Integration of isoelectric focusing with parallel sodium dodecyl sulfate gel electrophoresis for multidimensional protein separations in a plastic microfluidic [correction of microfludic] network

An integrated protein concentration/separation system, combining non-native isoelectric focusing (IEF) with sodium dodecyl sulfate (SDS) gel electrophoresis on a polymer microfluidic chip, is reported. The system provides significant analyte concentration and extremely high resolving power for separated protein mixtures. The ability to introduce and isolate multiple separation media in a plastic microfluidic network is one of two key requirements for achieving multidimensional protein separations. The second requirement lies in the quantitative transfer of focused proteins from the first to second separation dimensions without significant loss in the resolution acquired from the first dimension. Rather than sequentially sampling protein analytes eluted from IEF, focused proteins are electrokinetically transferred into an array of orthogonal microchannels and further resolved by SDS gel electrophoresis in a parallel and high-throughput format. Resolved protein analytes are monitored using noncovalent, environment-sensitive, fluorescent probes such as Sypro Red. In comparison with covalently labeling proteins, the use of Sypro staining during electrophoretic separations not only presents a generic detection approach for the analysis of complex protein mixtures such as cell lysates but also avoids additional introduction of protein microheterogeneity as the result of labeling reaction. A comprehensive 2-D protein separation is completed in less than 10 min with an overall peak capacity of approximately 1700 using a chip with planar dimensions of as small as 2 cm x 3 cm. Significant enhancement in the peak capacity can be realized by simply raising the density of microchannels in the array, thereby increasing the number of IEF fractions further analyzed in the size-based separation dimension.     

High-efficiency, two-dimensional separations of protein digests on microfluidic devices

High-efficiency, two-dimensional separations of tryptic digests were achieved using glass microfluidic devices. Following micellar electrokinetic chromatography (MEKC) separations in a 19.6-cm-long serpentine channel, the peptides were rapidly sampled into a 1.3-cm-long second-dimension channel, where they were separated by capillary electrophoresis (CE). The turns in the serpentine channel were asymmetrically tapered to minimize geometrical contributions to band broadening and to provide ample channel length for high-efficiency chromatographic separations. Analysis of rhodamine B injections routinely produced plate numbers of 230000 and 40000 in the first (MEKC) and second (CE) dimensions, respectively, corresponding to plate heights of 0.9 and 0.3 microm. The electric field strengths were 200 V/cm for MEKC and 2400 V/cm for CE. In analysis times less than 15 min, two-dimensional separation of bovine serum albumin tryptic digest produced a peak capacity of 4200 (110 in the first dimension and 38 in the second dimension). The system was used to identify a peptide from a tryptic digest of ovalbumin using standard addition and to distinguish between tryptic digests of human and bovine hemoglobin.     

Multichannel microchip electrophoresis device fabricated in polycarbonate with an integrated contact conductivity sensor array

A 16-channel microfluidic chip with an integrated contact conductivity sensor array is presented. The microfluidic network consisted of 16 separation channels that were hot-embossed into polycarbonate (PC) using a high-precision micromilled metal master. All channels were 40 microm deep and 60 microm wide with an effective separation length of 40 mm. A gold (Au) sensor array was lithographically patterned onto a PC cover plate and assembled to the fluidic chip via thermal bonding in such a way that a pair of Au microelectrodes (60 microm wide with a 5 microm spacing) was incorporated into each of the 16 channels and served as independent contact conductivity detectors. The spacing between the corresponding fluidic reservoirs for each separation channel was set to 9 mm, which allowed for loading samples and buffers to all 40 reservoirs situated on the microchip in only five pipetting steps using an 8-channel pipettor. A printed circuit board (PCB) with platinum (Pt) wires was used to distribute the electrophoresis high-voltage to all reservoirs situated on the fluidic chip. Another PCB was used for collecting the conductivity signals from the patterned Au microelectrodes. The device performance was evaluated using microchip capillary zone electrophoresis (mu-CZE) of amino acid, peptide, and protein mixtures as well as oligonucleotides that were separated via microchip capillary electrochromatography (mu-CEC). The separations were performed with an electric field (E) of 90 V/cm and were completed in less than 4 min in all cases. The conductivity detection was carried out using a bipolar pulse voltage waveform with a pulse amplitude of +/-0.6 V and a frequency of 6.0 kHz. The conductivity sensor array concentration limit of detection (SNR = 3) was determined to be 7.1 microM for alanine. The separation efficiency was found to be 6.4 x 10(4), 2.0 x 10(3), 4.8 x 10(3), and 3.4 x 10(2) plates for the mu-CEC of the oligonucleotides and mu-CZE of the amino acids, peptides, and proteins, respectively, with an average channel-to-channel migration time reproducibility of 2.8%. The average resolution obtained for mu-CEC of the oligonucleotides and mu-CZE of the amino acids, peptides, and proteins was 4.6, 1.0, 0.9, and 1.0, respectively. To the best of our knowledge, this report is the first to describe a multichannel microchip electrophoresis device with integrated contact conductivity sensor array.     

