Microchannel electrophoretic separation ofbiogenic amines by micellar electrokineticchromatography

Fast, efficient separation of most common biogenic amines was successfullyperformed on a glass microchip capillary electrophoresis device. The aminesputrescine, histamine, tyramine, cadaverine, phenethylamine, tryptamine, spermidineand spermine were derivatized prior to fluorescence detection with fluoresceinisothiocyanate. Separation was carried out using a channel length of 28 mm, a crosssection of 50 � 8 mm, and a field strength of 600 V/cm. After optimization of bufferelectrolyte conditions (120 mM boric acid, pH 9.4, modified with 40 mM SDS),fluorescein thiocarbamyl amine derivatives were successfully resolved. Analysis timewas as short as 75 s. Determination of the biogenic amines was achieved in soy saucesamples.

Keywords: Capillary electrophoresis / Microchip / Biogenic amines / Fluorescence detection /Micellar electrokinetic chromatography EL 3286

Isabel Rodríguez1

Hian Kee Lee1

Sam F. Y. Li2

1Department of Chemistry,National University ofSingapore, Singapore,Republic of Singapore

2Department of Chemistry,and Institute of MaterialsResearch and Engineering,National University ofSingapore, Singapore,Republic of Singapore

1 Introduction

In recent years, microfabrications have attracted consid-erable interest for their potential of miniaturization andintegration of an entire chemical or biological analysisprocess on a single device. Microfabrication offers thepossibility to miniaturize traditional analytical instrumen-tation with the advantages of speed, automation andvolumetric reduction of samples, reagents and waste. Theuse of capillary electrophoresis (CE) has recently beenextended to the microfabricated environment [1±3]. CEappears to be a promising technique to be integrated in asingle microfabricated device because of its experimentalsimplicity. Pumping action is achieved using electrokineticeffects and fluid flow is precisely controlled by regulatingthe applied potentials at the terminus of each micro-channel. Using electric fields to direct and control fluidflow eliminates the need for micromoving parts such aspumps and valves, allowing for a convenient integrationprocess of complete assays. Over the past six years,applications of microfabricated capillary electrophoresischips have been successfully demonstrated in the fieldsof free zone electrophoresis [4±6], synchronized cyclicelectrophoresis [7, 8], gel electrophoresis [9±12], open-

channel electrochromatography [13] and micellar electro-kinetic chromatography (MEKC) [14±16]. Monolithicdevices that integrate sample processing, chemicalreactions and analysis included free zone electrophoresiswith precolumn [17] or postcolumn [18, 19] reactors,devices which allow solvent mixing for isocratic andgradient elution MEKC [16], DNA restriction digestionswith fragment sizing [20], PCR amplification and electro-phoretic analysis [21], cell lysis, PCR amplification andelectrophoretic analysis [22]. Microfabricated capillaryelectrophoresis chips have found widespread applicationin the area of clinical analysis. Human serum proteinshave been separated and relatively quantified [23]. Serumcortisol [24], thyroxine [25], monoclonal antibodies andtheophylline [26] have been determined by competitiveimmunoassay. Identification of peptides and proteins [27]was achieved by the successful coupling of microchipswith an electrospray ionization mass spectrometer. Alarge number of applications can be found in the area ofDNA analysis [9±12, 20±22].

The separation and quantification of biogenic amines haslong been of importance due to their toxic effects [28, 29],and more recently due to their relation with certain typesof cancer [30]. These amines are designated as biogenicbecause they are formed by the action of livingorganisms. Food substances that have been preparedby fermentation process, or have been exposed tomicrobial contamination, are likely to contain amines.The excessive levels of biogenic amines in food productsmay be an indication of poor quality raw materials or poorhandling and can lead to adverse reaction such ashistamine poisoning [31]. Biogenic amines such astyramine and 2-phenylethylamine have been proposed

Correspondence: Dr. Sam F. Y. Li, Department of Chemis-try,National University of Singapore, Kent Ridge, Singapore119260, Republic of SingaporeE-mail: chmlifys@nus.edu.sgFax: +65-7791691

