Anal. Chem. 1991, 63,802-807
a02
ACKNOWLEDGMENT We thank Gary Sagerman and Steve Palistrant from the fabrication of equipment necessary for interfacing our SLM 48000 MHF to fiber-optic sampling. LITERATURE CITED (1) Lakowicz. J. R. Wnclples of fluorescence Spectroscopy; Plenum
Press: New York, 1983. (2) Warner, I . M.; Patony. G.; Thomas, M. P. Anal. Chem. 1985, 57, 463A-483A. (3) Bright, F. V. Anel. Chem. 1888, 60.1031A-1039A. (4) Lenac, 2.; Tomas, M. S. Swf. Sci. 1989, 275, 299-318. (5) Leimer, A.; Reinkch, H. Chem. phvs. Lett. 1988, 146, 320-324. (6) Kemnitz, K.; Nakashlma, N.; Yoshihara, K.; Matsunami. H. J. phvs. Chem. 1989, 93, 6704-6717. (7) Jameson, D. M.; Gratton, E.; Hall. R. D. Appl. Spectrosc. Rev. 1986, 20, 55-106. (8) Lakowicz, F. R.; Laczko, G.; Gryczynski, I.; Sqmacinski, H.; Wlczk. W. J. Ffwtochem. Ffwtobiol.. B : 8/01. 1988, 2 , 295-311. (9) Gratton. E.; Jameson. D. M.; Hall, I?. D. Ann. Rev. Biophys. Bioeng. 1984, 13, 105-124. (10) Bright. F. V.; Betts, T. A.; Litwiler, K. S. C . R . C . Crit. Rev. Anal. Chem. 1990, 27, 389-405. 111) . . Feddersen. 8. A.: Piston. D. W.; Gratton. E. Rev. Sci. Instrum. 1989, 60, 2929-2936. (12) Bright, F. V. Roc. SPIE 1988, 909, 23-28. (13) Bright, F. V.; Betts, T. A.; Litwiler, K. S. Anal. Chem. 1990, 6 2 , 1065-1069 . - - - . - -. .
(14) Lttwiier, K. S.; Catena, G. C.; Bright, F. V. Anal. Chim. Acta 1990, 237. 485-490. (15) Be&, T. A.; Catena, G. C.; Huang, J.; Lltwiler, K. S.; Zhang, J.; Zagrobelny, J.; Bright, F. V. Anal. Chlm. Acta, in press. (16) Lltwlier, K. S.; Bright, F. V. I n Chemical Sensors andMlcroinstrumentat!on; Munay, R. W., Dessey, R. E., Heineman, W. R., Janata. J., Seitz, W. R., Eds.; ACS Symposium Series 403; American Chemical
Society: Washington, DC, 1989; Chapter 25. (17) Zhu, C.; Bright, F. V.; Wyatt, W. A.; HieftJe, G. M. J . Electrochem. Soc.1989, 736, 567-570. (18) Bright, F. V.; Poirier, G. E.; Hieftje, G. M. Talanta 1988. 35, 113-118. (19) Blatt, E.; Launikonis, A.; Mau, A. W.; Sasse, W. H. Aust. J. Chem. 1987. 40, 1-12. (20) Bright. F. V. Appl. Spectrosc. 1988, 42, 1531-1537. (21) Litwiler, K. S.; Huang, J.; Bright, F. V. Anal. Chem. 1990, 62, 471-476. (22) Lttwiier, K. S.; Bright, F. V. Appl. Spectrosc. 1990, 4 4 , 1089-1092. (23) Aicala, J. R.; Gratton, E.; Predergast, F. G. Siophys. J . 1987, 51, 587-596. (24) Kuwabata, S.; Nakamura, J.; Yoneyama, H. J . Eiectroanai. Chem. 1989, 267, 363-373. (25) Moore, R. B.; Martin, C. R. Macromolecules 1888, 27, 1334-1339. (26) DeSiiva. A. P.; Gunaratne, H. Q. J. Chem. Soc., Chem. Commun. 1990, 2 , 166-168. (27) Sharma, A.; Wolfbeis. 0. S. Appl. Spectrosc. 1988, 42, 1009-1012. (28) Roe, J. N.; Szoka, F. C.; Verkman, A. S. Analyst 1990, 175, 353-358. (29) Marhold, S.; Koiier, E.;,Meyer, I.; Wolfbeis, 0. S. Fresenuis J . Anal. Chem. 1990, 336, 111-113. (30) Morf, W. E.; Seller, K.; Lehmann, 6.; Behringer, C.; Hartman, K.; Simon, W. Pure Appl. Chem. 1989, 61, 1613-1618. (31) Shahriari. M. R.; Zhou, Q.; Sigel, G. H. Opt. Lett. 1988, 73,407-409. (32) Schaffar, B. P.; Wolfbeis. 0. S. Roc. SPIE 1989, 990, 112-129. (33) Schaffar, B. P.; Wolfbeis, 0. S. Anal. Chim. acta 1989, 217, 1-9.
