkers, F. E. P.; Everaerts, F. M.; Verheggen, T. P. E. M. J. Chromatogr. 1979, 169, 11-20. (3) Tsuda T.; Mizuno, T.; Akiyama, J. Anal. Chem. 1987, 59, 799-800. (4) Operations Manual, Model 3850 Electropherograph, Isco, Inc., Lincoln, NE. (5) Deml, M.; Foret, F.; Bocek, P. J. Chromatogr. 1985, 320, 159-165. (6) Verheggen, T. P. E. M.; Beckers, J. L.; Everaerts, F. M. J. Chromatogr. 1988, 452, 615-622.
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relative to the liquid level at the other terminus,7 (b) application of pneumatic pressure at the inlet, or (c) application of negative pressure at the outlet, and (2) electroosmosis-electromigrationbased sample introduction.8 Both of the above methods have some disadvantages, which are discussed in detail in extant monographs.9 Briefly, pressureinduced injection can be dependent on the viscosity of the sample and the running buffer electrolyte and thence also dependent on temperature. The reproducibility of electroosmotic-electromigration (sometimes called electrokinetic) injection is relatively poorsthe extent to which different sample constituents are injected depends on their mobility, and the whole process is dependent on the composition of the sample matrix. With refinements of mechanical and temperature control, good reproducibility can be achieved for pressure-induced sample injection with the same type of samples. This is the most popular injection mode used in commercial instruments. The volume of the injected sample is a function of pressure difference, time of injection, dimensions of the capillary, and viscosities of the sample and the running electrolyte. When the viscosity of the sample to be injected varies, one may expect poor reproducibility. In the following, we introduce a new, viscosity-independent mode of sample injection in CZE. PRINCIPLES We have previously reported on how a circular wire loop can be formed at the tip of a capillary and a liquid film formed on such a loop.10 As shown in Figure 1, there are four general configurations for such a capillary-loop assembly: (a) one in which the plane of the loop is perpendicular to the tip of the capillary; (b) a variant of a, in which a stiffer and larger wire forms a parallel arm to the capillary so that the wire loop can be suspended from this wire at any desired angle and position with respect to the capillary tip; (c) one in which the plane of the loop is in the same plane as that of the capillary, with the capillary tip at the center of the loop; and (d) the same as c, except for the capillary tip being located at the perimeter of the loop. All of the above types can be constructed with a square-cut capillary end or a conical capillary tip. In previous work,10 we used configuration c with a square-cut 375 µm o.d. capillary tip. With a 2 mm diameter loop formed from a 100 µm diameter Pt wire, the volume of this loop is too large for the introduction of the whole content; (7) Tsuda, T.; Nomura, K.; Nakagawa, J. J. Chromatogr. 1983, 264, 385-392. (8) Jorgenson, J. W.; Lukacs, K. D. Anal. Chem. 1981, 53, 1298-1302. (9) Foret, F.; Krivankova, L.; Bocek, P. Capillary Zone Electrophoresis; VCH: New York, 1993; p 140. (10) Dasgupta, P. K.; Kar, S. Anal. Chem. 1995, 67, 3853-3860. 0003-2700/96/0368-1164$12.00/0
© 1996 American Chemical Society
Figure 1. Various modes of fabricating small loops at the tip of a capillary. The top row shows capillaries with square-cut ends, and the bottom row shows the same configuration with a tapered tip capillary. All work reported here was conducted with a device similar to that depicted in the lower right corner.
additionally, this geometry does not permit the introduction of the entire content of the film anyway. With all geometries other than d, the film ruptures at some point during the introduction, and the remaining liquid forms microdroplets on the wire support. Such introduction is neither quantitative nor especially reproducible. In contrast, with geometry d, especially when constructed with a conical tip capillary, we have observed that essentially the entire sample contained in the loop can be reproducibly introduced into the capillary. This can be induced either by gravity or, more rapidly, by applied pneumatic pressure. As long as the applied pressure is not excessive, the hydrophilic nature of a silica capillary prevents any air from entering into the capillary. Results of sample introduction from such a loop-based injector system are described in this article.
