NMR detection with multiple solenoidal microcoils for continuous-flow capillary electrophoresis

Nuclear magnetic resonance (NMR) spectroscopy represents a promising on-line detector for capillary electrophoresis (CE). The inherent poor sensitivity of NMR mandates the use of NMR probes with the highest mass sensitivity, such as those containing solenoidal microcoils, for CE/NMR hyphenation. However, electrophoretic current degrades the resolution of NMR spectra obtained from solenoidal coils. A new method to avoid microcoil NMR spectral degradation during continuous-flow CE is demonstrated using a unique multiple solenoidal coil NMR probe. The electrophoretic flow from a single separation capillary is split into multiple outlets, each possessing its own NMR detection coil. While the CE electrophoretic flow is directed through one outlet, stopped-flow, high-resolution NMR spectra are obtained from the coil at the other outlet. The electrophoretic flow and NMR measurements are cycled between the outlets to allow a continuous CE separation with "stopped-flow" detection. As a new approach for improving multiple coil probe performance, the magnetic field homogeneity is automatically adjusted (via the shim coils of the magnet) for the active coil. The multiple microcoil CE/NMR coupling has been used to analyze a <3 nmole mixture of amines while obtaining between 1 and 2 Hz line width, demonstrating the ability to avoid electrophoretic current-induced line broadening.     

Capillary isotachophoresis/NMR: extension to trace impurity analysis and improved instrumental coupling

Building upon its promising initial performance, the online coupling of capillary isotachophoresis (cITP) to nuclear magnetic resonance (NMR) is extended to trace impurity analysis. By simultaneously concentrating and separating dilute charged species on the basis of their electrophoretic mobility, cITP greatly facilitates NMR structural elucidation. cITP/NMR appears particularly attractive for identifying trace charged synthetic and natural organic compounds obscured by large excesses of other components. A 9.4 microL injection of 200 microM (1.9 nmol) atenolol in a 1000-fold excess of sucrose (200 mM) is analyzed by cITP/NMR. A microcoil, the most mass sensitive NMR probe, serves as the detector as it provides optimal NMR observation of the capillary-scale separation. cITP successfully isolates the atenolol from the sucrose while concentrating it 200-fold to 40 mM before presentation to the 30 nL observe volume microcoil, thereby enabling rapid 1H NMR spectral acquisition of atenolol (experimental time of 10 s) without obstruction from sucrose. For this particular probe and sample, the stacking efficiency is near the theoretical limit as 67% of the sample occupies the 1 mm long microcoil during peak maximum. A multiple-coil probe with two serial 1 mm long microcoils arranged 1 cm apart has been developed to facilitate peak trapping and sample band positioning during cITP/NMR.     

Monitoring temperature changes in capillary electrophoresis with nanoliter-volume NMR thermometry

Nanoliter-volume proton nuclear magnetic resonance (NMR) spectroscopy is used to monitor the electrolyte temperature during capillary electrophoresis (CE). By measuring the shift in the proton resonance frequency of the water signal, the intracapillary temperature can be recorded noninvasively with subsecond temporal resolution and spatial resolution on the order of 1 mm. Thermal changes of more than 65 degrees C are observed under both equilibrium and nonequilibrium conditions for typical CE separation conditions. Several capillary and buffer combinations are examined with external cooling by both liquid and air convection. Additionally, NMR thermometry allows nonequilibrium temperatures in analyte bands to be monitored during a separation. As one example, a plug of 1 mM NaCl is injected into a capillary filled with 50 mM borate buffer. Upon reaching the NMR detector, the temperature in the NaCl band is more than 20 degrees C higher than the temperature in the surrounding buffer. Such observations have direct applicability to a variety of studies, including experiments which utilize sample stacking and isotachophoresis.     

