Lowering the UV Absorbance Detection Limit in Capillary Zone

Over the course of a run, therefore, an electropherogram is generated for each diode in the array. Averaging the electropherograms generated from 1500...
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Anal. Chem. 1998, 70, 2629-2638

Lowering the UV Absorbance Detection Limit in Capillary Zone Electrophoresis Using a Single Linear Photodiode Array Detector Christopher T. Culbertson and James W. Jorgenson*

Kenan Laboratories of Chemistry, Department of Chemistry, The University of North Carolina, Chapel Hill, North Carolina 27599-3290

A new approach for lowering the UV absorbance detection limit in capillary electrophoresis is presented. This approach involves the use of a photodiode array in which each of the diodes in the array is treated as an independent detector. Over the course of a run, therefore, an electropherogram is generated for each diode in the array. Averaging the electropherograms generated from 1500 diodes in a diode array resulted in a signal-to-noise ratio 85 times that of an electropherogram generated from any one diode in the array. These signal-to-noise improvements are discussed, and the detection limits are compared to the detection limits obtained from a commercial single-point detector. The array detector improves the detection limit by a factor of 3.8 ((0.4). Capillary electrophoresis (CE) has rapidly become an important analytical technique for the separation of compounds ranging from small inorganic ions to large biological molecules. CE has many features which make it attractive for the separation of such compounds; these include rapid analysis times, high efficiencies, small sample volume requirements, and low solvent consumption. Because of these features, CE has been increasingly used as an alternative or complementary technique to high-performance liquid chromatography (HPLC). One of CE’s principal limitations, which has prevented even wider acceptance of the technique, is the rather poor concentration detection limits achievable when using UV absorbance detection. These limits typically range from 10-5 to 10-7 M depending upon the analyte being examined. Although fluorescence and electrochemical detection schemes with good mass and concentration detection limits exist for CE, UV absorbance detection has remained very popular because of its ease of implementation and wide applicability, especially for the detection of organic and biologically active compounds. Absorbance detection limits are poor in CE, as the capillary columns commonly used have inner diameters (i.d.’s) of only 25-75 µm. These small i.d.’s severely restrict the absorption path length. Due to the small sample volumes (i.e. short analyte plug lengths) post or off-column detection, which can provide substantially longer path lengths, is not a viable option as it is with HPLC. Several attempts have been made to develop instrumental methods to lower the on-column absorbance detection limits of CE by increasing the absorption path length. These attempts have S0003-2700(97)00654-9 CCC: $15.00 Published on Web 05/09/1998

© 1998 American Chemical Society

included the use of Z-cells,1-3 bubble cells,4-6 axial illumination with axial detection,7, 8 and multireflection cells.9 The use of bubble cells and Z-cells in 50 µm i.d. capillaries can lower detection limits by factors of 3 and 4, respectively. These lower detection limits, however, come at some expense to both efficiency and resolution.1-3,6 Axial illumination with axial detection and multireflection cells have not yet yielded detection limits which are lower than those of commercially available single-point detectors. Another way to attempt to lower the absorbance concentration detection limits is to increase the photon flux through the detection system. If the emission of photons from the excitation source is random and the detector is shot-noise-limited, the signalto-noise ratio will grow as the square root of the incident light intensity, thus lowering the detection limit.10 The most successful application of increasing the photon flux through the detection system has been the use of a sapphire-ball lens to focus more light through the capillary separation channel.1,3,11 Such lenses are now a standard part of many capillary cell holders used on commercial UV-vis absorbance detectors. Intense laser sources and precision alignment techniques have also been used to increase light throughput. Unfortunately, lasers tend to suffer from a high degree of flicker noise and come in a limited number of wavelengths. Precision alignment techniques, while helpful in certain instances, are problematic because they are tedious and timeconsuming to implement and difficult to reproduce precisely.12 Absorbance detection limits in CE may also be lowered through the use of signal averaging. However, making repetitive runs in CE to signal-average is not practical because analyte (1) Bruin, G. J. M.; Stegeman, G.; Van Asten, A. C.; Xu, X.; Kraak, J. C.; Poppe, H. J. Chromatogr. 1991, 559, 163-181. (2) Chervet, J. P.; Van Soest, R. E. J.; Ursem, M. J. Chromatogr. 1991, 543, 439-449. (3) Moring, S. E.; Reel, R. T. Anal. Chem. 1993, 65, 3454-3459. (4) Hewlett-Packard. Peak 1993, 2, 11. (5) Heiger, D. N.; Kaltenbach, P.; Sievert, H.-J. P. Electrophoresis 1994, 15, 1234-1247. (6) Xue, Y.; Yeung, E. S. Anal. Chem. 1994, 66, 3575-3580. (7) Xi, X.; Yeung, E. S. Anal. Chem. 1990, 62, 1580-1585. (8) Taylor, J. A.; Yeung, E. S. J. Chromatogr. 1991, 550, 831-837. (9) Wang, T.; Aiken, J. H.; Huie, C. W.; Hartwick, R. A. Anal. Chem. 1991, 63, 1372-1376. (10) Ingle, J. D., Jr.; Crouch, S. R. Spectrochemical Analysis; Prentice Hall: Englewood Cliffs, NJ, 1988. (11) Moring, S. E.; Colburn, J. C.; Grossman, P. D.; Lauer, H. H. LC-GC 1990, 8, 34-46. (12) Flint, C. D.; Grochowicz, P. R.; Simpson, C. F. Anal. Proc. 1994, 31, 117121.

