Theoretical Migration Model for Micellar Capillary Electrophoresis and Its Application to the Separation of Anionic Metal Complexes of HEDTC and CDTA

A mathematical model relating the effective mobility of an analyte in micellar capillary electrophoresis (MCE) to the concentration of surfactant and organic modifier in the background electrolyte (BGE) was derived. Effective mobility is expressed in terms of the electrophoretic mobility of the analyte, the partition coefficient of the analyte into the micelle, and the influence of organic modifier on these two factors. The performance of the model was evaluated using Cd(II), Pb(II), Co(II), Ni(II), Bi(III), Cu(II), and Hg(II) complexes of bis(2-hydroxyethyl)dithiocarbamate, all of which carry a partial negative charge, and Cd(II), Pb(II), Co(II), Ni(II), Bi(III), Cu(II), Hg(II), Fe(III), Ag(I), Tl(I), and Mn(II) complexes of trans-1,2-diaminocyclohexane-N,N,N',N'-tetraacetic acid, all of which are anionic having charges in the range -1 to -3. These analytes were separated in borate BGEs containing 10-50 mM sodium dodecyl sulfate and 0-20% (v/v) methanol. Nonlinear regression was used to derive parameters for the model from experimental data and these parameters were used to predict effective mobilities of the analytes. Predicted values of effective mobilties agreed with experimental values to within 3.1%. Values of parameters from the model equation are used to explain changes in separation selectivity observed at different BGE compositions and the model equation is shown to be applicable to computer-assisted optimization of the BGE composition, in MCE using a limited number of experiments.     

Predicting peak shape in capillary zone electrophoresis: a generic approach to parametrizing peaks using the Haarhoff-Van der Linde (HVL) function.

We have found that the Haarhoff-Van der Linde (HVL) peak function provides excellent fitting to the shapes of CZE peaks. Initially designed for overloaded peaks in gas chromatography, this function describes a Gaussian peak when there is no peak distortion, and a triangular peak when there is no diffusional peak broadening. As such, it is ideal for CZE peaks distorted by electromigration dispersion (EMD). Fitting peaks with this function gives four parameters: three of them can be related to the Gaussian peak that would have been obtained in case of no EMD; the last one is a measure of the peak distortion. Using moving boundary theory, this peak distortion parameter may readily be expressed in terms of analyte and background electrolyte mobilities and concentrations, electric field, and sample injection length. The variance of an HVL peak is shown to be described by a universal function, and a master equation is presented. The region where EMD adds less than 10% to the Gaussian variance is shown to be very narrowly spread around the mobility matching condition. Under typical CZE operating conditions with an analyte at 1% of the BGE concentration, significant peak distortion is always present. Because the total peak variance is not an addition of the Gaussian and triangular contributions, the HVL model and the methodology introduced here should always be used to correctly combine variances.     

Determination of shapes and maximums of analyte peaks based on solute mobilities in capillary electrophoresis

In capillary electrophoresis, the relative orders of mobilities of analyte, additive, and the complex formed determine the analyte peak shape in a way similar to the way the binding isotherms determine the peak shapes in chromatography. The three mobilities allow six possible orders; each produces a characteristic peak shape in CE. Equations describing the analyte migration in a CE system with the presence of mobility-changing additives can be implemented into computer programs to predict the migration times of the analyte peak maximums, and the predicted migration times agree well with the experimental results.     

Imprinted polymer-based sensor system for herbicides using differential-pulse voltammetry on screen-printed electrodes.

A sensor system for the herbicide 2,4-dichlorophenoxy-acetic acid has been developed based on specific recognition of the analyte by a molecularly imprinted polymer and electrochemical detection using disposable screen-printed electrodes. The method involves a competitive binding step with a nonrelated electrochemically active probe. For batch binding assays, imprinted polymer particles are incubated in suspension with the analyte and the probe, followed by centrifugation and quantification of the unbound probe in the supernatant. Two different compounds, namely 2,4-dichlorophenol and homogentisic acid, were tested as potential electroactive probes. Both compounds could be conveniently detected by differential-pulse voltammetry on screen-printed, solvent-resistant three-electrode systems having carbon working electrodes. Whereas 2,4-dichlorophenol showed very high nonspecific binding to the polymer, homogentisic acid bound specifically to the imprinted sites and thus allowed calibration curves for the analyte in the micromolar range to be recorded. An integrated sensor was developed by coating the imprinted polymer particles directly onto the working electrode. Following incubation of the modified electrode in a solution containing the analyte and the probe, the bound fraction of the probe is quantified. This system provides a cheap, disposable sensor for rapid determination of environmentally relevant and other analytes.     