Analysis of underivatized amino acids and their D/L-enantiomers by sheathless capillary electrophoresis/electrospray ionization mass spectrometry

Capillary electrophoresis/electrospray ionization-mass spectrometry (CE/ESI-MS) was applied to the analysis of underivatized amino acids and the separation of their D/L-enantiomers. Under full-scan mode, all standard protein amino acids were separated and detected at low-femtomole levels using a 130-cm-long, 20-microm-i.d., 150-microm-o.d. underivatized fused-silica capillary with 1 M formic acid as the background electrolyte. The CE/ESI-MS technique was also applied to the separation of L-arginine from L-canavanine (a close analogue of arginine where the terminal methylene linked to the guanidine group of arginine is replaced by an oxygen atom) in a complex mixture containing all standard protein amino acids. The utility of CE/ESI-MS in the analysis of real-world samples was demonstrated by the identification of two metabolic diseases (PKU and tyrosinemia) through blood analysis with minimal sample preparation. In addition, the on-line separation of 11 underivatized L-amino acids from their D-enantiomers was achieved by using a 30 mM solution of (+)-(18-crown-6)-2,3,11,12-tetracarboxylic acid as the background electrolyte.     

Dual microcoil NMR probe coupled to cyclic ce for continuous separation and analyte isolation

Capillary electrophoresis (CE)-nuclear magnetic resonance (NMR) spectroscopy combines the separation efficiency of CE and the information-rich detection capabilities of NMR. However, the temporally narrow CE peaks reduce NMR sensitivity and prevent on-line multidimensional NMR acquisitions. In this work, cyclic CE with multicoil NMR instrumentation is developed to perform CE in multiple closed loops. As a proof of concept, a two-loop five-junction capillary configuration creates two connected yet independently operable fluidic loops. With appropriate voltage switching, analytes can be directed as desired around or between the loops, and a particular analyte band can be parked in one NMR detector coil while CE continues in the second loop and monitored with a second NMR detector coil. The separation of a mixture of amino acids (Ala, Val, Thr) is achieved in two cycles. After one CE cycle, Ala is separated and COSY data are recorded in one loop while Val and Thr are separated in the second loop. At the end of the second cycle, both Val and Thr are separated and multidimensional NMR spectra acquired. With this instrumentation and appropriate protocols, two-dimensional NMR data acquisition and CE separation are achieved simultaneously.     

Portable microcoil NMR detection coupled to capillary electrophoresis

High-efficiency separation techniques, such as capillary electrophoresis (CE), coupled to a nondestructive nuclear magnetic resonance (NMR) spectrometer offer the ability to separate, chemically identify, and provide structural information on analytes in small sample volumes. Previous CE-NMR coupled systems utilized laboratory-scale NMR magnets and spectrometers, which require very long separation capillaries. New technological developments in electronics have reduced the size of the NMR system, and small 1-2 T permanent magnets provide the possibilities of a truly portable NMR. The microcoils used in portable and laboratory-scale NMR may offer the advantage of improved mass sensitivity because the limit of detection (LOD) is proportional to the coil diameter. In this work, CE is coupled with a portable, briefcase-sized NMR system that incorporates a microcoil probe and a 1.8 T permanent magnet to measure (19)F NMR spectra. Separations of fluorinated molecules are demonstrated with stopped- and continuous-flow NMR detection. The results demonstrate that coupling CE to a portable NMR instrument is feasible and can provide a low-cost method to obtain structural information on microliter samples. An LOD of 31.8 nmol for perfluorotributylamine with a resolution of 4 ppm has been achieved with this system.     

On-line coupling of capillary electrochromatography, capillary electrophoresis, and capillary HPLC with nuclear magnetic resonance spectroscopy

A novel capillary NMR coupling configuration, which offers the possibility of combining capillary zone electrophoresis (CZE), capillary HPLC (CHPLC), and for the first time capillary electrochromatography (CEC) with nuclear magnetic resonance (NMR), has been developed. The hyphenated technique has a great potential for the analysis of chemical, pharmaceutical, biological, and environmental samples. The versatile system allows facile changes between these three different separation methods. A special NMR capillary containing an enlarged detection cell suitable for on-line NMR detection and measurements under high voltage has been designed. The acquisition of 1D and 2D NMR spectra in stopped-flow experiments is also possible. CHPLC NMR has been performed with samples of hop bitter acids. The identification and structure elucidation of humulones and isohumulones by on-line and stopped-flow spectra has been demonstrated. The suitability of the configuration for electrophoretic methods has been investigated by the application of CZE and CEC NMR to model systems.     