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migration times are not sufficiently reproducible. Even if analyte migration times were sufficiently reproducible for signal averaging, the number of runs, and therefore the time it would take to make a reasonable signal-to-noise ratio increase, would be prohibitive. Signal averaging in CE, however, may also be accomplished over the course of a single run by detecting an analyte multiple times with multiple detectors as it migrates through the capillary. The analyte’s signals from all of the detectors can then be averaged together to increase the signal-to-noise ratio. If the detectors are shot-noise- or white (random)-noise-limited, then the improvement in the signal-to-noise ratio should be equal to the square root of the number of detectors.10 One of the difficulties with detecting an analyte multiple times as it passes through a capillary is that separations are dynamic events. Analyte bands are continually broadening through processes such as longitudinal diffusion, and the resolution between each pair of analyte bands is continually increasing. Multiple detection events must, therefore, be spaced as closely together as possible to minimize the adverse effects that these processes may have on signal averaging. For this reason, a linear diode array has been chosen to provide the multiple detectors needed in this experiment. Linear diode arrays consisting of several hundred to several thousand diodes are readily available from several instrument manufacturers. The individual diodes in these types of arrays generally range from 7 to 50 µm in width, so that the entire array is usually only a few centimeters long. Because each diode is capable of responding to light signals independently of its neighbors, each diode can generate an independent electropherogram over the course of a CE run just as any single-point detector can (Figure 1A). The number of electropherograms generated during a CE separation using such an array detector is, therefore, equal to the number of diodes in the array. Diode arrays have been used previously as both spectral5,13 and spatial imaging detectors14-27 for CE separation methods, and some limited forms of signal averaging have been proposed28 or performed using such array detectors. Wu and Pawliszyn have used diode arrays, both linear charge-coupled devices (CCDs) and CCD cameras, to develop imaging detectors for capillary isoelectric focusing (CIEF) based upon refractometry17,19-25 and absorption.17-19,22 After focusing the analytes, Wu and Pawliszyn were able to temporally signal-average, although it was limited to short periods of time due to the analyte cathodic drift typically (13) Kobayashi, S.; Ueda, T.; Kikumoto, M. J. Chromatogr. 1989, 480, 179184. (14) Sweedler, J. V.; Shear, J. B.; Fishman, H. A.; Zare, R. N. Anal. Chem. 1991, 63, 496-502. (15) Nilsson, S.; Johansson, J.; Mecklenburg, M.; Birnbaum, S.; Svanberg, S.; Wahlund, K.-G.; Mosbach, K.; Miyabayashi, A.; Larsson, P.-O. J. Capillary Electrophor. 1995, 2, 46-52. (16) Johansson, J.; Witte, D. T.; Larsson, M.; Nilsson, S. Anal. Chem. 1996. (17) Wu, J.; Pawliszyn, J. Analyst 1995, 120, 1567-1571. (18) Wu, J.; Pawliszyn, J. Electrophoresis 1995, 16, 670-673. (19) Wu, J.; Pawliszyn, J. Anal. Chem. 1994, 66, 867-873. (20) Wu, J.; Pawliszyn, J. J. Chromatogr. 1993, 652, 295-299. (21) Wu, J.; Pawliszyn, J. J. Liq. Chromatogr. 1993, 16, 3675-3687. (22) Wu, J.; Pawliszyn, J. J. Liq. Chromatogr. 1993, 16, 1891-1902. (23) Wu, J.; Pawliszyn, J. Electrophoresis 1993, 14, 469-474. (24) Wu, J.; Pawliszyn, J. Anal. Chem. 1992, 64, 2934-2941. (25) Wu, J.; Pawliszyn, J. Anal. Chem. 1992, 64, 224-227. (26) Thormann, W.; Mosher, R. A.; Bier, M. J. Chromatogr. 1986, 351, 17-29. (27) Thormann, W.; Tsai, A.; Michaud, J.-P.; Mosher, R. A. J. Chromatogr. 1987, 389, 75-86. (28) Kita, J.-i. (Shimadzu Corp.) U.S. Patent 5,303,021, 1994.

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Figure 1. (A) Independent generation by each diode in the diode array of its own electropherogram. The migration time of an analyte in each successive diode electropherogram is slightly longer as each successive diode is further from the injection point. (B) Simple time shift in the diode electropherograms relative to one another, allowing only one point on the diode electropherograms to be averaged properly, as the velocity of each analyte is different.

found with CIEF separations. Diode arrays have also been used in CZE as detectors for laser-induced fluorescence (LIF).14-16 Sweedler et al. set up a CCD camera in a time-delayed integration mode (TDI) to image the last few centimeters of a separation capillary. In the TDI mode, with the CCD arranged so that the direction of pixel shifts on the array is parallel to the capillary, the shift rate of the pixels can be synchronized with the migration velocity of an analyte in the capillary. This enables the analyte signal to be electronically integrated on the CCD chip over the time the analyte spends in the detection region. With this type of detection scheme, real-time signal averaging can be performed as analytes migrate through the imaged portion of the capillary. Signal-to-noise ratio increases of 2-5 were obtained using this method. Although TDI works well for fluorescence detection when the diodes are far from their saturation points, it cannot be used in absorbance detection schemes where the diodes are almost completely saturated over the integration time of each acquisition. In this paper some preliminary results will be presented in which 1500 diodes of a 2048-element linear photodiode array (PDA) detector have been signal-averaged to lower the concentration detection limits of UV absorbance detection in CE. Using such an array detector, a signal-to-noise ratio increase of 85 was generated over that of a single diode. This is slightly more than twice the increase predicted, assuming that the diodes are whitenoise-limited. The signal-to-noise ratio improvement gained