RECEIVED for review November 6 , 1990. Accepted January 18, 1991. This work was generously supported by CHE8921517 awarded by the National Science Foundation and 3M, Inc. K.S.L. also acknowledges support from the 1989 ACS Analytical Division Summer Fellowship sponsored by the Pittsburgh Conference.
On-Column Sample Gating for High-speed Capillary Zone Electrophoresis Curtis A. Monnig and James W. Jorgenson* Department of Chemistry, University of North Carolina, Chapel Hill, North Carolina 27599-3290
Hlgh-speed zone ekdrophoresls In a fused-slllca capillary Is described. Elevated electrlc fields and short caplllary lengths allow a mlxture of fluorescein lsothlocyanate (FITC) labeled amino aclds to be separated In tlmes as short as 1.5 8. Formatlon of the analyte zone at the head of the caplllary Is controlled by laser-Induced photolysis of a tagging reagent. Tht6 gathg procedure alkws rapkl and automated lntrodudkm of sample Into the caplllary. Ultlmately, Joule heating of the buffer IlmHs the speed and efflclency of the Separation.
INTRODUCTION Electrophoresis has been widely used for several decades as a method for separating ionized compounds. More recently, there has been growing interest in capillary electrophoresis (CE) as a general, high-efficiency means of separating complex mixtures. In capillary electrophoresis, the separation is carried out in a capillary tube with a typical inner diameter of 5-100 I.cm and a total length of 30-100 cm. The small radial dimensions of the capillary allow Joule heat to be dissipated efficiently, which in turn allows potentials as high as 30 kV to be applied across the length of the capillary. As a result, excellent separation efficiencies (>1 OOO OOO theoretical plates)
* Author t o whom correspondence should be addressed. Phone number: (919)966-5071. 0003-2700/9 I /0363-0802$02.50/0
have been reported for many compounds, often in analysis times as short as a few minutes. When compared with chromatographic separation procedures, CE can offer a significant improvement in both speed and efficiency for the separation of charged species. With a typical CE instrument, separation of a mixture usually requires between 5 and 30 min. Although this time is fast relative to many competitive procedures, it is slow relative to many chemical events. As a result, CE has not been used as a method for monitoring dynamic chemical systems. To gain this capability, it is necessary to increase the speed of the analysis. One of the motivating forces behind this interest is the possibility of using such high-speed devices in a coupledcolumn multidimensional separation instrument. The resulting multidimensional instrument would have a separating power far in excess of any single-dimension instrument. However, for a coupled-column instrument to be practical, the separation in the second (or higher order) dimension should have an analysis time that is short relative to the preceding dimension. If this condition is not met, resolution in the preceding dimension will be sacrificed. Consequently, the speed of the second dimension can play an important role in determining the overall analysis time of the multidimensional instrument. To address the need for increasingly rapid CE separations, we have begun investigating a method for increasing the speed of electrophoretic separations. In this paper we will demon@ 199 1 American Chemical Society
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strate that it is theoretically possible to greatly reduce CE analysis times without a loss of separation efficiency. Furthermore, we will describe an instrument that exploits these principles to obtain this enhanced separation speed.