Abbreviations: Cad, Cadaverine; FTC, fluorescein thiocar-bamyl; HEPT, height equivalent theoretical plate; His, Histamine;Phe, Phenethylamine; Put, Putrescine; Spd, Spermidine; Spn,Spermine; Tryp, Tryptamine; Tyr, Tyramine

� WILEY-VCH Verlag GmbH, 69451 Weinheim, 1999 0173-0835/99/0101-0118 $17.50+.50/0

118 Electrophoresis 1999, 20, 118±126

as the initiators of hypertensive crises in certain patientswith dietary-induced migraine. Biogenic amines can alsobe considered as carcinogenic for their capacity to reactwith nitrites and produce potential carcinogenic nitros-amines [29]. This paper describes the development of amicrochip electrophoretic separation method to analyzethe most important biogenic amines occurring in foodproducts. Optimal separation conditions were determinedto allow for the separation and detection of eightfluorescently labeled biogenic amines in less than 75 s.Application of the method to real samples was demon-strated for soy sauce.

2 Materials and methods

2.1 Instrumental

Separations were monitored using a lamp-based fluo-rescence detection system. An episcopic fluorescencemicroscope (DAS Mikroskop DMLS; Leica, Heerbrugg,Switzerland) and its accessories were used. A 50 W high-pressure mercury lamp was used as excitation radiation.The filter system consisted of an excitation filter (EX 450±490 nm), a dichroic mirror (DM 510 nm) and a barrier filter(BA 515 nm). The fluorescence emission was collectedwith a Leica PL FLUOTAR long working distanceobjective (magnification, 63; numerical aperture, 0.70).A silicon photodiode with a built-in amplifier (Model HC220-21; Hamamatsu, Bridgewater, NJ, USA) was at-tached to the trinocular housing of the microscope. Anintegrator C-R6A Chromatopac (Shimadzu, Kyoto, Japan)was used for signal recording. Two high-voltage CEpower supplies of 15 kV maximum output (Spellman HighVoltage Electronics, Plainview, NY, USA) were used forinjection and separation.

2.2 Reagents

Biogenic amine standards: putrescine (Put), histamine(His), cadaverine (Cad) dihydrochloride, tyramine (Tyr),tryptamine (Tryp), phenethylamine (Phe) hydrochloride,spermidine (Spd) trihydrochloride, spermine (Spn) tetra-

hydrochloride, 1,6-diaminohexane, fluorescein isothiocya-nate Isomer I (FITC), sodium dodecyl sulfate (SDS),dodecylbenzene sulfonic acid and boric acid werepurchased from Fluka (Buchs, Switzerland). Water waspurified with a Millipore-Q system (Millipore, Bedford, MA,USA). C18 cartridges (SPE-3) were purchased fromWaters (Mildford, MA, USA). All chemicals were ofanalytical reagent grade. Soy sauce samples wereobtained from a local retailer.

2.3 Microchip fabrication and operation

The glass microchannels were fabricated using standardphotolithographic and wet chemical etching techniquesdescribed previously [9]. The channel structure anddimensions are shown in Fig. 1. To form a close networkof channels, a cover plate with laser-drilled holes wasbonded over the etched channels. Prior to thermalbonding, the top plate with holes and the bottom platewith the etched channels were submerged in hot H2SO4/H2O2 for 5 min and rinsed thoroughly with Millipore water.Then the surfaces of both plates were hydrolyzed in diluteNH4OH solution, rinsed again with Millipore water, anddried with N2 gas. Subsequently, the pair of glass plateswas placed between two pieces of well-polished vitreouscarbon (Lecarbon Lorraine, Grenvillers, France) andannealed in a Ney model 2-525 programmable furnace(Barkmeyer Division, CA, USA). The temperature was620oC for 4 h, followed by natural cooling of the furnace toroom temperature. The process was repeated if neces-sary. After bonding, pipette tips were affixed on the holesusing epoxy glue to form reservoirs. Platinum electrodesinserted in these reservoirs provided electrical contactfrom the power supply and the electrolyte solution in thereservoirs. Sample solution was filled in reservoir 1, therest of the reservoirs were filled with buffer electrolyte. Forthe injection of a sample plug, 300 V potential was appliedbetween reservoirs 1 and 3 for 5 s. Subsequently,analysis was carried out between reservoirs 2 and 4 byapplying 3000 V. The distance from the injection point tothe detection window was 28 mm. The linear range of the