through the use of signal averaging with an array detector is discussed, and a comparison of this detector to a commercial, single-point absorbance detector is also made. THEORY Signal Averaging. The electropherograms generated at each diode, which will be referred to henceforth as single-diode electropherograms, can be averaged to produce a signal-to-noise ratio enhancement of the analytes in the run. Signal averaging, however, cannot be performed by simply summing the signals at a particular time point for all of the single-diode electropherograms, because any particular analyte has a slightly longer migration time in each successive single-diode electropherogram (Figure 1A). The increase in migration time at each successive diode is due to the increased distance from the injection point. In addition, timeshifting the single-diode electropherograms relative to one another will not correctly signal-average all of the analytes, as each analyte migrates through the detector array at a different velocity, as shown in Figure 1B. For any particular velocity correction, therefore, only one analyte band would be signal-averaged properly. To perform the signal averaging properly, the first single-diode electropherogram is used as the base electropherogram. The velocity (v) of any analyte which enters this first diode detector at migration time t1 is easily determined, as the distance between the first diode and the injection point is known. After the velocity of an analyte has been determined, its migration time (tn) in any subsequent single-diode electropherogram (n) can be determined, as the distance between each diode in the array is also known. This is shown by the equation

tn ) t1 +

(n - 1)d vM

(1)

where v is equal to the velocity of an analyte in the capillary as determined from the first diode electropherogram, M is the absolute value of the image magnification, and d is the diode width. Once the migration time of an analyte is known for each singlediode electropherogram, the data points which contain the analyte signal in all of the single-diode electropherograms can be averaged using the equation

σ ) x2Dt

∑A

n)1

N

(3)

where D is the diffusion coefficient and t is the time at which the analyte band is measured. The fractional difference in the standard deviation (∆σ/σi) of the peak between the time that it enters and leaves the imaged area of the capillary can be expressed as

∆σ σf - σi ) σi σi

(4)

where σi and σf are the standard deviations of the peak as it enters and as it leaves the imaged portion of the capillary, respectively. Substituting eq 3 into eq 4 and simplifying gives

∆σ xtf ) -1 σi t

xi

(5)

where ti is the time at which the analyte band enters the imaged portion of the capillary and tf is the time at which the analyte band leaves the imaged portion of the capillary. The distance from the injection point to the beginning (li) and to the end (lf) of the imaged section of the capillary can be substituted for ti and tf, respectively, as the velocity (v ) l/t) of the peak is known. This substitution results in the following modification to eq 5:

∆σ xlf ) -1 σi l

xi

(6)

The length of the capillary imaged (∆l) is given by

∆l ) lf - li

N

ASA )

the detector. The magnitude of this band broadening can easily be calculated as shown below. Normally in CE, because of the essentially flat flow profile, longitudinal diffusion alone is responsible for the irreducible minimum amount of band broadening that can be expected. The magnitude of this diffusion, expressed as the standard deviation (σ) of the analyte bandwidth, is given by the Einstein equation

(7)

tn

(2)

In this equation, ASA is the signal-averaged absorbance value, N is the number of diodes in the array, and Atu is the absorbance value at migration time tn for single-diode electropherogram n. The procedure can be generalized to every data point on the first diode electropherogram, so that all of the points in the electropherogram are signal-averaged, not just points containing analyte peaks. This will result in a complete signal-averaged electropherogram. Using this generalized approach, no a priori determination of the number or position of analyte bands is necessary. Analyte Band Broadening through the Array. Although the diodes in the array are spaced closely together, there will still be some band broadening of the analytes as they pass through

Solving for lf, substituting into eq 6, and simplifying gives

∆σ ) σi

x

∆l +1-1 li

(8)

Equation 8 shows that the band broadening (∆σ) of any analyte band as it migrates through the imaged portion of the capillary is determined by the ratio of the imaged length of the capillary to the preimaged length (e.g. the distance from the injection point to the beginning of the imaged portion of the capillary), given a constant analyte velocity. When the imaged portion of the capillary is kept to less than 10% of the preimaged length, the band broadening which will occur as the analyte travels through the imaged portion of the capillary can be limited to less than 6% and, therefore, should not adversely affect the signal averaging. Analytical Chemistry, Vol. 70, No. 13, July 1, 1998

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For the photodiode array used below, the saturation charge for each diode, as determined from data given by the instrument manufacturer, is about 9.3 pC or 59 million electrons.29 The shot noise for a fully saturated diode should therefore be 7600 electrons, which gives a signal-to-noise ratio (nj e/σe) of 7600. Conversion of this value into an absorbance shot-noise limit (σA) can be performed using the equation

σA )

Figure 2. Relative peak width increase as a function of the preimaged capillary length for three different imaged lengths.