Contact conductivity detection in poly(methyl methacrylate)-based microfluidic devices for analysis of mono- and polyanionic molecules

An on-column contact conductivity detector was developed for the analysis of various mono- and polyanionic compounds separated by electrophoresis chips fabricated in poly(methyl methacrylate) (PMMA) using hot embossing techniques from Ni electroforms. The detector consisted of a pair of Pt wires (127 microm diameter) with an end-to-end spacing of approximately 20 microm and situated within the fluidic channel. The waveform applied to the electrode pair was a bipolar pulse with a frequency of 5.0 kHz and was used to reduce the charging current from measurement so that the current recorded at the end of one pulse is more representative of the solution conductivity. Using the detector, separations of amino acids, peptides, proteins, and oligonucleotides were demonstrated. For the amino acids and peptides, free-solution zone electrophoresis was performed. A calibration plot for the amino acid alanine was found to be linear from approximately 10 to 100 nM in a carrier electrolyte consisting of 10 mM triethylamonium acetate. The concentration detection limit was found to be 8.0 nM, with the corresponding mass detection limit equal to 3.4 amol (injection volume = 425 pL). The protein separations with conductivity detection were performed using MEKC, in which the carrier electrolyte contained the anionic surfactant sodium dodecyl sulfate (SDS) above its cmc. Near baseline resolution was achieved in the PMMA microchip for a solution containing 8 different proteins. In the case of the DNA fragments, capillary electrochromatography was used with a C18-modified PMMA chip and a carrier electrolyte containing an ion-pairing agent.     

Sweeping with electrokinetic injection and analyte focusing by micelle collapse in two-dimensional separation via integration of micellar electrokinetic chromatography with capillary zone electrophoresis

A novel integrated concentration/separation approach involving online combination of sweeping with electrokinetic injection and analyte focusing by micelle collapse (AFMC) with heart-cutting two-dimensional (2D) capillary electrophoresis (CE) in a single capillary was developed for analysis of Herba Leonuri and mouse blood samples. First, a new sweeping with an electrokinetic injection preconcentration method was developed to inject a large volume sample solution and significantly enhance detection sensitivity. Then, the preconcentration scheme was integrated to the 2D-CE to provide significant analyte concentration and extremely high resolving power. The sample was preconcentrated by sweeping with electrokinetic injection and separated in first dimension micellar electrokinetic chromatography (MEKC). Then, only a desirable fraction of the first dimension separation was transferred into the second dimension of the capillary by pressure and further analyzed by capillary zone electrophoresis (CZE) acting as the second dimension. As the key to successful integration of MEKC and CZE, an AFMC step was integrated between the two dimensions to release analytes from the micelle interior to a liquid zone and to overcome the sample zone diffusion caused by mobilization pressure. The injected sample plug lengths for flavonoids under 15 kV for 60 min were experimentally estimated as 546 cm. The dual concentration methods resulted in the increased detection factors of 6000-fold relative to the traditional pressure injection method. The relative standard deviation (RSD) values of peak height, peak area, and migration time were 2.7-4.5%, 1.9-4.3%, and 4.7-6.8% (n = 10), respectively. The limits of detection (S/N = 3) were in the range of 7.3-36.4 ng/L, and the theoretical plate numbers (N) were in the range of 1.7-4.3 × 10(4) plates/m. This method has been successfully applied to determine flavonoids in Herba Leonuri and postdosing mouse blood samples. The pharmacokinetic study also demonstrated that the proposed concentration/separation method was convenient and sensitive and would become an attractively alternative method for online sample concentration and separation in complex samples.     