EXPERIMENTAL SECTION Fabrication of the Capillary. Both mechanical11 and chemi12 cal procedures have been described for shaping the tip of a fused silica capillary. We chose the chemical etching procedure. Approximately 1 cm length of the polyimide coating was burned off at one end, and the opposite side of the capillary was connected with a distilled-water-filled syringe. The bare end was inserted in a concentrated HF solution (∼29 M). While the HF etched the outside of the capillary, water was made to flow slowly through the capillary into the HF to prevent enlargement of the capillary bore at the tip. In our experience, flowing a gas instead of water for this purpose is not entirely satisfactory; with gas flow, some of the HF diffuses into the capillary and etches the inner surface. While the application of water flow prevents this, the effluent water dilutes the HF at the tip, and the etching rate is much lower at the tip itself. Consequently, the etched capillary has the appearance of a mace, i.e., the terminal 1 mm or so is poorly etched and (11) Kriger, M. S.; Cook, K. D.; Ramsey, R. S. Anal. Chem. 1995, 67, 385-389. (12) Fishman, H. A.; Amudi, N. M.; Lee, T. T.; Scheller, R. H.; Zare, R. N. Anal. Chem. 1994, 66, 2318-2329.
retains close to the original dimensions. Beyond this “neck” the capillary is etched the most; this gradually tapers out to the original diameter over ∼3-4 mm. After ∼1.5 h, the etching was stopped, the capillary was thoroughly flushed with water, and ∼2 mm of the unevenly etched larger end portion was cut off. This left a tip that reaches a terminal outer diameter of ∼140 µm from the original diameter of ∼375 µm over a length of 2.5-3 mm. We have worked with a number of different wire loops of different loop and wire diameter and wire material coupled to a conical tip capillary such as that described above. The data reported here were collected with a loop made from a nickel wire, 50 µm in diameter. The loop itself was fabricated under a microscope, wrapping the wire around the tip of a pin. The loop was affixed at the capillary tip by wrapping around and then applying some epoxy adhesive to prevent the loop from slipping off the tip. After complete assembly, the loop appeared to be not exactly circular but elliptical (500 µm × 700 µm), with the longer axis situated along the capillary axis. Finally, the capillary was cut to the desired length (56 cm), and the detection window was made at 8 cm from the end opposite to the loop. Results obtained with other, mostly larger, loops made with Pt and Nichrome wires were different in the amount of sample injected but were used in the same manner. Instrumentation. The fully automated CZE apparatus was assembled in-house from commercially available components. A Linear Model 206 PHD UV/visible photometric detector (Linear Instruments/Spectra-Physics) was used with PC-based data acquisition and processing using a DAS-16 A/D board (Keithley/ Metrabyte) and software written in-house. The high-voltage power supply was CZE2000 (Spellman High Voltage, Plainview, NY). Fused silica capillaries (75 µm i.d., 375 µm o.d., Polymicro Technologies) were used for separation. The loop-bearing terminus (injection end) of the capillary was mounted in a Plexiglas head that could be moved in both the horizontal and the vertical planes by pneumatically operated cylinders (1/8 in. diameter piston, double-acting cylinders; Clippard Instruments, Cincinnati, OH) operated by air pressure supplied by solenoid valves that are in turn controlled by a Micromaster LS 100A programmable microcontroller (Minarik Electric, Los Angeles, CA). The injection end of the capillary can thus be made to address various solution vials placed in a Model 2110 fraction collector (Bio-Rad, Richmond, CA). The specific vial to be addressed by the capillary is brought into position by sending one or more position advance signals to the fraction collector from the microcontroller. (The movement of the vials in this inexpensive fraction collector is, however, unidirectional; back-and-forth movement, as in a true random access autosamplers, is not possible.) The Plexiglas head containing the capillary injection end also holds the high-voltage electrode adjacent to the capillary tip. To perform a pressure injection from a sample-filled loop, the head is