Figure 2 shows the effect of the preimaged length of capillary on the three imaged lengths in terms of the amount of band broadening expected. Photodiode Arrays vs Charge-Coupled Devices. A PDA is used as the detector in this setup instead of a CCD because the linear PDA has a much larger electron well capacity. The electron well depth of each diode in the PDA used in this experiment is about 59 million electrons, whereas most diodes in high-performance linear CCD’s are limited to electron well depths of only about 0.3 million electrons.29 If the diodes in both types of devices are shot-noise-limited, which is not a difficult condition to meet, the dynamic range of the PDA is 10-20 times greater than that of the CCD, given the same readout rates. This extra dynamic range is important when performing absorbance measurements, where a small difference between two large numbers is measured. The larger the dynamic range, the smaller the difference that can be detected, and therefore, the lower the initial absorbance detection limit in each single-diode electropherogram. Although the PDA generally has a higher background (dark) current than a CCD, the PDA is usually shot-noise-limited at high light intensities, as will be the case when it is being used for absorbance detection.29 CCD pixels can be binned to improve their dynamic range, but this comes at the expense of spatial resolution. Calculation of PDA Shot Noise. Determining the shot noise for this detector is important, as this is the noise which ultimately determines the minimum noise level for the system. Shot noise is generally defined as the combined noise associated with the random generation of photons from the excitation source and the random generation of electrons in the photodiode. The shot noise (σe) for the instrument described in this paper can be determined using the equation

σe ) xηn j p ) xn je

(9)

where η is the quantum efficiency of the photodiode, nj p is the number of photons striking the diode, and nj e is the number of photoelectrons generated at the diode junction.30 2632 Analytical Chemistry, Vol. 70, No. 13, July 1, 1998

0.43σe Tn je

(10)

where T is the transmittance.10 The shot-noise limit for each diode at its saturation point, assuming T is equal to 1, should be 5.6 × 10-5 AU. To make actual absorbance measurements, however, the diodes should not be completely saturated; rather, they should be 85-90% saturated to allow room to compensate for uncontrollable variables which can change over the period of data acquisition. For diodes in the array at 85-90% saturation, the expected shot-noise detection limit should be between 5.8 × 10-5 and 6.1 × 10-5 AU. It should be pointed out, however, that for low analyte absorbance values a shot-noise limited detection system may not be realized, as signal flicker noise often predominates.10 EXPERIMENTAL SECTION Capillary Electrophoresis with the PDA Detector. CE was performed using a Spellman 1000R high-voltage power supply (Plainview, NY). It was interfaced to a Power Macintosh 8100/ 110 using a National Instruments (Austin, TX) NB-MIO-16XL-42 multifunction I/O board. All code used to operate the high-voltage power supply for the analyte injections and electrophoretic runs was written in-house using National Instruments LabVIEW 3.1.1. The fused silica separation capillaries (Polymicro Technologies Inc.; Phoenix, AZ) were 52 µm i.d. (nominal), 350 µm o.d., and 91 cm in length. The capillary was imaged for 10.24 cm between 76 and 86.24 cm from the injection point. The polyimide was removed from this section of capillary using fuming sulfuric acid. Injections were made from the high-voltage (anode) end of the capillary. This end of the capillary was enclosed in a Plexiglas box to protect the operator from accidental shock. To allow the imaging to take place as close as possible to the end of the capillary, the capillary was grounded remotely by inserting the capillary end into a PEEK OPTI-LOK tee (Optimize Technologies, Portland, OR), which was connected to a buffer reservoir by 0.020 in. (0.51 mm) i.d. PEEK tubing from Alltech (Deerfield, IL). A grounded platinum electrode was present in this remote reservoir, and the current through the capillary was monitored using a microammeter which was inserted in the ground line. Less than 0.1% of the voltage is dropped through the PEEK tubing which connects the reservoir to the tee because of its large i.d. compared to the separation capillary i.d. Array Detection System. The PDA detector constructed for the experiments described below is shown in Figure 3A. The light source, filter, capillary holder, iris diaphragm, and lens were all contained in a light-tight aluminum box attached to a laser breadboard (TMC; Peabody, MA). All optical components were (29) High Performance Digital CCD Cameras; Princeton Instruments: Trenton, NJ, 1995. (30) Photomultiplier Handbook; RCA Corp., Lancaster, PA, 1980.

Figure 3. (A) Generalized schematic of the instrumental setup for imaging a CE capillary with a photodiode array. (B) Schematic of capillary holder and slit. The capillary is held in place between two PVC plates. These plates also serve as the slit.

centered 13.4 cm above the laser table. The light source, a 254 nm Mercury Pen-Ray lamp (model 3-SC-9) from UVP (San Gabriel, CA), had a lighted length of 23 cm, was run using a 60 Hz stabilized power supply, and was partially encased in an aluminum block to help maintain a stable temperature. The light passed through a 254 nm interference filter which was fabricated in a custom size (5.08 × 10.16 cm) by Omega Optical, Inc. (Battleboro, VT). The transmission efficiency was 12%. The lamp was warmed for 1 h before runs were performed. The imaged portion of the capillary was held between two PVC plates (Figure 3B) which served both to hold the capillary in place and to serve as a slit with which to cut out stray light. A shallow groove, ∼50 µm deep, was etched into each plate to hold the capillary firmly in place. The transmitted light from the capillary passed through an aperture created by an iris diaphragm (Melles Griot; Rochester, NY) and a single, biconvex, 10.16 cm diameter fused-silica lens (f ) 10.1 cm; F/# ) 1) obtained from Oriel (Stratford, CT). This focusing system created an inverted image of the capillary at a nominal magnification of -0.5 on the face of the PDA, which had been mounted on the light-tight box. At this magnification, 50 µm contiguous segments of capillary are imaged onto sequential 25 µm diode elements. Optically, the spatial resolution along the section of capillary imaged is 50 µm. Electrophoretically, the minimum distance that can be discerned between any two peak maxima is 100 µm. The analyte peaks are expected to be about 5 mm wide; therefore, adequate sampling across a peak should be realized. Because of the peak widths