Capillary
THEORY In an earlier paper Jorgenson and Lukacs (1) developed a set of equations which describe the efficiency (N) and speed ( t )of capillary electrophoretic separations. These equations are
N = pV1/2D
(1)
t = Ll/pV
(2) where p is the effective electrophoretic mobility of the analyte, V is the total applied voltage, Vl is the voltage drop between the point of sample introduction and detection, L is the total length of the capillary in which the separation is being performed, 1 is the length of the capillary between the point of injection and the point of detection, N is the number of theoretical plates, D is the diffusion constant for the species, and t is the time required for the analyte to migrate to the detector. When discussing high-speed separations, it is desirable to develop a new equation that expresses the number of theoretical plates (N) which can be obtained in a unit period of time (t). This new equation can be derived from the two previous formulas:
N/T = p2V1V/2D1L This expression can be simplified to
(3)
2
T
20
(4)
Thus, to maximize the number of theoretical plates obtained in any given time, the voltage to length ratio (electric field strength) should be maintained at as high a level as possible. Nickerson and Jorgenson (2) demonstrated the utility of elevated field strengths by separating eight amino acids in less that 70 s. In practice, what ultimately limits the possible field strength is Joule heating of the capillary. Overheating of the capillary will lead to the formation of gas bubbles in the buffer that eventually results in electrical breakdown and arcing in the capillary. Overheating of the capillary also leads to temperature gradients in the buffer that are recognized to produce broadened peaks (3). As these difficulties were not considered in the derivation of eq 4, the relationship between the indicated parameters are likely to change. To develop an expression for power dissipation in the capillary, fist the current passing through the capillary (i) and the effective resistance of the buffer-filled capillary (R) must be calculated. Equations 5 and 6 allow these values to be defined in terms of funda-
i = Vrr2/pL
(5)
R = pL/rr2 (6) mental parameters, where i is the current passing through the capillary, p is specific resistance of the buffer filling the capillary, and r is the radius of the central channel. By substitution, the power dissipation in the capillary can be calculated as shown in eq 7. A more useful parameter is the P = i2R = VZrr2/pL (7) power dissipated per unit length of capillary. Dividing through eq 6 by the column length (L) gives the following expression:
P / L = (V/L)2rr2/p (8) Interestingly, the ratios N l t and P I L are both proportional to ( V / L ) 2 .Thus, for all other factors remaining constant, N l t m PIL. As a general rule, for a passively cooled column, we have found that a power dissipation less than 1 W/m of
Capillary
Support
-
803
;;;;roostnotic
I
I “r’
w Capillary ith -T-Gating
Beam
Polyimide
-Probe
Beam
Figure 1. Diagram of capillary mount showing the relative position of the capillary, the “gating” and the “probe” laser beams.