vertically lifted, horizontally translated to the top of an empty polypropylene enclosure equipped with a pressurization port, and then lowered. The underside of the Plexiglas head housing the capillary is lined with a silicone gasket so that a pneumatic seal is formed when it rests on the polypropylene enclosure. The rate at which the loop is lifted out of a solution determines both the amount of the solution lifted out by the loop and the reproducibility of such a process. Stable values are obtained as long as the rate of withdrawal is slow (total time for the 5.1 cm movement of the vertical actuator, g1.27 s. This was controlled by adjusting the length and bore of Analytical Chemistry, Vol. 68, No. 7, April 1, 1996
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the air exit tube connected to the solenoid valve governing the pneumatic actuator. For pressure injection from the loop, accurate control of the applied pneumatic pressure at a relatively low level is necessary. A precision pressure-regulating valve (Fairchild Inc.) in conjunction with a Magnehelic gauge (Dwyer Instruments Inc., Michigan City, IN) and a solenoid valve were used for maintaining constant pressure for injection. A ballast volume of 500 mL was incorporated between the pressure regulator and the solenoid valve to minimize pressure shocks. All operational steps were automatically carried out by the microcontroller. Chemicals. N,N-Dimethylformamide (DMF, Fisher Scientific), N,N,N ′,N ′-tetramethyl-p-phenylenediamine dihydrochloride (TPPDA, Eastman Organic Chemicals, Rochester, NY), sodium dodecyl sulfate (SDS, Sigma), sucrose (Fisher), and starch (Baker & Adamson) were used for the preparation of test solutions. All solutions, electrolytes, and standards were prepared in distilled, deionized water. Operation. The capillary tip and loop are first washed by dipping in the running electrolyte. The capillary is then filled with the running electrolyte by pressurization from a fresh vial. The loop is washed with sample by lowering the empty loop into the sample vial. The loop is then filled for injection using a fresh sample vial and withdrawing at a slow rate, as specified above. Partial or total injection of the contents of the loop is then accomplished by transferring and lowering the head on the pressurization chamber and applying pneumatic pressure for a desired period (typically, several seconds). The capillary tip is rinsed by dipping in a carrier electrolyte vial and then put into a second vial of carrier electrolyte to commence electrophoresis. (To avoid contamination problems, any change from the sample to the running electrolyte solution (or vice versa) was always conducted in the manner stated above, i.e., via at least one intermediate rinse vial that contains the same solution as that into which the capillary is to be placed in the next step.) Electrophoretic separation is conducted in a constant voltage mode at an applied voltage of 16.8 kV. Experiments are conducted at laboratory temperature, 23 ( 1.5 °C. Detection is performed at 214 nm for DMF and at 254 nm for TPPDA and other analytes, with a data acquisition rate of 10 Hz. RESULTS AND DISCUSSION Applied Pneumatic Pressure and Injection Period. An interesting phenomenon related to the hydrophilic nature of a silica capillary is that a significant amount of motive force is actually necessary to introduce an air bubble into an aqueoussolution-filled capillary. If one end of a solution-filled fused silica capillary rests in a vial and the other end (not entrained in a solution containing vial) is lifted, it will be readily realized that the free end of the capillary must be raised far above the equipotential point for any flow to actually occur (for an air bubble to be introduced at the free end). The minimum pressure necessary to introduce an air bubble into a liquid-filled capillary depends on the diameter of the capillary and the surface tension of the sample solution.13 In the case of water and a silica capillary of 75 µm internal diameter, as long as the applied pressure does not exceed 0.5 psi, no air is introduced. Injections can also be made from the loop by establishing a hydrostatic height difference. However, in our specific experi(13) Adamson, A. W. Physical Chemistry of Surfaces; John Wiley & Sons: New York, 1976; p 21.