Micelle stacking in micellar electrokinetic chromatography

In order to understand the role of stacked micelles in sample preconcentration, it is necessary to understand the factors that contribute to the micelle stacking phenomenon. Various MEKC background electrolyte (BGE) solutions were prepared in the presence of Sudan III in order to monitor the micelle stacking phenomenon in the anionic sodium dodecyl sulfate and sodium cholate micelle systems. The data show that micelle stacking is a dynamic process that is strongly dependent upon the relative conductivities of the sample matrix and BGE, the relative column length of the sample plug, and the mobilities of the ions involved in the stacking process regardless of electric field conditions (i.e., field-amplified stacking, sweeping, or high-salt stacking). Conditions under which micelle stacking can be expected to occur are presented, and the extent of micelle stacking is quantified. The micelle stacking phenomenon is correlated to the separation performance of a series of neutral alkaloids. It is shown that neutral analytes migrate rapidly through the evolving stacked micelle region in the initial moments of the separation. As a consequence of this transient interaction, analytes with small retention factors spend less time in the stacked micelle region and experience lower stacked micelle concentrations than analytes with large retention factors that spend more time in the growing stacked micelle region. It is also demonstrated that the extent of analyte enrichment generally increases with injection length, by facilitating greater interaction time with stacked micelles; however, enrichment will eventually plateau with increasing injection length as a function of an analyte's affinity for the micelle. Finally, it is shown that, in contrast to conventional wisdom, a range of long injection plugs exist where separation efficiency can be dramatically improved due to analyte interaction with an actively growing stacked micelle region.     

Preconcentration, separation, and indirect detection of nonfluorescent analytes using fluorescent mobility markers

We present a method to achieve separation and indirect detection of nonfluorescent species using fluorescent mobility markers. This technique leverages isotachophoresis (ITP) for both preconcentration and separation. We employ a leading electrolyte (LE), trailing electrolyte (TE), and a set of fluorescent markers of mobilities designed to bound those of nonfluorescent analytes of interest. Fluorescent markers and nonfluorescent analytes are initially mixed homogenously and ITP is initiated. The dynamics of isotachophoresis cause the analyte and fluorescent marker mixture to segregate into respective zones between the LE and TE in the order of reducing mobility. Unlabeled analytes are detected as gaps (regions with local minimums in intensity) in the fluorescent signals of mobility markers. We have successfully demonstrated preconcentration, separation, and detection of unlabeled amino acids serine, glycine, and phenylalanine; and of acetic acid, aspartic acid, and 3-phenylpropionic acid. We show detection of 12 microM concentration of analytes with signal-to-noise ratio of 4.0 and with a high degree of repeatability. We discuss methods for encoding mobility marker identity using marker fluorescence intensity level and alternating fluorescence emission wavelengths. We present example experimental results of fluorescence intensity level encoding.     

Electrokinetic injection for stacking neutral analytes in capillary and microchip electrophoresis

An on-column mechanism for electrokinetically injecting long sample plugs with simultaneous stacking of neutral analytes in capillary electrokinetic chromatography is presented. On-column stacking methods allow for the direct injection of long sample plugs into the capillary, with narrowing of the analyte peak width to allow for an increase in the detected signal. Low-pressure injections (approximately 50 mbar) are commonly used to introduce sample plugs containing neutral analytes. We demonstrate that injection can be accomplished by applying an electric field from the sample vial directly into the capillary, with neutral analytes injected by electroosmotic flow at up to 1 order of magnitude faster than the corresponding pressure injections. Since stacking occurs simultaneously with electrokinetic injection, stacking is initiated at the capillary inlet, resulting in an increased length of capillary remaining for separation. Reproducibility obtained for peak height and peak area with electroosmotic flow injection is comparable to that obtained with the pressure injection mode, while reproducibility of analysis time is markedly improved. Electrokinetic stacking of neutral analytes utilizing electroosmotic flow is demonstrated with discontinuous (high conductivity, high mobility) as well as continuous (equal conductivity, equal mobility) sample electrolytes. Injecting neutral analytes by electroosmotic flow affords a 10-fold or greater decrease in analysis times when capillaries of 50-microm i.d. or smaller are used. This stacking method should be exportable to dynamic pH junction stacking and electrokinetic chromatography with capillary arrays. Equations describing this electrokinetic injection mode are introduced and stacking of a neutral analyte on a microchip by electrokinetic injection using a simple cross-T channel configuration is demonstrated.     