Dynamic labeling during capillary or microchip electrophoresis for laser-induced fluorescence detection of protein-SDS complexes without pre- or postcolumn labeling.

The analysis of proteins under denaturing conditions is routinely performed with SDS-polyacrylamide gel electrophoresis. The automated capabilities of CE, use of nongel sieving matrixes, and on-line optical detection by either ultraviolet (UV) absorption or laser-induced fluorescence (LF) promise to revolutionize this method. While direct on-line detection of proteins is possible as a result of their intrinsic ability to absorb light in the UV part of the spectrum (detection sensitivity comparable to Coomassie Blue staining of gels), LIF provides more powerful detection but requires pre- or postcolumn fluorescence labeling of the proteins. The development of a protocol analogous to that used for double-stranded DNA analysis, where fluorescent intercalating dyes are simply included in the separation medium, would simplify size-based protein analysis immensely. This would avoid the complications associated with covalent modification of the proteins but still exploit the sensitivity of LIF detection. We demonstrate that this is possible with CE and microchip detection by incorporating, into the run buffer, a fluorescent dye that interacts hydrophobically with protein-SDS complexes. Key to this is a dye that fluoresces significantly when bound to protein-SDS complexes but not when bound to SDS micelles. Comparison of electropherograms from CE-based denaturing protein analysis with UV and LIF detection indicates that the presence of the fluor does not alter separation of the proteins. Moreover, comparison with electropherograms generated from microchip electrophoresis with LIF detection shows that equivalent patterns can be obtained. Despite the unoptimized nature of this separation system, a dynamic labeling protocol that allows for LIF detection for proteins is attractive and has the potential to circumvent the tedious labeling steps typically required.     

A 15-s protein separation employing hydrodynamic force on a microchip

We report here a novel pressurization technique for microchip electrophoresis that enables 15-s separation of protein mixtures extracted from biological samples. Although pressure-driven flow is usually parabolic flow, pressurization prior to electrophoresis separation produced a plug flow and achieved a dramatic migration time reduction without compromising resolution. Sample plugs were pushed forward by pressurization after loading the sample but before electrophoresis separation, in the absence of an electric potential. Higher pressures enabled higher speed separation; furthermore, the resolution could be easily controlled using an optimal pressure. In addition, the slow medium-pressurization technique enabled 2-D separation in only a single channel on a microchip. Utilizing this technique, 12 samples of complex protein mixture extracted from a human T lymphoblastic cell line, Jurkat cells, were separated within 15 s in a single run using a 12-microchannel array. In addition, target proteins from Jurkat cells were detected within this time. This novel pressurization technique on a microchip will offer enormous advantages for proteome analysis over commonly used 2-D electrophoresis.     

Microchip devices for high-efficiency separations

We have fabricated a 25-cm-long spiral-shaped separation channel on a glass microchip with a footprint of only 5 cm x 5 cm. Electrophoretic separation efficiencies for dichlorofluoroscein (DCF) on this chip exceeded 1,000,000 theoretical plates and were achieved in under 46 s at a detection point 22.2 cm from the injection cross. The number of theoretical plates increased linearly with the applied voltage, and at a separation field strength of 1,170 V/cm, the rate of plate generation was approximately 21,000 plates/s. The large radii of curvature of the turns minimized the analyte dispersion introduced by the channel geometry as evidenced by the fact that the effective diffusion coefficient of DCF was within a few percent of that measured on a microchip with a straight separation channel over a wide range of electric field strengths. A micellar electrokinetic chromatography separation of 19 tetramethylrhodamine-labeled amino acids was accomplished in 165 s with an average plate number of 280,000. The minimum resolution between adjacent peaks for this separation was 1.2.     