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Figure 2. Dependence of the observed peak area upon injection period. Applied pressure is constant at 0.45 psi. The vertical dimension of the error bars represents (1 standard deviation (n ) 3-8 in each case). Conditions: sample, 0.01% aqueous N,N-dimethylformamide; electrolyte, 2.5 mM Na2B4O7; 5 mM NaH2PO4; 56 cm long 75 µm i.d. capillary; 16.8 kV applied.
mental arrangement, it was difficult to establish a large enough hydrostatic height difference that would permit a desirably fast sample introduction rate. If the sample introduction rate is too slow, one may encounter potential problems with the evaporation of the loop contents. We therefore conducted injections with an applied pneumatic pressure fixed at e0.5 psi. The results are shown in Figure 2 for a sample composed of 0.01% DMF and a carrier electrolyte composed of 2.5 mM Na2B4O7 and 5 mM NaH2PO4. The finite y-intercept indicates that some sample is introduced due to capillary forces or interfacial diffusion. Beyond this, and up to a period of 5 s, the amount injected increases linearly with time (linear r2 ) 0.9981). After about 10 s injection time, the peak area becomes invariant with respect to injection period, at least up to 60 s, the maximum time studied. With a dye incorporated in the sample to aid visualization, we also visually observe that the loop contents are entirely gone by ∼7 s. If the applied pressure is significantly higher than 0.5 psi, air is introduced into the capillary if quantitative injection is attempted, and separation becomes impossible due to the presence of air in the capillary. Good reproducibility was observed both for a fixed injection period of 20 s (rsd 1.6%, n ) 8) and for different injection periods (10-30 s) lumped together, as long as this period was greater than the minimum period necessary to achieve quantitative injection (rsd 1.07%, n ) 19). Many repeats of hydrodynamic injections of DMF were made, and rsd typically ranged between 1 and 2%. The observed reproducibility with the loop injection technique for a real multicomponent sample containing sulfanilamide, 1-naphthalenesulfonate, benzoate, 2,6-naphthalenedisulfonate, and phthalate with the same borate/phosphate carrier electrolyte was somewhat poorer, rsd 3.6, 3.7, 2.8, 2.8, and 3.2% (n ) 8), respectively, for the cited analytes. A typical electropherogram is shown in Figure 3. The observed rsd values for the same analytes by hydrodynamic injection were also higher relative to DMF as sample and ranged from 2.3 to 4.1%.
Figure 3. Representative electropherogram obtained upon quantitative injection from a 12 nL loop equipped capillary. Conditions as in Figure 2.
Injected Volume. The volume of the loop content is obviously dependent on the size of the loop. If the liquid volume is represented as a flat elliptical disk, with the disk thickness equal to the wire diameter, the liquid volume in the loop described for the present experiments will be 13.7 nL. Examination of this filled loop under a video microscope is difficult due to evaporation problems, but examination of somewhat larger loops clearly shows the shape of the liquid film to be slightly biconcave, rather than a flat disk. The actual volume should, therefore, be somewhat less than the value cited above. We have not found a satisfactory method for evaluating the volume of the loop contents from first principles. Surface tension forces can suggest the maximum amount of liquid a horizontal wire loop or ring can just lift against gravity (the De Nou¨y tensiometer13 works on this principle), but this is a static calculation and greatly overpredicts the amount of liquid actually held in our loop. The actual injected volume can be experimentally determined, however, by comparing the peak area obtained with that obtained by hydrodynamic injection under a known set of conditions. The injected volume in the latter case can be calculated on the basis of first principles. When the volume of the loop is determined on the basis of the hydrodynamic injection data, any spontaneous injection should be taken into account. In our system, the relationship of the peak area with the hydrodynamic injection period was linear (r2 ) 0.9999, n ) 10) and could be expressed by the relationship
peak area (arbitrary units) ) 17.535 × injection period (s) + 232.8 (1)
The intercept represents the signal due to the amount spontaneously injected. Taking this into account, the volume of the sample injected with the loop system was thence determined to be 12.9 ( 0.35 nL. Effect of Changes in Sample Surface Tension. Based on the earlier work of Marmur,14 Fishman et al.12 have shown that not only is some amount of the sample spontaneously injected