Peak shape distortions in the capillary electrophoretic separations of strong electrolytes when the background electrolyte contains two strong electrolyte co-ions

A series of 25 mM phosphate buffer background electrolytes were prepared from phosphoric acid and mixtures of lithium hydroxide and tetrabutylammonium hydroxide as pH adjusters and sources of background electrolyte co-ions. These background electrolytes were used for the capillary electrophoretic separation of quaternary ammonium analytes. Abnormally distorted peaks, different from the simple characteristic triangular peaks usually attributed to electromigration dispersion, were observed. In order to understand the origin of the greatly distorted peaks, capillary electrophoretic separations with two co-ion background electrolytes were numerically simulated using a mathematical model of the electrophoretic process. Generalized peak shape rules were derived from the simulations which can be used to predict the shape of the analyte, co-ions, and counterion concentration peaks, as well as the local electric field strength changes. Abnormal peak shape and peak disappearance can occur when the analyte peak and the noncomigrating system peaks overlap.     

A family of single-isomer, sulfated gamma-cyclodextrin chiral resolving agents for capillary electrophoresis. 1. Octakis(2,3-diacetyl-6-sulfato)-gamma-cyclodextrin

The first member of the single-isomer, sulfated gamma-cyclodextrin family, the sodium salt of octakis(2,3-diacetyl-6-sulfato)-gamma-cyclodextrin (ODAS-gamma CD) has been synthesized, analytically characterized, and used to separate, by capillary electrophoresis, a variety of neutral, acidic, basic, and amphoteric enantiomers in low pH background electrolytes. The anionic effective mobilities of the neutral and anionic analytes were found to increase with the concentration of ODAS-gamma CD. For weakly binding cationic analytes, the effective mobilities went from cationic high values, through zero, to increasingly larger anionic values as the concentration of ODAS-gamma CD was increased. For the strongly complexing cationic analytes, the effective mobilities became anionic even at very low ODAS-gamma CD concentrations and became smaller as the ionic strength of the background electrolyte increased with the increasing ODAS-gamma CD concentration. Separation selectivity followed the predictions of the charged resolving agent migration model: for neutral analytes it decreased as the concentration of ODAS-gamma CD was increased. For cationic analytes, selectivities were found to increase as the cationic effective mobilities approached zero, then decreased as the concentration of ODAS-gamma CD was increased further. The extent of peak resolution that could be realized with ODAS-gamma CD strongly depended on the magnitude of separation selectivity and the normalized electroosmotic flow mobility. ODAS-gamma CD proved to be a broadly applicable chiral resolving agent.     

Mechanistic study on analyte focusing by dynamic pH junction in capillary electrophoresis using computer simulation

Dynamic pH junction is an on-line preconcentration method in capillary electrophoresis (CE) based on electrokinetic focusing of weakly ionic analytes with in large sample volumes in a multisection electrolyte system. In this report, experiments and computer simulations were performed to gain a better insight of the analyte focusing mechanism when a dynamic pH junction was used. A computer program, SIMUL, was used to simulate the band-narrowing process of a group for phenol derivatives under optimized buffer conditions, which were compared with experimental results. Computer simulations revealed the formation of a sharp moving pH boundary within the sample zone causing efficient focusing of long plugs of weakly acidic analytes based on their pKa. These studies offered useful information for understanding the band-narrowing process by control of the depth and lifetime of the moving pH boundary as a function of analyte pKa, sample pH, and injection length. The change in pH of the sample within the capillary was also estimated by measuring the absorbances of an analyte at two different wave-lengths. Optimization of analyte focusing resulted in enhanced detection responses of about 60-450-fold in terms of peak heights for some phenol derivatives' relation to conventional injections. Dynamic pH junction represents a novel approach to control band dispersion (peak width) and selectivity (mobility) of specific analytes for high-resolution CE separations.     