Condensation nucleation light scattering detection for capillary electrophoresis

We describe two means for interfacing condensation nucleation light scattering detection to capillary electrophoresis (CE). With the first method, a fused-silica capillary was used for the separation and the CE was grounded through a Nafion membrane that also connected the system to a microconcentric pneumatic nebulizer. Limits of detection (LODs) for underivatized amino acids were at the low microgram per milliliter level, and separation efficiencies were ～9 times lower than the optimum predicted for these species based on the injection plug width and axial dispersion by diffusion. LODs were limited by background nonvolatiles resulting from dissolution of fused silica at the high pHs used for the separations. An alternate system employed PEEK capillaries which acted as the separation capillary and also as the inner nebulizer capillary. In this case, the exit end of the capillary was coated with conductive paint which extended to the tip of the nebulizer, was in contact with the CE buffer, and was grounded to complete the CE circuit. Response was nonlinear and the separation efficiency of this system was somewhat lower than that for the Nafion membrane system. Response as peak heights for all of the amino acids and peptides studied was nearly identical on a mass basis. With this system, much lower background signals were obtained, and as a result, LODs for underivatized amino acids and peptides were below the 1 μg/mL level, corresponding to less than 10 pg or less than 100 fmol injected. Both systems were fairly simple, effective means to generate aerosols with the low flows of CE and should be applicable to interfacing of other aerosol-based detectors with CE.     

Double-chained surfactants for semipermanent wall coatings in capillary electrophoresis

The double-chained cationic surfactant didodecyldimethylammonium bromide (DDAB) was found to form more stable coatings onto the walls of CE capillaries than similar single-chained surfactants such as cetyltrimethylammonium bromide (C16TAB). After removing DDAB from the buffer, the reversed EOF decreased only 3% over 75 min under continuous electrophoretic conditions. Also, the reversed EOF is 60% greater for DDAB than for C16TAB at pH 2. This greater coating stability is associated with a different aggregate structure for the surfactant at the capillary surface. The more homogeneous coating and greater surface coverage provided by DDAB allows the excess surfactant to be flushed from the capillary prior to performing electrophoretic separations. Separations of a basic protein mixture yielded quantitative recoveries, efficiencies ranging from 560,000 to 750,000 plates/m, and migration time reproducibility of 0.8-1.0% RSD (n = 10). This performance is similar to that of adsorbed cationic polymers (Polybrene, polyethyleneimine) but is achieved using a coating procedure that is over 10 times faster.     

Capillary electrophoresis with in-capillary solid-phase extraction sample cleanup

An in-capillary, solid-phase extraction (SPE)-capillary electrophoresis (CE) method, with not only high preconcentration factor but also no adsorption on the inner capillary wall of absorbing species in real complex samples, has been developed with a hole-opened capillary. The SPE sorbents approximately 3 mm in length was packed in the inlet end of the capillary. A hole approximately 30 microm in diameter was opened after the sorbents on the capillary. Sample solutions were loaded from the inlet end, and the sample wastes flowed out from the hole. After a certain time of the sample loading, a 1.5-mm-long methanol plug was introduced from the inlet end and forced to pass by the sorbents and the hole. Then, a separation voltage was applied between the hole and the outlet end of the capillary to carry out normal CE. When the sample loading time was 120 min, CE peak heights of the 2,4-dichlorophenol and 2,4,5-trichlorophenol were proportional to their concentration in a range of 0.08-5 ng/mL, and their detection limits were 25 and 17 pg/mL, respectively. A 16,000-fold sensitivity enhancement was obtained for CE of the chlorophenols with only a little decrease in CE separation efficiency. It was also demonstrated with the mixture of the chlorophenols and a surfactant cetyltrimethylammonium bromide that the present method could eliminate the adsorption problem of absorbing species on the inner wall during sample loading. Furthermore, the SPE-CE was directly applied to determination of chlorophenols on the level of 0.02 ppb in downstream water of a river, and the results agreed well with those obtained with off-line SPE-HPLC experiments.     