Surface modification of the channels of poly(dimethylsiloxane) microfluidic chips with polyacrylamide for fast electrophoretic separations of proteins

The electrophoresis of proteins was investigated using poly(dimethylsiloxane) (PDMS) microfluidic chips whose surfaces were modified with polyacrylamide through atom-transfer radical polymerization. PDMS microchips were made using a glass replica to mold channels 10 microm high and 30 microm wide, with a T-intersection. The surface modification of the channels involved surface oxidation, followed by the formation of a self-assembled monolayer of benzyl chloride initiators, and then atom-transfer radical polymerization to grow a thin layer of covalently bonded polyacrylamide. The channels filled spontaneously with aqueous buffer due to the hydrophilicity of the coating. The resistance to protein adsorption was studied by open-channel electrophoresis for bovine serum albumin labeled with fluorophor. A plate height of 30 microm, corresponding to an efficiency of 33 000 plates/m, was obtained for field strengths from 18 to 889 V/cm. The lack of dependence of plate height on field strength indicates that there is no detectable contribution to broadening from adsorption. A 2- to 3-fold larger plate height was obtained for electrophoresis in a 50-cm polyacrylamide-coated silica capillary, and the shape of the electropherogram indicated the efficiency is limited by a distribution of species. The commercial capillary exhibited both reversible and irreversible adsorption of protein, whereas the PDMS microchip exhibited neither. A separation of lysozyme and cytochrome c in 35 s was demonstrated for the PDMS microchip.     

High throughput profiling of charge heterogeneity in antibodies by microchip electrophoresis

A high throughput microchip capillary zone electrophoresis (CZE) method was developed for the analysis of charge heterogeneity in antibodies. The method utilizes high speed microchip electrophoresis separation and is well-suited for high throughput charge profiling of antibodies during process and formulation development. The method involves derivatization of protein molecules with Cy5 N-hydroxysuccinimide ester (NHS-ester), which does not change the protein charge profile and enables fluorescence detection on a commercial microchip instrument. The sample preparation can be performed in 96-well microtiter plates within 1 h, and each sample analysis takes only 80 s. Protein charge variants with a pI difference of 0.1 can be readily resolved in the 12.5 mm microfluidic channel. Charge profiles similar to those obtained using conventional CZE technology were found for all antibodies tested (pIs in the range of 7.5-9.2). The separation efficiency corresponds to 1.2 × 10(4) theoretical plates (1.0 μm plate height). Assay performance is assessed by demonstrating specificity, carryover, linearity, limit of detection, and precision.     

Microchip capillary electrophoresis with an integrated indium tin oxide electrode-based electrochemiluminescence detector

This paper describes an indium tin oxide (ITO) electrode-based Ru(bpy)3(2+) electrochemiluminecence (ECL) detector for a microchip capillary electrophoresis (CE). The microchip CE-ECL system described in this article consists of a poly(dimethylsiloxane) (PDMS) layer containing separation and injection channels and an electrode plate with an ITO electrode fabricated by a photolithographic method. The PDMS layer was reversibly bound to the ITO electrode plate, which greatly simplified the alignment of the separation channel with the working electrode and enhanced the photon-capturing efficiency. In our study, the high separation electric field had no significant influence on the ECL detector, and decouplers for isolating the separation electric field were not needed in the microchip CE-ECL system. The ITO electrodes employed in the experiments displayed good durability and stability in the analytical procedures. Proline was selected to perform the microchip device with a limit of detection of 1.2 microM (S/N = 3) and a linear range from 5 to 600 microM.     

Atmospheric pressure photoionization for enhanced compatibility in on-line micellar electrokinetic chromatography-mass spectrometry

Atmospheric pressure photoionization (APPI) is presented as a novel means for the combination of micellar electrokinetic chromatography (MEKC) and mass spectrometry (MS). The on-line coupling is achieved using an adapted sheath flow interface installed on an orthogonal APPI source. Acetone or toluene is added as dopant to the sheath liquid to enhance analyte photoionization. It is demonstrated that with APPI signal suppression and interferences by the surfactant sodium dodecyl sulfate (SDS) and nonvolatile buffers can be circumvented. This implies that MEKC conditions can be selected independently from MS detection. Moreover, it is shown that both polar and apolar compounds can be photoionized, thereby also facilitating the analysis of compounds that are not amenable to electrospray ionization. Consequently, the MEKC-APPI-MS system can provide effective separation and detection of compounds of diverse character in one run using background electrolytes containing up to 50 mM SDS. Concentration limits of detection derived from extracted-ion traces (full scan mode) of test compounds were approximately 1 microg/mL, and the detection sensitivity remained unaffected during 1 day of continuous use. Overall, the system features are very favorable for applications such as drug impurity profiling as is illustrated by the analysis of mebeverine and related compounds (both charged and neutral) at the 0.25% (w/w) level.     