capillary produces negligible broadening of sample zones. The key to increasing the speed of a CE separation is to establish a set of experimental conditions in which the electric field is maintained at as high a level as possible but overheating and thermally induced zone dispersion is inconsequential. Equation 7 indicates that the power dissipation may be kept within acceptable limits simply by reducing the radius of the column. Although this procedure is very effective at minimizing power dissipation in the column, it can increase the difficulty of finding a suitable detector to record the passage of the analyte zone. Although power dissipation is an important consideration when operating at high potential fields, other problems have also deterred the development of high-speed CE instrumentation. The physical size of many commercially available detectors restricts their use with short lengths of capillary. Fortunately, this limitation can often be overcome by redesign of the instrument. A more troublesome problem is that tradiational methods of sample introduction (electromigration, hydrostatic pressure, etc.) are relatively slow and unwieldy with “short” capillary lengths. To exploit the high-speed potential of capillary electrophoresis, the sample introduction procedure should be automated to ensure both reproducible injections and ease of use. Furthermore, it is desirable to be able to make these injections while the capillary is maintained at operating voltage. Otherwise, time will be lost slewing the high-voltage power supply. Several automated methods of sample introduction can be easily envisioned, including microvalve gating, high-speed flow diversion (4, 51, and electrochemical modulation. In this work we demonstrate a fourth procedure which we call on-column optical gating. Typically, in most capillary-based separations instruments, the sample is introduced as a “plug” of material at one end of the column and allowed to traverse the column where the separation occurs. With on-column optical gating, the components in the mixture to be determined are first tagged with a fluorescent molecule and then continuously introduced into one end of the column. Near the entrance of the capillary a laser is used to photodegrade the tag and thus render the material undetectable to a fluorescence detector that is located further along the column (see Figure 1). A sample zone is generated by momentarily preventing the laser from striking the column, and thereby allowing a small amount of tagged material to pass intact. Because the sample modulation is optical rather than mechanical, temporally narrow plugs of
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+
On-Column Optical Injection
Voltage Drop Across This Portion of the Capillary for a 25 kV Driving Potential
n n
(2.1 kV)
(22.7 kV)
(0.2 kV)
n 0
2
4
6
u u
8
10
12
Figure 4. Diagram demonstrating the principle of coupling capillaries of different diameters. The resistor equivalent circuit to the left of the capillary lists the resistances calculated for each section of the capillary. The capillary dimensions are those used for the experiments described in the text.
Time ( s e c )
Figure 2. Diagram showing the temporal relationship between the intensity of the "gating" beam and the fluorescence signal generated at the "probe" laser beam.
Spectral Selection Computer multiplier 1
I
Figure 3. Block diagram of "fast" capillary electrophoresis instrument.
material can be introduced into the column. Furthermore, the injection can be made while the capillary is maintained a t the operating voltage. Separation of the tagged species ~ C U E in the column region between the point of sample gating and the point of detection. Thus, a fluorescence signal will be recorded a t the detector channel a t some time delay from the interruption of the gating beam. This temporal relationship is illustrated in Figure 2.
INSTRUMENTATION Sample Introduction. Figure 3 shows a diagram of the instrument used for high-speed electrophoretic separations. An argon ion laser (Model 70-2, Coherent Inc.) operating at less than 1 W of power at 488.0 nm was focused into the central channel of the capillary with a fused-silica lens (f = 75 mm, Oriel Corp.) to photodegrade the fluorescent species. Intensity modulation of the laser beam was accomplished with an acoustooptic modulator (Model AOM-30 modulator and Model DE-30X VCO driver, IntraAction Corp.). Previously, Hirschfeld (6) demonstrated that the molecule fluorescein could be efficiently photolyzed with relatively modest laser powers (- 12 kW cm-*). These power densities are easily achieved by focusing a low-power continuous-wave laser into the capillary column. Consequently, the work reported here employed molecules that could be easily labeled with the fluoresceinderivative, fluorescein isothiocyanate (FITC). The electrophoretic separation proceeds in much the same way as conventional CE with the effective column length (1) being the distance between the gating beam and the fluorescence detector. Capillary Columns. It becomes increasingly difficult to work with elevated electric fields as the capillary column length is reduced. In particular, spontaneous breakdown in air may occur,