expected, the resolution between any pair of peaks should be limited electrophoretically, not optically. The 2048-element photodiode array and its accompanying ST116 controller were purchased from Princeton Instruments (Trenton, NJ). The diodes in the array were 25 µm in width and 2500 µm in height. The controller was interfaced to the same Macintosh PowerPC which was responsible for controlling the CE power supply. This interfacing was accomplished using a National Instruments NB-DMA-2800 GPIB board. All code used to operate the PDA through its ST-116 controller was written inhouse using National Instruments LabVIEW 3.1.1. The transmitted light data collected at the PDA over the course of a run was converted to absorbance values using the tenth acquisition as the blank reference. This acquisition was obtained 1 s after the PDA began taking data. The PDA detector was kept at 0 °C using a thermoelectric cooling unit. Data Acquisition Using a PDA. Very large data sets are generated for each CE run using the 2048-element PDA detector. A typical 30 min run with a data acquisition rate of 10 Hz generates 147 Mb of data. The final signal-averaged electropherogram, however, only requires as much disk space as a typical CE run made on a single-point detector for 30 min at 10 Hz (i.e. 72 kb). At the present time, because of buffer limitations in the PDA controller, all of the data collected during a run must temporarily be stored in the computer RAM. To keep the initial raw data sets to a manageable size, data acquisition was limited to the 15 min period of time during which the analyte peaks were expected to migrate through the detector. CE with Single-Point Detection. For the single-point detection comparisons, a Linear UVis 200 detector outfitted with an on-column capillary detection cell was used (Thermoseparations; Freemont, CA). The detector rise time was set at 0.3 s. The separation capillaries were 52 µm i.d. (nominal) and 100.1 cm in length with the detection window located at 82.5 cm. The current through the capillary was also monitored using a microammeter which was inserted in the ground line. Separation Conditions. All electrophoretic separations were performed in pH 7.35 100 mM Na0.5HEPES buffer. The injection and run conditions for all separations were the same. The analytes were injected at the anode for 10 s at 10 kV (∼100 V/cm) and run at 30 kV (∼300 V/cm). Prior to each set of runs, the capillary was washed with a solution composed of 67% (v/v) 1 M sodium hydroxide and 33% (v/v) 2-propanol for 15 min. This was followed by a water rinse and a buffer rinse for 15 min each. Reagents. The nucleic acids Adenosine 5′-monophosphate (AMP), guanosine 5′-monophosphate (GMP), thymidine 5′-monophosphate (TMP), and cytidine 5′-monophosphate (CMP) and Na0.5HEPES ((N-(2-hydroxyethyl)piperazine-N′-(2-ethanesulfonic acid)) hemisodium salt) were purchased from Sigma Chemical Co. (St. Louis, MO) and used without further purification. All solutions were prepared using distilled deionized water obtained from a Barnstead Nanopure System (Dubuque, IA). Procedures. The nucleic acids were weighed using a microbalance, transferred to a 50 mL volumetric flask, and dissolved in running buffer. The nucleic acids were serially diluted to the appropriate concentrations just prior to making a run. Analytical Chemistry, Vol. 70, No. 13, July 1, 1998

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Figure 4. Raw data image of capillary. Only the 1500 diodes between the two grayed out sections were used for signal averaging.

Figure 5. Two-dimensional gray scale image of the separation of four nucleic acids - GMP, AMP, TMP, and CMPsat concentrations of 110, 200, 110, and 100 µM, respectively.

Data Analysis. Data analysis was performed using LabVIEW 3.1.1, IGOR Pro 2.04 (Wavemetrics, Lake Oswego, OR), and Spyglass Transform 3.02 (Spyglass, Inc.; Champaign, IL.). All peaks were fit to Gaussian distributions. The rms noise in all of the electropherograms was obtained using a section of baseline near one of the analyte peaks. This baseline section was of the same width as the peak of interest. RESULTS AND DISCUSSION Raw Data Images. A typical raw data image of the capillary obtained using the PDA is shown in Figure 4. As can be seen from Figure 4, the light intensity near the edges of the array drops significantly. The simple optical setupsa 4 in. (10.16 cm) diameter fused-silica biconvex lensswas not able to satisfactorily image the entire 10 cm section of the capillary onto the array. Therefore, only the diodes between 250 and 1750 were used for the results shown below. The average saturation value for these diodes was 71.4%. The shot-noise absorbance value calculated from the average percent saturation using eq 10 was 6.6 × 10-5 AU. Several spikes can be seen in this image. These spikes are due to nicks and/or optical flaws in the capillary itself or from small shavings of PVC left behind from the grooving process on the capillary holder. They are permanent features of the system and, therefore, can be effectively canceled out when a reference image of the capillary is taken during the electrophoretic run. These small absorbance spikes were used to produce the best focused image of the capillary. Signal-to-Noise Improvements. Figure 5 shows the separation of four nucleic acidssGMP, AMP, TMP, and CMPsat concentrations of 110, 200, 110, and 100 µM, respectively. This two-dimensional plot is a result of sequentially placing the electropherograms which were obtained from each diode next to one another. To simplify the figure, the peak intensities are shown in gray scale, where the greater the absorbance, the deeper the shade of gray. The electrokinetic velocities of the analytes, or the slopes of the analyte streaks as seen in Figure 5, range from 0.044 cm/s for GMP to 0.038 cm/s for CMP. Figure 6 shows five single-diode electropherograms produced from the data taken at diodes 255, 650, 1000, 1350, and 1745. Signal-to-noise ratios for GMP and AMP average 10 ((2) and 16 ((3), respectively (Table 1). The smaller analyte peaks (CMP, TMP) have signal-to-noise (S/N) ratios of only about 4, which is 2634 Analytical Chemistry, Vol. 70, No. 13, July 1, 1998