Chromatographic preconcentration coupled to capillary electrophoresis via an in-line injection valve

A preconcentration-capillary electrophoresis (CE) system using a small precolumn in combination with an in-line injection valve is presented. The advantage of the present design is the ability to perform the sample preconcentration fully independently from the CE separation and to prevent sample matrix and washing solvents from entering the CE capillary. With a micro injection valve, sample could be effectively introduced into the CE system in an in-line fashion without seriously affecting the CE separation efficiency. Breakthrough volume, desorption efficiency, and elution volume for the C18 microcolumn (5 x 0.5 mm i.d.) were established, yielding values of 750 microL, 70%, and 0.9-1.1 microL, respectively, using enkephalin peptides. The time between the start of the desorption of the analytes from the precolumn and the injection into the CE system was also studied in order to achieve optimal sensitivity and separation efficiency. The performance of the complete system was demonstrated by the preconcentration and separation of an enkephalin mixture. Using a sample volume of 250 microL and a CE injection voltage of -15 kV for 12 s, linearity was observed over 2 orders of magnitude, and detection limits (S/N = 3) were in the 5-10 ng/mL range. A 1000-fold sensitivity enhancement is obtained using this setup, as compared to a regular CE setup. For 100 ng/mL samples, repeatabilities (RSDs) of migration time and peak area were 1.2 and 11%, respectively.     

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.     

Analysis of major protein-protein and protein-metal complexes of erythrocytes directly from cell lysate utilizing capillary electrophoresis mass spectrometry

The separation and detection of the major protein-protein and protein-metal complexes of erythrocytes directly from cell lysate under native conditions has been accomplished for the first time using capillary electrophoresis electrospray ionization-mass spectrometry (CE/ESI-MS). All three major protein-protein and protein-metal complexes in human red blood cells (RBCs) with a concentration dynamic range of approximately 3 orders of magnitude were successfully detected. Intact complexes detected in lysed RBCs included carbonic anhydrase II (CAII-Zn at approximately 0.8 amol/cell) complexed with its zinc cofactor, carbonic anhydrase I (CAI-Zn at approximately 7 amol/cell) complexed with its zinc cofactor, and hemoglobin A (Hb-tetramer at approximately 450 amol/cell)a tetramer formed by two alpha-beta-subunits and four heme groups. The average molecular weights measured for these complexes were consistent with their theoretical values within 0.01% mass accuracy. The use of Polybrene as a self-coating reagent in conjunction with ammonium acetate at pH approximately 7.4, narrow capillary for high separation efficiency, and forward polarity CE to avoid acid production at the tip of the capillary were overriding experimental factors for successful analysis of protein complexes. Diluting the lysed blood sample in ammonium acetate for a minimum of 6 h before injecting the sample into the CE was essential for obtaining the mass accuracy consistent with their theoretical average molecular weights. At physiological pH, the mass spectrum of the electrophoretic peak of Hb-tetramer included a small amount of the monomers and Hb-dimer. The migration time and peak profile of these species were almost identical to that of the tetramer, indicating that they are formed from decomposition of the Hb-tetramer during the ESI process. A separate electrophoretic peak for the Hb-dimer was only detected when the pH of the BGE was lowered from 7.4 to approximately 6.6. Running CE in forward polarity mode was essential for detection of the intact Hb-tetramer as well as CAI-Zn and CAII-Zn complexes. Under forward polarity mode, CE outlet/ESI shared electrode acts as the cathode of the CE circuit and the anode (positive voltage for positive ions) of the ESI circuit, thereby maintaining approximately neutral pH at the CE outlet/ESI electrode. In addition, under forward polarity mode, CAII-Zn and CAI-Zn migrated ahead of Hb-tetramer, avoiding being masked by 562x and 64x, respectively, molar excess of Hb-tetramer.     