Combination of analyte protectants to overcome matrix effects in routine GC analysis of pesticide residues in food matrixes

Analyte protectants were previously defined as compounds that strongly interact with active sites in the gas chromatographic (GC) system, thus decreasing degradation, adsorption, or both of coinjected analytes. In this study, we evaluated various combinations of promising analyte protectants for the volatility range of GC-amenable pesticides using GC/quadrupole mass spectrometry (MS) and 1-microL hot splitless injection for sample introduction. A mixture of ethylglycerol, gulonolactone, and sorbitol (at 10, 1, and 1 mg/mL, respectively, in the injected samples) was found to be the most effective in minimizing losses of susceptible analytes and significantly improving their peak shapes (due to reduction of peak tailing). When added to final sample extracts and matrix-free calibration standards alike, these analyte protectants induced a similar response enhancement in both instances, resulting in effective equalization of the matrix-induced response enhancement effect even after a large number of fruit and vegetable extract injections. As compared to matrix-matched standardization, the analyte protectant approach offers a more convenient solution to the problems associated with calibration in routine GC/MS analysis of pesticide residues and possibly other susceptible analyte types in diverse samples. Moreover, the use of analyte protectants also substantially reduced another adverse matrix-related effect caused by gradual build-up of nonvolatile matrix components in the GC system, thus improving ruggedness and, consequently, reducing need for frequent maintenance.     

Capillary electrophoresis with lamp-based wavelength-resolved fluorescence detection for the probing of protein conformational changes

Native protein fluorescence spectra encompass information on protein conformation. In this study, capillary electrophoresis (CE) combined with lamp-based wavelength-resolved fluorescence detection (wrFlu) is presented as a novel tool for the analysis of protein mixtures and the monitoring of protein unfolding. The CE-wrFlu system provides three-dimensional data (time, emission wavelength, intensity) from which electropherograms and accurate emission spectra of separated proteins can be extracted. For model proteins, linear detector responses (peak height vs concentration) were obtained (R(2) > 0.96) with detection limits (LODs) in the 6-32 nM range. The minimum protein concentration required for precise determination of the maximum emission wavelength by CE-wrFlu was about 15 times the LOD. Unfolding of various model proteins was induced by protein incubation and analysis in background electrolyte (BGE) containing 7.0 M urea. CE-wrFlu of the unfolded species revealed peaks with clear red-shifted spectra, which adequately corresponded to reference spectra obtained on a standard spectrophotometer. Moreover, unfolded proteins showed a significant decrease in effective electrophoretic mobility (after correction for BGE viscosity) due to the increase of their molecular hydrodynamic radii. It is concluded that the CE-wrFlu system provides two independent indicators for changes in protein folding and will allow the simultaneous assessment of protein purity and conformation.     

Dielectric friction in capillary electrophoresis: mobility of organic anions in mixed methanol-water media

The mobilities of a series of organic carboxylates and sulfonates, ranging in charge from -1 to -4, were investigated by capillary electrophoresis using buffers containing 0 to 75% (v/v) methanol. Effective mobilities were measured at a series of ionic strengths, and were extrapolated to zero ionic strength using Pitts' equation to yield absolute mobilities. Generally, higher-charged ions were more strongly influenced by ionic strength, as predicted by the Pitts' equation. Some differences in the ionic strength effects for anions of like charge were observed and were consistent with the relaxation effect. The absolute mobilities of anions were altered by the addition of methanol to the buffer. Analytes with higher charge-to-size ratios were slowed to a greater extent than were ions with lower charge-to-size. As a result, dramatic changes in relative mobility were observed, such as a reversal in migration order between anions of -1 and -4 charge at 75% methanol and 20 mM ionic strength. The mobility changes caused by the addition of methanol are attributed to dielectric friction. Mobilities in the methanol-water solutions were found to depend on analyte charge-to-size and solvent dielectric relaxation time (tau) and were inversely dependent upon solvent dielectric constant (epsilon), as predicted by the Hubbard-Onsager mobility model.     