observed when the electric field exceeds 3000-4000 V cm-'. As a result, special attention must be directed toward electrically isolating the buffer reservoirs. We overcome this problem by coupling capillaries of different diameters to concentrate the electric field into a short section of small-diameter capillary. This "coupled-column" technique is illustrated in Figure 4. The capillary column used in the work was constructed from 99 cm of a capillary with an inner diameter of 150 pm and 4 cm of capillary with an inner diameter of 10 pm. The equivalent electrical circuit is shown to the left of the column diagram. Analysis of this circuit indicates that approximately 90% of the voltage drop occurs over the 4 cm of 10 pm i.d. capillary. Consequently, it is possible to generate electric fields in excess of 5OOO V cm-' in this short length of capillary. This should be compared with the 300-400 V cm-' that is typically employed with CE. In light of the preceding discussion, it is easy to see how this elevated electric field can be used to shorten the time of analysis. However, the overall length of the coupled capillary is the same as the capillary used in the traditional CE analysis, so problems with isolating the high voltages are minimized. A final problem which must be considered is resistive heating of the buffering medium. As discussed previously, Joule heating of the capillary must be minimized if separation efficiency is to be maintained. Capillary diameters between 5 and 15 pm seem to provide a good compromise between capillary temperature control and ease of use with a fluorescence detector. Detection and Signal Processing. A small fraction (-4%) of the laser power was split from the gating beam and directed into the capillary to form a fluorescence detector (the probe beam in Figure 1). The resulting fluorescence signal was collected with a microscope objective (16X, Melles Griot) and then spectrally isolated with a monochromator (Model H-10, Instruments SA Inc.) and band-pass filter (Omega Optical). The resulting photon flux was converted to an electrical signal with a photomultiplier tube (Model R1527-03, Hamamatsu) and a high-speed amplifier (Model 427, Keithley Instruments). A LabVIEW (National Instruments) program running on the Macintosh I1 computer acquired the data through a laboratory interface board (Model NB-MIO-16XL-42, National Instruments) configured with a 16-bit analog to digital interface. This same program was used for data processing and storage. Peak parameters (theoreticalplates, retention time, peak widths, etc.) were derived from statistical moments that were calculated with a second LabVIEW program. Procedures. Solutions were prepared in the following manner. First 1 mL of FITC/acetone solution (6.1 mM) was added to 3 mL of 3 mM solution of each amino acid in a pH 9.2 carbonate buffer. This mixture was allowed to react at room temperature for at least 3 h. This mixture was further diluted with the mobile phase (pH 9.2, carbonate buffer) to obtain the desired concentration of labeled product. Before use, all solutions were passed through a 0.22-pm filter to remove particulates. Sample was introduced into the capillary by electrophoretic migration. Although this sample introduction procedure necessarily selects for those species with the highest electrophoretic mobility, for all molecules studied we found the injected amount adequate for our investigations. Before data collection was ini-
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0
4
2
8
6
Field (kV/cm) Power (mW) F@we 5. Plot showing the effect of gating beam power on the fraction of fluorophore photolyzed.
Figure 6. Plot of the current passing through the coupled capillary as a function of the electric field in the smalldiameter capillary. The buffer solution was 30 mM carbonate at pH 9.2.
tiated, the sample was passed into the column for several minutes to equilibrate the capillary with the buffer solution, in order to better stabilize electroosmotic flow.
RESULTS AND DISCUSSION The success of on-column optical gating relies upon its ability to efficiently photodegrade the fluorescently labeled compound as it passes through the gating beam. To assess the degree to which complete photobleaching was observed, a bare fused silica capillary (10 pm i.d.) was positioned in the instrument and FITC-labeled arginine was forced through the capillary at a constant velocity (0.19 cm SI). The relationship between probe beam power and the fraction of the fluorophore photolyzed is illustrated in Figure 5. These data suggest that a t least two processes are involved in the photolysis of fluorescein. The first mechanism is fast and irreversible. This accounts for the rapid drop in fluorescence intensity observed a t low laser powers. The second mechanism is less sensitive to laser power and accounts for the approximately 10% remaining fluorescence observed even at high laser powers. Unfortunately, this persistent fluorescence introduces a background upon which all of our signals must be observed. Not surprisingly, this background increased the noise which limits the dynamic range and precision of the measurements. However, there is no reason to believe that other fluorophores would suffer from these same limitations. This possibility is currently being investigated. For the studies presented here, we elected to continue to use fluorescein because of its excellent spectral match with our laser. Figure 6 shows the measured current passing through the coupled capillary column as a function of the electric field in the short capillary. Significant deviations from linear behavior are observed by the time the field strength has reached 3 kV cm-’. This behavior is indicative of Joule heating of the buffer. Figure 7 supports this hypothesis by plotting the temporal standard deviation of the sample zone as a function of the electric field. A minimum zone width is observed when the applied voltage is 2.0 kV cm-I. This problem can be partially overcome by reducing the current passing through the capillary. This is easily accomplished by lowering the concentration of the supporting electrolyte in the buffer or by further reducing the diameter of the capillary. In Figure 8 the temporal standard deviation of the peak for FITC-labeled arginine is plotted as a function of the sample introduction time (the time the “gating” laser beam is deflected away from the capillary) for a capillary with a length ( I ) of
0
1
2
3
4
5
Electric Field (kV/cm) Flgure 7. Standard deviation of the fluorescein peak as a function of the electric field in the smalldiameter capillary. Column length ( I ) was 1.2 cm, and the buffer was the same as listed in the caption of Figure 6.