A microfabricated fluidic device for performing two-dimensional liquid-phase separations

A microfabricated fluidic device that combines micellar electrokinetic chromatography and high-speed open-channel electrophoresis on a single structure for the rapid automated two-dimensional analysis of peptides has been devised and demonstrated. The microchip operates by rapidly sampling and analyzing effluent in the second dimension from the first dimension. Second-dimension analyses are performed and completed every few seconds, with total analysis times of less than 10 min for tryptic peptides. The peak capacity of the two-dimensional separations has been estimated to be in the 500-1000 range. The orthogonality of the separation techniques, an important factor for maximizing peak capacity or resolution elements, was verified by examining each technique independently for peptide separations. The two-dimensional separation strategy was found to greatly increase the resolving power over that obtained for either dimension alone.     

Electrophoresis separation in open microchannels. A method for coupling electrophoresis with MALDI-MS

The separation of biological mixtures in open micro-channels using electrophoresis with rapid and simple coupling to mass spectrometry is introduced. Rapid open-access channel electrophoresis employs microchannels that are manufactured on microchips. Separation is performed in the open channels, and the chips are transferred to a matrix-assisted laser desorption/ionization (MALDI) source after the solvent is evaporated. The matrix (2,5-dihydroxybenzoic acid) is placed in the solution with the run buffer before the separation of the analyte components. After separation, the solvent is evaporated and the microchip is ready for MALDI-MS analysis. The microchip is placed directly into a specially designed ion source of an external source Fourier transform mass spectrometry instrument. Separation of simple mixtures containing oligosaccharides and peptides is shown.     

低电压电泳芯片及其在生化样品分析中的应用

为了解决微流控电泳芯片集成化问题,设计并制作出一种具有管道两侧微阵列电极结构的硅-PDMS复合低电压电泳芯片.通过电路控制程序在微侧壁阵列电极上施加交替循环的低电压,以实现芯片微管道中低电压电泳过程;并对硅-PDMS芯片的电绝缘性、伏安曲线及电渗流等性能进行了测试和评价.以pH为10.0、10mmol/L的硼砂作为缓冲体系,分离场强150V/cm、切换时间3s的条件下,完成了10-4mol/L的苯丙氨酸和精氨酸的低电压电泳分离,分离度达1.6,实现了两种氨基酸的完全分离.在此基础上,将系统用于牛血清白蛋白和α-乳白蛋白的分离,并初步实现了该两种蛋白质的芯片电泳分离.     

Performance of SU-8 microchips as separation devices and comparison with glass microchips

Effective analytical performance of native, all-SU-8 separation microdevices is addressed by comparing their performance to commercial glass microdevices in microchip zone electrophoresis accompanied by fluorescence detection. Surface chemistry and optical properties of SU-8 microdevices are also examined. SU-8 was shown to exhibit repeatable electroosmotic properties in a wide variety of buffers, and SU-8 microchannels were successfully utilized in peptide and protein analyses without any modification of the native polymer surface. Selected, fluorescent labeled, biologically active peptides were baseline resolved with migration time repeatability of 2.3-3.6% and plate numbers of 112,900-179,800 m(-1). Addition of SDS (0.1%) or SU-8 developer (1.0%) to the separation buffer also enabled protein analysis by capillary zone electrophoresis. Plate heights of 2.4-5.9 microm were obtained for fluorescent labeled bovine serum albumin. In addition, detection sensitivity through SU-8 microchannels was similar to that through BoroFloat glass, when fluorescence illumination was provided at visible wavelengths higher than 500 nm. On the whole, the analytical performance of SU-8 microchips was very good and fairly comparable to that of commercial glass chips as well as that of traditional capillary electrophoresis and chromatographic methods. Moreover, lithography-based patterning of SU-8 enables straightforward integration of multiple functions on a single chip and favors fully microfabricated lab-on-a-chip systems.     

Microchip capillary electrophoresis with electrochemical detection

A novel microchip capillary electrophoresis system with electrochemical detection, using the replaceable microelectrode, is first reported. This kind of electrode can be fabricated in general laboratories and can be replaced quickly with electrodes of different materials according to the requirements of experiments. The end-column electrochemical detection on microchip CE was achieved by fixing the working electrode (such as carbon fiber, Pt, or Au, etc.) through a guide tube on the end of the separation channel. The experiment results indicate that the alignment of the electrode with the channel outlet can be carried out accurately and reproducibly, and therefore, the detection device has low noise and good reproducibility. The detection limit of dopamine is 2.4 x 10(-7) M, which is the lowest result reported so far. The separation and detection of dopamine, 5-hydroxytryptamine and epinephrine using carbon fiber and Pt microdisk electrodes within 50 s was successfully performed.     