Electrophoresis 1999, 20, 118±126 Microchannel MEKC of biogenic amines 119

Figure 1. Layout of the micro-fabricated channels. Channelsreferred to in the text areidentified by numbers. The sep-aration channel is 50 mm wide,45 mm long; the injection chan-nel is 30 mm wide, 7 mm long; allthe channels are 8 mm deep.Access holes are 1 mm indiameter. The point of fluores-cence detection is marked withan arrow.

CE

and

CE

C

current-voltage for the microchip used was tested with theworking buffer. Figure 2 shows the current-voltage curvesobtained for the injection and separation channels. Forthe separation channel, deviation from linearity occurredat 6 kV, which corresponds to an electric field of 1260 V/cm. This is one order of magnitude greater than istypically used in CE. For the injection channel, themaximum voltage which could be applied was 1 kVbefore arcing occurred between electrodes.

2.4 Derivatization, extraction and separationconditions

Fluorescein thiocarbamyl amine derivatives (FTC-amines) were obtained according to the proceduresdescribed previously [32, 33]. A 2.5 � 10±3

M solution ofFITC was prepared in acetone. Solutions containing10±4

M of each amine were prepared in 20 mM boratebuffer (pH 10). One hundred mL of each of the aminesolution was allowed to react with 10 mL of FITC solutionduring a period of 5±12 h in the dark at room temperature.Samples were diluted in the run buffer to the requiredconcentration before injection. Microchannels were con-ditioned for use by filling first with water and thensubsequently flushing with 1 M NaOH, water and electro-lyte buffer. Regeneration of the channels was performedby rinsing the capillary with 1 M NaOH followed by waterand run buffer. Biogenic amines were extracted from soysauce samples by solid-phase extraction in C18 car-tridges using dodecylbenzene-sulfonic acid as ion pairreagent. Soy sauce sample was diluted two times withacetate buffer and the pH was adjusted to five. Two mL ofthe diluted sample were passed through a C18 cartridgeto clear of unwanted components. One mL of the eluatewas then mixed with 1 mL of a 1.5 mM dodecylbenzene-

sulfonic solution. then 2 mL of this solution were passedthrough another C18 cartridge. After washing with 5 mLacetate buffer at pH 5, the amines were eluted with 2 mLof methanol. The solvent was evaporated in an N2 streamand the sample was dissolved in 1000 mL borate buffer(pH 10). The extracted amines were derivatized by adding10 mL of FITC solution to 100 mL of the extract. Prior toinjection, the derivatized extract was diluted ten times andthe internal standard was added. The separation of theamines was performed by MEKC. The separation bufferconsisted of 120 mM borate buffer (pH 9.4) and 40 mM

SDS.

3 Results and discussion

3.1 Optimization of FTC-amine separationconditions

The effects of the concentration and pH of the boratebuffer and concentration of SDS in the buffer electrolytewere studied in order to determine the conditions leadingto the best separation of the FTC-amines. Borate buffersof concentrations in the range of 60±150 mM were tested.As the buffer concentration was increased, improvementin the resolution of the FTC-amines was observed as aconsequence of the decrease in electroosmotic mobilitywith the ionic strength. Optimum concentration of bufferelectrolyte was found at 120 mM of borate. Furtherincreasing the buffer concentration did not improveresolution significantly but resulted in excessively longmigration times. Optimum pH of the borate buffer wasfound by keeping the borate concentrate at 120 mM andSDS concentration at 40 mM. The optimum pH wasdetermined to be in the range of 9.4±9.6 and was, inprinciple, dictated by the nature of the FTC-amine

120 I. Rodríguez, H. K. Lee and S. F. Y. Li Electrophoresis 1999, 20, 118±126

Figure 2. Current-voltage curvesfor the injection and separationchannels with 120 mM borate buffer(pH 9.4) and 40 mM SDS.