1.2 cm and an electric field of 3.3 kV cm-’. These data demonstrate that temporally small sample zones must be injected into the capillary to obtain a minimum peak width. Figure 9 shows the electropherogram acquired when a solution containing FITC derivatives of three amino acids (Arg, Phe, Glu) was introduced into the instrument. For this analysis, the distance between the “gating” and the “probe” beams was 1.2 cm. The electric field was maintained a t 3.3 kV cm-’. The elution times for these three species are 0.62, 0.91 and 1.33 s, respectively. The number of theoretical plates for these peaks ranged between 5000 and 7000. These data can be compared with the separation of amino acids reported by Cheng and Dovichi (7). In that report, the elution time for the three amino acids shown in Figure 9 ranged between 13 and 24 min with peak efficiencies of approximately 400 000 theoretical plates. Clearly, we have sacrificed much of the efficiency of the separation to increase the overall speed of analysis. Heat buildup in the capillary likely has contributed to some of the width of the peak observed in this figure. In principle, reducing this excess heat should allow even higher separation efficiencies to be obtained. T o regain some the efficiency sacrificed in Figure 9, the operating conditions were modified. Figure 10 shows the
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ANALYTICAL CHEMISTRY, VOL. 63, NO. 8, APRIL 15, 1991 Zone Length (mm) 0.24
0.0
0.48
0.72
0.96
1.20
1.44
I " " l " " l " " I " " l " " I " " I " " I 200 400 6.00 8.00
000
1000
12.00
1400
Time (s) Figure 10. Electropherogram of a mixture of FITC-labeled amlno aclds. Sample introduction time was 50 ms. The electric field in the small diameter capillary was maintained at 1.75 kV cm-'. Column length (I) was 4.0 cm. Anaiyte concentrations are 8 pM for each species. The buffer solution was the same as listed in the caption of Figure 6.
0
20
40
80
60
100
120
Sample Introduction Time (ms) Figure 8. Standard deviation of the FITGlabeled arginlne peak as a function of the sample introduction time. The electric field in the smalldiameter capillary was maintained at 3.3 kV cm-'. Column length (I) was 1.2 cm, and the buffer was the same as listed in the caption of Figure 6.
7-Arg
I !I'
1
FTC-Phe
(i
FTC-GIu
I 0
1000
2000
3000
Time (ms) Electropherogram of a mixture of FITGIabeled amlno acids. Sample introduction time was 40 ms. The electric field in the smaicdiemeter capillary was maintained at 3.3 kV cm-'.Column length ( I ) was 1.2 cm, and the buffer was the same as listed in the caption of Figure 6. Analyte concentrations are 10 pM for each species. Figwe 9.