Fluorescence correlation spectroscopy for rapid multicomponent analysis in a capillary electrophoresis system

We describe a new technique for performing multicomponent analysis using a combination of capillary electrophoresis (CE) and fluorescence correlation spectroscopy (FCS), which we refer to as CE/FCS. FCS is a highly sensitive and rapid optical technique that is often used to perform multicomponent analysis in static solutions based on the different diffusion times of the analyte species through the detection region of a tightly focused laser beam. In CE/FCS, transit times are measured for a mixture of analytes continuously flowing through a microcapillary in the presence of an electric field. Application of an electric field between the inlet and outlet of the capillary alters the transit times, depending on the magnitude and polarity of the applied field and the electrophoretic mobilities of the analytes. Multicomponent analysis is accomplished without the need to perform a chemical separation, due to the different electrophoretic mobilities of the analytes. This technique is particularly applicable to ultradilute solutions of analyte. We have used CE/FCS to analyze subnanomolar aqueous solutions containing mixtures of Rhodamine 6G (R6G) and R6G-labeled deoxycytosine triphosphate nucleotides. Under these conditions, fewer than two molecules were typically present in the detection region at a time. The relative concentrations of the analytes were determined with uncertainties of ～10%. Like diffusional FCS, this technique is highly sensitive and rapid. Concentration detection limits are below 10(-)(11) M, and analysis times are tens of seconds or less. However, CE/FCS does not require the diffusion coefficients of the analytes to be significantly different and can, therefore, be applied to multicomponent analysis of systems that would be difficult or impossible to study by diffusional FCS.     

Electrokinetic stacking injection of neutral analytes under continuous conductivity conditions

In capillary electrokinetic chromatography, neutral analytes can be injected by electroosmotic flow directly from a sample matrix into a separation buffer containing an electrokinetic vector with an opposite mobility. Analytes are injected at the velocity of electroosmotic flow but are retained at the interface of the sample matrix co-ion and separation buffer micelle zones as analyte/micelle complexes. A simple electrokinetic chromatography system containing sodium dodecyl sulfate as the micellar agent with borate as the buffering electrolyte included in the separation buffer and in the sample matrix to provide continuous conductivity was investigated. Concentrations of the micelle, methanol, and borate in the separation buffer were explored to increase maximum injection length of neutral analytes. Reducing the analyte velocity in the separation buffer without substantially decreasing the velocity of the analyte during injection from the sample vial allowed greatly extended sample plug injection lengths. It is presently possible to inject sample solvent volumes equivalent to approximately 7 effective capillary lengths (180 cm) with a 50-microm-i.d. capillary (24.5 cm effective capillary length), total volume of sample injection approximately 3.5 microL Equations describing the injection process and maximum injection lengths for this mode of stacking in electrokinetic capillary chromatography are introduced. The result of this work leads to a postulated generalization of electrokinetic stacking injection maximums for electrophoretic processes, and the concept of orthogonal analyte stacking/injection systems is discussed.     

Design and characterization of a high-power laser-induced acoustic desorption probe coupled with a fourier transform ion cyclotron resonance mass spectrometer

We report here the construction and characterization of a high-power laser-induced acoustic desorption (LIAD) probe designed for Fourier transform ion cyclotron resonance mass spectrometers to facilitate analysis of nonvolatile, thermally labile compounds. This "next generation" LIAD probe offers significant improvements in sensitivity and desorption efficiency for analytes with larger molecular weights via the use of higher laser irradiances. Unlike the previous probes which utilized a power-limiting optical fiber to transmit the laser pulses through the probe, this probe employs a set of mirrors and a focusing lens. At the end of the probe, the energy from the laser pulses propagates through a thin metal foil as an acoustic wave, resulting in desorption of neutral molecules from the opposite side of the foil. Following desorption, the molecules can be ionized by electron impact or chemical ionization. Almost an order of magnitude greater power density (up to 5.0x10(9) W/cm2) is achievable on the backside of the foil with the high-power LIAD probe compared to the earlier LIAD probes (maximum power density approximately 9.0x10(8) W/cm2). The use of higher laser irradiances is demonstrated not to cause fragmentation of the analyte. The use of higher laser irradiances increases sensitivity since it results in the evaporation of a greater number of molecules per laser pulse. Measurement of the average velocities of LIAD-evaporated molecules demonstrates that higher laser irradiances do not correlate with higher velocities of the gaseous analyte molecules.     