A low-makeup beveled tip capillary electrophoresis /electrospray ionization mass spectrometry interface for micellar electrokinetic chromatography and nonvolatile buffer capillary electrophoresis

A robust interface has been developed for interfacing micellar electrokinetic chromatography (MEKC) and nonvolatile buffer capillary electrophoresis (CE) to electrospray ionization mass spectrometry (ESI-MS). The interface consists of two parallel capillaries for separation (50 microm i.d. x 155 microm o.d.) and makeup (50 microm i.d. x 155 microm o.d.) housed within a larger capillary (530 microm i.d. x 690 microm o.d.). The capillaries terminate in a single tapered tip having a beveled edge. The use of a tapered beveled edge results in a greater tip orifice diameter (75 microm) than in a previous design from our laboratory (25 microm) that used a flat tip. While maintaining a similar optimum flow rate and consequently similar sample dilution, a 75-microm beveled emitter is more rugged than a 25-microm flat tip. Furthermore, the incorporation of a sheath liquid capillary allows the compositions of the final spray solution to be controlled. The application of this novel CE/ESI-MS interface was demonstrated for MEKC using mixtures of triazines (positive ion mode) and phenols (negative ion mode). The ability to perform CE/ESI-MS using a nonvolatile buffer was demonstrated by the analysis of gangliosides with a buffer consisting of 40 mM borate and 20 mM alpha-cyclodextrin.     

Effective and reproducible capillary electrophoretic separation of thiols under conditions where exceptionally high current is generated

There is an escalating interest in the role of endogenous nitric oxide (NO) in biological systems and how this chemical regulates physiology in normal and disease states. In biological systems, the cellular concentration can be estimated, in the simplest form, by accounting for NO and its common metabolites, nitrate and nitrite. However, since NO is also known to interact with other chemical entities, such as thiols, it would be valuable to have a rapid qualitative assay that could account for thiol binding and S-N bond cleavage in the presence of different reducing agents. A separation buffer consisting of 10 mM phosphate, 10 mM HCl, and 250 mM KCl is shown to be adequate for the separation of glutathione, nitrosylated glutathione, and glutathione disulfide solubilized in 2 M HCl. The current observed under these separation conditions (249 microA at 11 kV) is extremely high by capillary electrophoresis (CE) standards, with a total power (current x voltage/capillary length) calculated to be in excess of 7 W/m. While this exceeds the approximately 1.0 W/m recommended by previous studies as a maximum for CE-based separations, we demonstrate that effective CE separation of thiols can, in fact, be accomplished under these conditions with acceptable reproducibility, provided that buffer depletion issues are addressed.     

Flow counterbalanced capillary electrophoresis using packed capillary columns: resolution of enantiomers and isotopomers

A method with the ability to increase greatly both the resolution and efficiency of a given capillary electrophoretic system is described. This method differs from traditional capillary electrophoresis (CE) in that a counterflow is induced in the direction opposite to the electrokinetic migration of the analyte. This has the effect of extending not only the time the analytes migrate in the electric field but also the effective length and the effective applied voltage of the system. Previous work in our group with flow counterbalanced capillary electrophoresis has utilized an open tube of small inner diameter to reduce peak broadening caused by hydrodynamic flow. Narrow-diameter capillaries (5-10 microm) restricted analysis to fluorescent analytes and laser-induced fluorescence detection. The method described here uses a capillary of much larger inner diameter (75 microm) that has been packed with nonporous silica particles. The packing material reduces the amount of band broadening caused by pressure-induced flow relative to that experienced in an open tube. A larger diameter capillary allows the detection of analytes by UV absorption, not only eliminating the need to tag analytes with fluorescent tags but also allowing for the detection of a much broader range of analytes. The system was evaluated by studying the separations of several enantiomers using only beta-cyclodextrin as the chiral selector. The system was also used to resolve the two naturally occurring isotopes of bromine and to resolve phenylalanine from phenylalanine-d8. Relative to traditional CE, large improvements in resolution and separation efficiency have been achieved with this method.     

Experimental characterization of end-column electrochemical detection in conjunction with nonaqueous capillary electrophoresis

An end-column electrochemical detector arrangement for capillary electrophoresis (CE) based on a 75-microm-i.d. capillary and a 25-microm microdisk electrode is characterized. The investigations were carried out using a nonaqueous (acetonitrile-based) buffer and ferrocene model compounds which offer high reliability for voltammetric measurements. The positioning of the microdisk electrode relative to the capillary outlet is the most important parameter for optimization of detection performance as it determines the characteristics of mass transport toward the electrode and the effect of ohmic potential drop resulting from the electrophoretic current on the actual detection potential. On the basis of spatially resolved studies, it was concluded that for the detection system used the microdisk electrode should be placed in a central position relative to the capillary outlet at a distance within the range of 75-100 microm. The presence of a high-voltage electric field had no negative effect on baseline noise, which was demonstrated by comparison of capillary flow injection based on gravity flow and CE experiments. Even a faster stabilization of the baseline was observed by increasing the separation voltage.     