electropherogram obtained when these same compounds where electromigrated in a field of 1750 V cm-' over a distance of 4 cm. The efficiencies of the three peaks ranged from 70000 to 90 000 theoretical plates which corresponds to approximately 2 X lo6 plates/m and a HETP of 0.5 pm. Arginine was found to be separated at a rate equivalent to 12000 theoretical plates/s. Equally important, the peak capacity of the electropherogram has been dramatically increased. CONCLUSIONS The applications for "fast" CE instrumentation are many and varied. Potential applications include a means for monitoring "fast" chemical events, a device for quickly obtaining estimates of molecular parameters (i.e., diffusion coefficients (8), mobilities, molecular charge, etc.), a "fast" second dimension in a multidimensional separation scheme (9),or a multiplex separation instrument (10, 11). Clearly,
this list is not all inclusive but is only meant to give an idea of the scope of applications for these devices. As advances in instrumentation continue to be made, it is likely that many of the problems we have encountered with this f i t generation instrument will be overcome. Although chromatographic methods of analysis may benefit from some of the technology which will be developed for fast CE instruments, it will be more difficult for chromatography to match the speed-efficiency capabilities of capillary electrophoresis. We have begun work in the application of this technology to capillary chromatographic systems (12). One future avenue of investigation which appears to hold great promise is the construction of a complete CE instrument on a single substrate wafer ("an instrument on a chip"). Several advantages are likely to result from this departure from typical CE instrument form, particularly when constructing a high-speed instrument. Recent advances in the micromachining of miniature valves and gates should allow more universal methods of sample introduction and detection to be employed. This would avoid many of the problems associated with on-column optical gating and fluorescence detection. Another possible advantage is that column thermostating would become much easier. Integrated cooling systems for silicon substrate circuits have demonstrated the ability to efficiently handle power dissipations a factor of 50 in excess of the noncooled substrate (13). If this same technology were applied to capillary electrophoresis, this could directly translate into the ability to operate at higher potential fields, with a corresponding increase in separation analysis speed and efficiency. Additionally, the ability to manufacture instruments in a batch mode would allow the cost of manufacturing to be spread over many instruments and thereby lower the cost per device. The work presented here provides only a first glimpse into this promixing venue. However, it seems clear that the advantages made possible by miniaturization could provide the key to allow this technology to compete favorably with current electrophoresis instruments.
ACKNOWLEDGMENT We thank Gary M. Hieftje of Indiana University for the loan of the acoustooptic modulator used in this work. LITERATURE CITED (1) Jorgenson. J. W.; Lukacs. K. D. Anal. Chem. 1981. 53, 1298. (2) Nickerson. B.; Jorgenson, J. W. M7C & CC, J . /-/@I Resdfd. Chromet o g . Chromatogr. Commun. 1888. 7 1 . 533. (3) Lukacs, K. D. Ph.D. Dksertatkn, mersity of North Carolina at Chapel Hili, 1983. (4) Annino, R.; Gonnord, M.; Guiochon. G. Anal. Chem. 1979, 57. 379. (5) Cram, S. P.; Chesler, S. N. J . Chrometogr. 1974, 99,287. (6) Hwschfeld, T. Appl. Opt. 1876, 75,3135. (7) Cheng. Y.; Dovichi, N. J. Science 1988, 242, 562. (8) Walbroehl, Y.; Jorgenson, J. W. J . M/crocolumn Sep. 1888, 7 , 41. (9) Bushey, M. B.; Jorgenson, J. W. Anal. Chem. 1980, 62. 978.
a07
Anal. Chem. 1991, 63,807-810 (10) Phillips, J. B. Anal. Chem. 1080, 52, 468A. (11) Smii, ti. C. Trends Anal. Chem. 1983. 2 , 1. (12) Monnig, C. E.; Dohmeier, D. M.; Jorgenson. J. W. Following article in this- Issue. (13) Angell, J. B.; Terry, S. C.; Barth, P.W. Sci. Am. 1983, 248, 44.
____
RECEIVED for review July 30, 1990. Revised manuscript re-
ceived December 27,1990. Accepted January 3,1991. This research has been supported in part by the National Institute of Health (Grant GM39515). the National Science Foundation (Grant CHE-8912926), and the National Science Foundation Postdoctoral Fellowship Program in Chemistry (Grant CHE-9044317).