Assessing the peak capacity of IMS-IMS separations of tryptic peptide ions in He at 300 K

Two-dimensional ion mobility spectrometry (IMS-IMS) coupled with mass spectrometry is examined as a means of separating mixtures of tryptic peptides (from myoglobin and hemoglobin). In this study, we utilize two distinct drift regions that are identical in that each contains He buffer gas at 300 K. The two-dimensional advantage is realized by changing the structures of the ions. As ions arrive at the end of the first drift region, those of a specified mobility are selected, exposed to energizing collisions, and then introduced into a second drift region. Upon collisional activation, some ions undergo structural transitions, leading to substantial changes in their mobilities; others undergo only slight (or no) mobility changes. Examination of peak positions and shapes for peptides that are separated in the first IMS dimension indicates experimental peak capacities ranging from approximately 60 to 80; the peak shapes and range of changes in mobility that are observed in the second drift region (after activation) indicate a capacity enhancement ranging from a factor of approximately 7 to 17. Thus, experimental (and theoretical) evaluation of the peak capacity of IMS-IMS operated in this fashion indicates that capacities of approximately 480 to 1360 are accessible for peptides. Molecular modeling techniques are used to simulate the range of structural changes that would be expected for tryptic peptide ions and are consistent with the experimental shifts that are observed.     

Analytical reliability of mobile-phase recycling in liquid chromatography

Although it is not officially condoned, mobile-phase (MP) recycling has become a widespread practice that is not well documented in terms of its effects on sample quantification. MP was spiked with three different concentrations of two analytes, tartaric acid and sodium citrate, to simulate MP recycling. These MPs were used to analyze eight different concentrations of these analytes in standard solutions. When analyte concentration in the MP exceeds that in the sample, a vacancy (negative) peak is observed, and when its concentration in the MP is equal to that in the sample, no peak is observed for that analyte. The slopes of the linear regression lines for standards in MPs with different concentrations of analyte did not change, although the gamma-intercept values decrease with increasing concentration of analyte in the MP. These results show that the concentration of analytes in recycled MP can be determined by comparing the absolute value of the gamma-intercept of the linear regression line with the corresponding peak area from the linear regression line for clean (solute-free) MP, provided other chromatographic parameters do not change. Suggestions are made for determining when recycled MP should be discarded.     

Capillary zone electrophoresis in nonaqueous solvents in the presence of ionic additives

Capillary zone electrophoresis (CZE) in nonaqueous media and in the presence of ionic additives has been successfully applied to the determination of compounds that differ only slightly in their electrophoretic mobilities. Triazine herbicides of environmental interest were chosen as test compounds because they behave as very weak bases. CZE separation of these analytes (especially chlorotriazines) in aqueous solution is difficult due to the low pH required for their conversion into protonated cationic form (HA(+)). However, in mixed nonaqueous solvents, 50% (v/v) acetonitrile-methanol, the acid-base characteristics of these compounds are modified, yielding the protonated ionic species that is susceptible to migration when subjected to an electric field. A noteworthy increase in separation selectivity and resolution can be achieved by using ionic additives. Thus, in this mode of capillary zone electrophoresis, separation is based on ionic interactions between the charged analytes and the ionic additive present in the separation medium. These interactions contribute to enhancing mobility differences and to improving analyte separation. For the separation of chloro- and methylthiotriazines, 10 mM perchloric acid in 50% (v/v) acetonitrile-methanol and 20 mM SDS proved to be satisfactory, providing high resolution in short analysis times. The selectivity achieved was found to depend on the degree of association of the analyte with the ionic additive in the nonaqueous medium. This permits manipulation of the selectivity of the electrophoretic separations as a function of the type and concentration of the ionic additive and of the nature of the nonaqueous medium employed.     

Coupling photochemical reaction detection based on singlet oxygen sensitization to capillary electrochromatography

Despite the impressive separation efficiency afforded by capillary electrochromatography (CEC), the detection of UV-absorbing compounds following separation in capillary dimensions remains limited by the short path length (5-75 microm) through the column. Moreover, analytes that are poor chromophores present an additional challenge with respect to sensitive detection in CEC. This paper illustrates a new photochemical reaction detection scheme for CEC that takes advantage of the catalytic nature of type II photooxidation reactions. The sensitive detection scheme is selective toward molecules capable of photosensitizing the formation of singlet molecular oxygen (1O2). Following separation by CEC, UV-absorbing analytes promote groundstate 3O2 to an excited state (1O2) which reacts rapidly with tert-butyl-3,4,5-trimethylpyrrolecarboxylate, which is added to the running buffer. Detection is based on the loss of pyrrole. The reaction is catalytic in nature since one analyte molecule may absorb light many times, producing large amounts of 1O2. The detection limit for 9-acetylanthracene, following separation by CEC, is approximately 6 x 10(-9) M (S/N = 3). Optimization of the factors effecting the S/N for four model compounds is discussed.