Figure 6. Five single-diode electropherograms generated at diodes (A) 255, (B) 650, (C) 1050, (D) 1350, and (E) 1745. These slices are taken from Figure 5. Part F shows the noise power spectrum obtained from the baseline data of the electropherogram in C, between 34.7 and 38.1 min.

near the detection limit of 7 × 10-4 AU (3 × S/N). For the diode electropherograms shown the average rms noise was 2.2 × 10-4 AU. This was typical for all of the diodes in the array and is approximately 3.3 times the expected shot-noise limit of 6.6 × 10-5 AU. The excess noise is believed to be due to signal flicker noise associated with the mercury Penray lamp, as evidenced by a noise power spectrum obtained from the baseline data of Figure 6C, between 34.7 and 38.1 min, which shows a significant amount of 1/f noise (Figure 6F). To properly perform the signal averaging on the diode electropherograms, as discussed in the theory section above, the “exact” distance from the injection point to the point imaged by

Table 1. Signal-to-Noise (S/N) Ratio Improvement Gained through Signal Averaging for Each of the Four Nucleic Acids at the Concentrations Indicated av single-diode signal-averaged concn electropherogram electropherogram S/N ratio a analyte (µM) S/N ratio ((SD) S/N ratio improvement GMP AMP TMP CMP

110 200 110 100

10 ((2) 16 ((3) 4.2 ((0.9) 3.8 ((0.9)

av S/N ratio improvement a

1379 880 364 310

85 85 89 82 85 ((3)

SD, standard deviation.

Figure 8. (A) Signal-averaged electropherogram of the 1500 singlediode electropherograms shown in Figure 5. (B) Noise power spectrum of the baseline of the electropherogram in (A) between 34.7 and 38.1 min.

Figure 7. Theoretical plate count for the four nucleotides as a function of the length to the detection window.

the first diode must be known. This distance can be measured quite accurately, but the equation assumes that the analytes begin electromigrating from the very end of the capillary, at their limiting velocities, as soon as the separation potential is applied. This is not strictly true under experimental conditions, as the injection potential, the injection time, and the slew up time for the power supply both during the injection and at the beginning of the run must be taken into account. In addition, the analyte velocities (slopes in the two-dimensional electropherograms) will also be affected by errors in determining the magnification of the optical system as shown in eq 1. To find the “corrected” capillary length to the detection window and, therefore, the correct velocities for the analytes, signal averaging was performed using a range of hypothetical capillary lengths around the actual distance to the detection window. The hypothetical length which gave rise to the electropherogram with the greatest theoretical plate counts for the analytes in the separation was considered the “corrected” length. The optimal value for this length, as can be seen from Figure 7 is 74.5 cm, while the measured distance from the 250th diode to the injection point was 77.25 cm. The theoretical plate count at 74.5 cm is 11% better than that at 77.25 cm. It might also be noted from Figure 7 that the value for the length to the

capillary window need not be extremely accurate, for there is a “plateau” in theoretical plate numbers within +0.5 cm of the best value. Figure 8A shows the signal-averaged electropherogram of all 1500 single-diode electropherograms using the best length-todetector value obtained from Figure 7. The rms noise for this electropherogram is 2.5 × 10-6 AU. The noise in the signalaveraged electropherogram has been reduced by a factor of 84 over that of any single-diode electropherogram. Assuming the best case scenario for signal averagingsthat the noise in the single-diode electropherograms is random in naturesa noise decrease of only 39 is expected after signal averaging over the 1500 single-diode electropherograms. The noise improvement is 2.2 times that predicted. A possible explanation for this greater than expected noise reduction is the fact that the signals are being averaged over both time and space simultaneously. As can be seen by the analyte streaks in Figure 5, averaging the analyte signal requires taking data points which are both time- and spaceshifted from each other. The noise power spectrum (Figure 8B) of the signal-averaged electropherogram shows that the 1/f noise, between 0.1 and 2.5 Hz, which is seen in the noise power spectrum of the single-diode electropherogram (Figure 6F) has virtually been eliminated (note that the noise power scale has been enlarged by 1000× compared to Figure 6F). This is important, for when the S/N ratio is measured the noise is determined from baseline lengths equal to the width of the peak of interest. The two-dimensional averaging effectively cancels most of the excess noise above the shot noise in the frequency region in which the noise for the S/N ratio is measured. As mentioned above, if each individual diode’s shot-noise limit is 6.6 × 10-5 AU, the expected noise after signal averaging 1500 times is 1.7 × 10-6 AU. The experimentally obtained value of 2.5 × 10-6 AU is less than 50% greater than the ultimate signal-averaged shot-noise limit. The Analytical Chemistry, Vol. 70, No. 13, July 1, 1998