into a hydrophilic silica capillary, but also the extent of this injection is dependent on the forces associated with the silica surface and the sample. We prepared 0.01% DMF as a sample in pure water and in 1.6 and 8 mM SDS solutions (respective surface tensions, 72, 60, and 40 dyn/cm). The same hydrodynamic injection procedure was used for injecting the samples in each case with the capillary filled with the same borate/phosphate electrolyte previously mentioned. The resulting DMF peak areas (arbitrary units, rsd in parentheses, n ) 4 in each case) are 949 ( 30 (3.2%), 926 ( 10 (1.1%), and 893 ( 30 (3.4%); the trend of decreasing peak areas with decreasing sample surface tension is evident. In the loop sampling system, the effect of surface tension is not known a priori. The resulting DMF peak areas (n ) 4 in each case), 444 ( 27 (6.1%), 431 ( 18 (4.2%), 468 ( 12 (2.6%), showed somewhat poorer precision but were indistinguishable from one another on the basis of a t-test. However, we found that if the rate of the withdrawal of the sampling loop from the sampling solution is too fast, changes in sample surface tension may have a readily discernible effect. For example, in a similar experiment with a DMF analyte prepared in dilute SDS solutions, when the withdrawal speed was increased (5.1 cm movement in ∼0.5 s), the peak areas obtained were 1620 ( 42, 1830 ( 72, and 1945 ( 86 for 0, 4, and 8 mM SDS solutions, respectively (n ) 5 each; these results are statistically different). Effect of Changes in Sample Viscosity. The effect of sample viscosity on the amount injected can be complex due to the interplay of various factors. It should be noted that the sample occupies a very small portion of the total capillary length. If the viscosity of the carrier electrolyte solution is varied, the results are predictable: the amount of sample injected under otherwise constant conditions varies inversely with the viscosity of the carrier electrolyte solution (which must be displaced to accommodate the sample). In contrast, experimenting with a conventional system with hydrodynamic injection, we noted to our surprise that the dependence of the injected sample volume upon sample viscosity did not follow a predictable trend when the sample’s viscosity was altered by the addition of glycerol. A number of factors, such as changes in mixing due to osmotic flow, alterations in the capillary surface, strong perturbation of laminar flow profile at the sample-electrolyte interface, etc., may be involved. Any or all of these can be responsible for the apparently anomalous increase of the injected sample volume with increasing sample viscosity. Establishing specific reasons for the observations made with specific viscosity modifying agents was beyond the scope of this work and was not attempted. We found, however, that when sucrose is used to alter viscosity, the injected sample volume in a conventional hydrodynamic injection system does decrease with increasing sample viscosity, at least up to 8% (w/v) sucrose content. The cationic test solute TPPDA was used as a probe in conjunction with a 10 mM NaH2PO4 electrolyte, so it could migrate ahead of the sample matrix and thus not be affected by the viscosity of the original sample zone during the bulk of its journey to the detector. With conventional hydrodynamic injection, the respective peak areas for 0.01% TPPDA in 0%, 5%, and 8% sucrose solution (viscosity 1, 1.141, and 1.242 cP, respectively) were found to be 1297 ( 8 (0.6% rsd), 1222 ( 2 (0.2%), and 1104 ( 20 (1.8%) (n ) 4 each). The same samples produced respective signals of 1225 ( 81 (6.6%), 1259 ( 25 (2.0%), and 1164 ( 14 (1.2%) (n ) 4 (14) Marmur, A. J. Colloid Interface Sci. 1988, 122, 209-219; 1989, 130, 288289; Chem. Eng. Sci. 1989, 44, 1511-1517.
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each) with the loop/pneumatic pressure injection system. It is clear that the viscosity dependence in the latter system is smaller, although precision may be poorer. CONCLUSIONS We have described here a novel injection technique that shows little dependence on sample surface tension and viscosity. In the present format, it is not difficult to make individual loops, but it will obviously be difficult to produce a commercial device with a reproducible volume from one loop to another. However, with advances in silica fabrication technology, it should be possible to form a reproducible loop of fused silica directly at the end of the capillary, providing a constant volume injection method.
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ACKNOWLEDGMENT This research was supported by the U.S. Environmental Protection Agency through R821117-01-0 and by Dionex Corp. However, this manuscript has not been subject to review by these agencies, and no endorsement should be inferred.
Received for review November 14, 1995. January 15, 1996.X
Accepted
AC951123I X
Abstract published in Advance ACS Abstracts, February 15, 1996.