Sample Gating in Open Tubular and Packed Capillaries for High-speed Liquid Chromatography Curtis A. Monnig, Daniel M. Dohmeier, and James W. Jorgenson* Department of Chemistry, University of North Carolina, Chapel Hill, North Carolina 27599-3290
A new llquid chromatography Instrument is described that employs small-diameter capillary columns and an optlcaliy controlled sample gatlng procedure to separate a mlxture of fluoresceln lsothlocyanate (FITC) labeled amines In as few as 6 8. Effidency of the separath, expressed as the nunber of theoretical plates, is observed to increase linearly with column length and thus analysts tlme. The combination of short analysts t h e and automated sample introductlon allows signal averaging to be used to enhance the precision of the measurement.
INTRODUCTION There is growing interest in developing microcolumn separation procedures that are capable of both rapid and efficient separation of complex mixtures. This trend is perhaps best exemplified by the surge of interest in capillary electrophoresis (CE). Unfortunately, many chemical species cannot be readily separated by electrophoretic procedures. For these molecules, chromatographic procedures are often the separation methods of choice. However, chromatographic procedures as they are usually practiced are configured to achieve maximum separation efficiency, with time of analysis being a secondary consideration. To address what we perceive to be a growing interest in "fast" separation procedures, we are investigating a new method of fast liquid chromatography. High-performance liquid chromatography (HPLC) usually requires a minimum of several minutes and often can take as long as several hours for complete separation. Consequently, most analysts do not associate liquid Chromatography with the rapid analysis of mixtures. In the limited number of occasions when a rapid analysis of mixtures has been performed by LC, the reduced analysis time is usually achieved by forcing the sample mixture though the column rapidly. This approach sacrifices much of the separation efficiency to gain an incremental improvement in analysis time. A second obvious approach would be to shorten the column length; this, however, will also lead to a decrease in efficiency. This loss in efficiency can be partially offset by reducing the chromatographic dimensions (the particle size in a packed column or the capillary diameter in an open tubular column). Unfortunately, this miniaturization process introduces a new set of problems that must be overcome before the utility of the technique can be demonstrated. *To whom correspondence should be sent.
In this report, we describe a new instrument for high-speed chromatographic analysis of complex mixtures. Specifically, we exploit the excellent column efficiency of open tubular liquid chromatography (OTLC) to maintain peak separation during a "fast" liquid chromatographic separation. Additionally, we will describe a novel sample gating procedure which minimizes zone broadening during the sample introduction step and allows simple automation of the analysis procedure.
THEORY To achieve acceptable peak resolution during a "fast" chromatographic separation it is necessary to minimize the processes that broaden the sample zone. The variance of the sample zone as recorded by a detector can be considered to be the sum of the variances produced during sample injection (u~~,.,~), migration on the column (uzCo1), and detection (a2d&).
It is desirable, under normal operating conditions, that oncolumn zone broadening be the predominant factor which determines the variance of the recorded peak. Fortunately, column-related zone-broadening mechanisms can be minimized in several ways. Although packed column beds are used in the vast majority of liquid chromatography instruments, it is well-known that open tubular columns can provide superior separations (1,2). Not surprisingly, excellent separations have been reported for OTLC in analysis times as short as a few minutes (3,4). Zone broadening can also be minimized by increasing the length to diameter ratio of the column. Finally, the experimental conditions can be adjusted so that the peak capacity factor ( k ? is as small as is practical to minimize broadening introduced by slow kinetics, which may impede the transfer of molecules into and out of the stationary phase. One obvious means to reduce the time required for an analysis is to shorten the length of the column. Unfortunately, changing the length of the column can introduce new factors that degrade the separation performance. First and foremost, to retain the efficiency of the separation, the solute in the mobile phase must be equilibrated with the stationary phase an equivalent number of times as in the longer column. This can be realized by simply reducing the distance the solute must diffuse to reach the stationary phase. For OTLC this is accomplished by reducing the diameter of the column. With packed columns, smaller stationary-phase particles can be used to minimize this diffusion distance. An equally important observation is that as the column length is shortened, zone
0003-2700/91/0363-0807$02.50/00 1991 American Chemical Society