Fluorescence correlation spectroscopy excited with a stationary interference pattern for capillary electrophoresis

Using a combination of capillary electrophoresis (CE) and patterned fluorescence correlation spectroscopy (patterned FCS), we have developed a new technique for performing electrophoretic analysis independently of the initial length of injected analyte plugs. In t histechnique, which is abbreviated as CE/patterned FCS, fluorescent analyte molecules dispersed continuously in a capillary migrate through a stationary interference pattern created by two intersecting excitation laser beams, and their fluorescence emission is monitored. We prove theoretically that the power spectrum of fluctuations in the fluorescence intensity gives a virtual electropherogram. The profile of the electropherogram and the number of theoretical plates are in general obtained by using analytical methods. Characterizing the capillary length within the excitation beams as the effective length, we compare CE/ patterned FCS with conventional CE. Numerical simulations on capillary gel electrophoresis of DNA predict that the optimized CE/patterned FCS is superior to conventional CE when the effective length is shorter than 1 cm. The experimental feasibility of this technique is demonstrated in the fluorometry of TOTO-1-stained DNA. For an effective length of 740 microm, a maximum number of plates of 7400, and a resolution of 1.0 were obtained with a one-component injection of pUC18 DNA and a two-component injection of pUC 18 DNA and lambda DNA, respectively.     

A metal nebulizer capillary electrophoresis/Fourier transform infrared spectrometric interface

A capillary electrophoretic (CE) system has been successfully interfaced to a Fourier transform infrared spectrometer. The advantage of such an interface is that analytes may be detected and often unequivocally identified without analyte derivatization. The interface consists of a stainless steel tube in which the CE capillary is placed and the two are held in contact with the use of a metal tee. A solvent elimination approach is used with the interface, so that analytes are deposited onto an infrared transparent window, that is, CaF2, and measured with the use of an infrared microscope. A critical component of this design is to provide an electrical connection at the end of the CE column to permit stable separations that allow for efficient transport of the sample onto the window. The interface produces an aerosol that is directed at the surface of the infrared transparent window. The use of a volatile electrolyte, along with the flow of helium, allows for partial evaporation of the electrolyte in flight and complete evaporation of the solvent and electrolyte on the surface of the window to produce a "dry", or neat, analyte deposit.     

Selective detection of biogenic amines using capillary electrochromatography with an on-column derivatization technique

A selective detection method for biogenic amines present in highly complex matrixes was devised by employing both electrokinetic injection and on-column-derivatization capillary electrochromatographic methods. The on-column derivatization capillary electrochromatography system was evaluated by use of a capillary column (total length of 45 cm, effective length of 25 cm) fabricated using a 100-mcirom (i.d.) fused-silica capillary tube packed with 5-microm (i.d.) ODS particles that were tolerant of an alkaline environment. The column was filled with a run buffer consisting of a derivatization reagent, o-phthalaldehyde/2-mercaptoethanol, in a mixture of borate buffer (pH 10). After electrokinetic injection of a mixture of five biogenic amines (histamine, serotonin, tyramine, putrescine, cadaverine) as a test sample, the free amines entered into the anodic site of the capillary column and started to travel along the column, during which time the analytes reacted with the derivatization reagent, separated out, and were detected with an absorbance at 340 nm when high voltage was applied to the column. When this system was applied to a mixture containing 5 biogenic amines and 17 amino acids, the 5 biogenic amines plus arginine selectively entered into the capillary with the electrokinetic injection and were observed on the electrochromatogram, but none of the amino acids lacking arginine were detected. The designated method was also tested for its ability to determine the presence of biogenic amines in the crude extracts obtained from two types of aged fish.