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Table 2. Theoretical Plate and Resolution Comparisons between Single-Diode Electropherograms and the Signal-Averaged Electropherogram single-diode concn electropherogram signal-averaged (µM) ((SD)a,b electropherogram GMP-theoretical plates AMP-theoretical plates TMP-theoretical plates CMP-theoretical plates resolution GMP-AMP resolution AMP-TMP resolution TMP-CMP

110 200 110 100

180 000 ((50 000) 170 000 ((30 000) 190 000 ((60 000) 170 000 ((80 000)

181 000 156 000 156 000 146 000

5.7((0.6) 5.9((0.9) 3.0((0.4)

5.7 5.6 2.9

a The theoretical plate counts were determined by averaging the results from each analyte over 16 single-diode electropherograms spaced approximately 100 diodes apart. The resolutions were determined by averaging the results over five single-diode electropherograms spaced approximately 350 diodes apart. b SD, standard deviation.

actual signal-to-noise improvements for the four nucleic acids can be seen in Table 1. The average improvement for these analytes, as expected from the noise reduction above, is a factor of 85 ((3) with an RSD of 3%. Table 2 shows the average theoretical plate counts for each analyte in the single-diode electropherograms and the plate counts for the analytes in the signal-averaged electropherogram. The average theoretical plate counts for the single-diode runs were obtained from 16 diode electropherograms spread approximately 100 diodes apart. The large experimental error associated with the plate counts is due to the low signal-to-noise ratio in these electropherograms. No loss of efficiency was seen, within experimental error, between the signal-averaged run and the average result from the single-diode electropherograms, even though a band broadening of about 5% was expected to occur as the analyte bands migrated through the capillary section imaged by the array. Table 2 also shows that there is no loss in resolution, within experimental error, among the peaks after the signal averaging has been performed. The failure to see the signalaveraging effects on either the efficiency or the resolution is most likely due to the large experimental errors associated with fitting the noisy analyte peaks in the single-diode electropherograms. Analytes need not be initially detectable in the single-diode electropherograms to benefit from the signal-averaging process. Figure 9A shows the single-diode electropherogram from diode 1000 for the separation of the four nucleic acidssGMP, AMP, TMP, and CMPsat concentrations of 11, 20, 11, and 10 µM, respectively. In this single-diode electropherogram no analyte peaks can be seen. Figure 9B shows the signal-averaged electropherogram obtained. It has been median-filtered-baselinesubtracted to remove low-frequency baseline fluctuations.31, 32 The rms noise decreased from 1.9 × 10-4 AU in the singlediode electropherogram to only 2.9 × 10-6 AU in the medianfiltered-baseline-subtracted signal-averaged run. This was a decrease of 66-fold: 1.7 times better than the 39-fold decrease (31) Moore, A. W., Jr.; Jorgenson, J. W. Anal. Chem. 1993, 65, 188-191. (32) Culbertson, C. T. Ph.D. Dissertation, University of North Carolina, Chapel Hill, 1996.

2636 Analytical Chemistry, Vol. 70, No. 13, July 1, 1998

Figure 9. (A) Single-diode electropherogram generated at diode 1050 for the separation of four nucleic acidssGMP, AMP, TMP, and CMPsat concentrations of 11, 20, 11, and 10 µM, respectively. (B) Median-filtered-baseline-subtracted signal-averaged electropherogram generated for the separation shown in (A). (C) Equivalent separation performed on a commercial Linear 200 UVis single-point detector.

expected assuming that the diodes were white-noise limited. It is again less than twice that of the ultimate signal-averaged shotnoise limit. The increase in the signal-to-noise ratio could not be measured in this analyte concentration range because the signals were below the detection limits for the single-diode electropherograms. The signal-to-noise ratio of the four analyte peaks in the signal-averaged electropherogram can be seen in Table 3. The equivalent separation performed on a Linear 200 UVis single-point detector can be seen in Figure 9C. This trace has also been median-filtered-baseline-subtracted using a 300-point filter width. The average S/N ratio improvement for the analyte peaks obtained using the array detector is 3.8 ((0.4) times that obtained from the Linear single-point detector (Table 3). The plates obtained using the diode array detector are slightly higher than those obtained using the commercial single-point detector (Table 3). To probe the actual detection limits achievable using signal averaging, the separation of the four nucleic acidssAMP, GMP, TMP, and CMPsat concentrations of 1.1, 2.0, 1.1, and 1.0 µM, respectively was performed. Figure 10 shows the signal-averaged electropherogram generated from this separation. It has been median-filtered baseline-subtracted using a filter width of 300 points. At this point the GMP and AMP analyte peaks can clearly be seen, although with some distortion. The peak height for AMP is 3.1 × 10-5 AU. The peaks for TMP and CMP can also be seen,

Table 3. PDA Detector Signal-to-Noise Improvement over the Commercial Linear UVis 200 Detector with a Capillary Holder Cell analyte

S/N ratio for the signal-averaged run

S/N ratio for the linear detector

GMP AMP TMP CMP

62.8 108.4 25.8 22.8

15.0 27.4 6.9 7.0

av S/N improvement

S/N improvement 4.2 4.0 3.7 3.2

theor plates for the signal-averaged run

theor plates for the linear detector

201 000 193 000 187 000 223 000

133 000 107 000 115 000 109 000

3.8((0.4)