I-shaped microchannel array chip for parallel electrophoretic analyses

We demonstrate electrophoresis in I-shaped microchannels with a new design and operation principle. Unlike the conventional T- or cross-shaped microchannels, the simple I-shaped design makes it straightforward to integrate parallel microchannels with electrodes onto a microchip. The operation of the I-shaped microchannels has been enabled by the autonomous solution filling technique, which exploits the high gas solubility in poly(dimethylsiloxane) (PDMS). We fabricated an I-shaped microchannel array (IMA) chip by integrating 12 independent microchannels and 2 electrodes onto a 3 cm x 2 cm area in a PDMS-glass hybrid microchip. For autonomous regulation of stable sample plugs in all the microchannels, we discovered that O2 plasma treatment of the PDMS-made reservoirs is effective. On the IMA chip, size-dependent separation of double-stranded (ds) DNA and sequence-specific separation of single-stranded DNA were achieved. Specifically, 10 fragments in a 100-1000-bp dsDNA ladder were separated using hydroxyethylcellulose as a sieving matrix within a separation length of 2 mm, and polymerase chain reaction products of the wild-type K-ras gene and its point mutant were separated using a probe DNA-poly(dimethylacrylamide) conjugate on the basis of affinity capillary electrophoresis. The IMA chip presented here opens up a new possibility of large-scale integration of microchannels for high-throughput electrophoretic analyses.     

A fluorescence detection scheme for capillary electrophoresis of N-methylcarbamates with on-column thermal decomposition and derivatization

This paper describes a fluorescence detection method for N-methylcarbamate (NMC) pesticides in micellar electrokinetic chromatography (MEKC) separation. Fulfillment of the fluorescence detection hinged on the discovery that quaternary ammonium surfactants (particularly cetyltrimethylammonium bromide, CTAB), besides serving as hydrophobic pseudophases in MEKC, are also capable of catalyzing the thermal decomposition of NMCs to liberate methylamine. Thus, a multifunctional MEKC medium consisting of borate buffer, CTAB, and derivatizing components (o-phthaldialdehyde/2-mercaptoethanol) was formulated, which allowed first normal MEKC separation, subsequent thermal decomposition, and finally in situ derivatization of NMCs. With careful optimization of the operation conditions, fluorescence detection of 10 NMC compounds was achieved, with column efficiencies typically higher than 50,000 and detection limits better than 0.5 ppm. The present work represents an unprecedented effort in capillary electrophoresis (CE), in which an intact capillary was consecutively utilized as chambers for separation, decomposition, derivatization, and detection, without involving any interfacing features. The success in the implementation of such a detection system resulted in strikingly simple instrumentation as compared with the traditional postcolumn fluorescence determination of NMCs by reversed-phase HPLC. Similar protocols should be workable in the determination of a wide range of pesticides and pharmaceuticals in CE formats.     

In-channel electrochemical detection for microchip capillary electrophoresis using an electrically isolated potentiostat

A new electrode configuration for microchip capillary electrophoresis (CE) with electrochemical (EC) detection is described. This approach makes it possible to place the working electrode directly in the separation channel. The "in-channel" EC detection was accomplished without the use of a decoupler through the utilization of a specially designed, electrically isolated potentiostat. The effect of the working electrode position on the separation performance (in terms of plate height and peak skew) of poly(dimethylsiloxane)-based microchip CEEC devices was evaluated by comparing the more commonly used end-channel configuration with this new in-channel approach. Using catechol as the test analyte, it was found that in-channel EC detection decreased the total plate height by a factor of 4.6 and lowered the peak skew by a factor of 1.3. A similar trend was observed for the small, inorganic ion nitrite. Furthermore, a fluorescent and electrochemically active amino acid derivative was used to directly compare the separation performance of in-channel EC detection to that of a widely used laser-induced fluorescence (LIF) detection scheme. In this case, it was found that the plate height and peak skew for both detection schemes were essentially equal, and the separation performance of in-channel EC detection is comparable to LIF detection.