Figure 10. Signal-averaged electropherogram generated from 1500 single-diode electropherograms for the separation of four nucleic acidssGMP, AMP, TMP, and CMPsat concentrations of 1.1, 2.0, 1.1, and 1.0 µM, respectively.

but they are effectively lost in the residual lower frequency noise, for which the median-filtered baseline subtraction was not completely able to compensate. The rms noise decreased from 2.9 × 10-4 AU in the singlediode electropherogram to only 2.8 × 10-6 AU in the signalaveraged electropherogram. The noise decreases by a factor of 104, which is 2.65 times that predicted. The value is again less than twice that of the ultimate signal-averaged shot-noise limit. The equivalent separation performed on a Linear UVis 200 singlepoint detector shows no identifiable analyte peaks. Calibration curves for each of the four analytes from the signalaveraged electropherograms were generated. The correlation coefficients (r2) are 0.9998 or better for each analyte curve. The slopes (m) of the log-log fits to the data are 1.000 for GMP, 1.007 for AMP, 0.997 for CMP, and 0.881 for TMP. With the exception of TMP, the calibration curves for all of the analytes can be considered linear over the range of concentrations studied. It might be noted here that the signal-averaging process introduces some low-frequency noise into the signal-averaged electropherogram. The noise is introduced because the lamp is referenced only once during the course of a run. Therefore, both spatial and temporal fluctuations in the lamp over the course of a run cannot, at present, be compensated for. This noise interferes with peak determination at low analyte concentration levels. Although median-filtered background subtraction worked satisfactorily to remove most of this low-frequency noise, this technique cannot be used in more complex separations. For this reason a dual-diode array is presently being constructed to better compensate for spatial and temporal fluctuations in the source intensity. A system which is capable of continuously monitoring the source fluctuations over the course of a run should eliminate most of the introduced low-frequency noise fluctuations and remove the need for median filtering.

Figure 11. Capillary images of 200 µM AMP as it enters and leaves the detection region: (A) image taken at 30.20 min; (B) image taken at 32.62 min.

Capillary Images. In addition to using the PDA for signal averaging, its imaging properties can be used to examine changes in an analyte band as it migrates through the detection region in the capillary. Two of the most interesting changes to study are those of the analyte band’s total area and standard deviation as a function of position in the detection region. The analyte peak area may change as the analyte migrates through the detection window due to photobleaching. This may occur as the analyte band is exposed to intense excitation source radiation for much longer periods of time than in conventional single-point detectors. Examination of the change in peak standard deviation is important to ensure that the analyte bands are not broadening to the extent that they might interfere with the signal averaging. In addition, the change in the standard deviation should, also, give an indication of how good the focus is across the entire imaged portion of the capillary. If the focus is not the limiting factor, it should be possible to measure diffusion coefficients as the analyte bands electromigrate through the capillary. Because of the limited sensitivity of each individual diode in the array, rather high analyte concentrations are required for imaging purposes. Parts A and B of Figure 11 show AMP at a Analytical Chemistry, Vol. 70, No. 13, July 1, 1998

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to a varying focus across the capillary, however, cannot be ruled out as causes for either the overall decrease in peak area seen or the scatter in the data points in the figure. Figure 12B shows the change in the analyte band standard deviation with image position. The data are again quite scattered (r ) -0.23), but the same slightly negative slope to the data can be seen. The data, however, show that the peak broadening should not adversely affect the signal averaging and from the results presented above, it does not seem to. At the present time, however, it is not possible to more accurately examine the bandbroadening effects, which occur as the band migrates through the detection window, because of the scatter in the data points.

Figure 12. (A) Variation in peak area of AMP as it migrates through the detection window. (B) Variation in peak height of AMP as it migrates through the detection window.

concentration of 200 µM as it is entering and leaving the imaged portion of the capillary. It takes about 2.5 min for the analyte to migrate completely through the detection region. The average signal-to-noise ratio taken from 19 equally spaced images over this time was 28.9 ((1.5). The change in peak area with image position for these 19 images is shown in Figure 12A. Although the data are somewhat scattered (r ) - 0.25), a linear fit to the data shows a decrease of about 5% in area as the peak migrates through the detection region. Because of the initially low signal-to-noise ratio, much of the scatter seen in Figure 12A may be due to errors in fitting the peaks to Gaussian distributions. Photobleaching and effects due

2638 Analytical Chemistry, Vol. 70, No. 13, July 1, 1998

CONCLUSIONS In this paper it has been clearly shown that signal averaging in a spatial manner can be accomplished along a capillary tube without a loss of efficiency or resolution among the analyte peaks. The S/N improvements are twice that expected. This better than expected improvement may be due to the randomization of the lamp flicker noise, which occurs as the result of signal averaging in both the spatial and temporal domains. The detection limits for using the array detector are 3.8 ((0.4) times lower than can be obtained using a commercially available detector with an oncolumn capillary cell (Table 3), or about the same as what can be obtained using the bubble cell or Z-cell. The detection limit, at present, is constrained by the initial detection limit of each diode in the array. Improving the detection limit of these diodes by increasing their electron well capacity should further lower the detection limit of the signal-averaged electropherogram. ACKNOWLEDGMENT This work has been supported by NSF grant CHE-9215320. C.T.C. has been supported by The American Chemical Society Division of Analytical Chemistry fellowship sponsored by GlaxoWellcome. Received for review June 24, 1997. Accepted January 7, 1998. AC970654Z