complexes. Buffer pH affects the charge of both channelwall surface and FTC-amines, thereby affecting theelectroosmotic flow, mobilities of FTC-amines and micellephase, as well as wall interactions. Figure 3 shows thechange in apparent mobilities of the FTC-amines as afunction of the buffer pH. The FTC-amines were broughtpast the detector by the EOF since the dissociation of thecarboxylic group of FITC (pK 7.9) provides the FTC-amines with net negative charges under the conditionsused. Therefore, apparent mobilities of the FTC-aminesincreased as the buffer pH was increased from 9 to 9.6mainly due to the increase of the EOF. At pH 9,efficiencies and resolutions of the FTC-amines werepoor. FTC-Spd and FCT-Spn were not detected. At pH9.2, efficiencies and resolutions of FTC-amines wereinsufficient and FTC-Spd and FCT-Spn showed verybroad peaks. The low efficiencies observed at lower pHcould be attributable to channel wall interactions of theFTC-amines. Wall adsorption was probably a conse-quence of the incomplete reaction between FITC and theamines. Due to the sluggish reaction, nonderivatized

amines at lower pH could be adsorbed onto the negativelycharged wall and consequently reduced the EOF. Inaddition, negatively charged FTC-amines can interactlocally with the modified channel wall. At higher pH theseinteractions were avoided and sharper, more efficientpeaks were obtained. At pH 9.4 and above, adequateefficiencies were obtained. At pH 9.4, resolution wasoptimum, whereas at pH 9.6 some peak pairs were notcompletely resolved. The decrease in resolution at pH 9.6may be attributed to the increase of the magnitude of theEOF.

Partitioning of the FTC-amines with the micellar phasewas an essential mechanism for the successful separa-tion of the biogenic amines. The addition of SDSsignificantly influenced the migration order and resolutionof the FTC-amines. Figure 4 shows the apparentmobilities of the FTC-amines as a function of the SDSconcentration in the buffer electrolyte. As can be seen,without the addition of SDS, the FTC-amines werearranged in three groups with the same apparent

Electrophoresis 1999, 20, 118±126 Microchannel MEKC of biogenic amines 121

Figure 3. Variation of the appa-rent mobilities of the FTC-amines as a function of thebuffer electrolyte pH.

Figure 4. Variation of the appa-rent mobilities of the FTC-amines as a function of theSDS concentration in the bufferelectrolyte.

mobilities. Therefore, poor resolution was achievedamong the FTC-amines. Addition of 20 mM of SDS tothe buffer electrolyte improved resolution in general butthe peak pairs of FTC-His and FTC-Tyr were notresolved. Concentrations of surfactant between 30 mM

and 50 mM were optimum to achieve greatest resolution.Concentrations of SDS above 60 mM resulted in loss ofresolution between FTC-Tyr, FTC-Cad and FTC-Phepeaks. Typically, for the determination of the fundamentalparameters of MEKC the migration time of the micelle, tm,and the migration time of the bulk solution, to, aredetermined by using marker compounds such as SudanIII and methanol. On a microchip-CE with fluorescencedetection these determinations are not straightforward.Therefore, we resorted to an approximation. The migra-tion time window, tm/to, for the buffer system wasmeasured in a fused-silica capillary using Sudan III asmicellar marker and dimethylsulfoxide as EOF marker.The parameter to was measured in the microchip byindirect fluorescence detection using 10 mM fluorescein asbuffer additive and methanol as marker for the electro-osmotic flow. With these values, the tm in the microchipwas estimated (data are presented in Table 1). Capacityfactors for the FTC-amines were calculated using thisestimated value. Since the FTC-amines were negativelycharged at the working pH, the capacity factors werecalculated following the model of Khaledi et al. [34]. Thismodel takes into account the partitioning process as wellas mobilities of analytes and reflects more accurately thebehavior of acidic solutes in MEKC. For this purpose themigration times of the FTC-amines in absence of themicelles were used as to. The results are shown in Table1. Amines that showed higher capacity factors are the

polyamines which can react with two, three, or fourmolecules of FITC. FTC-Spd and FTC-Spn after deriva-tization with three and four molecules of FITC, respec-tively, experienced great gains in hydrophobicity andshowed high capacity factors. The capacity factors ofFTC-Spd and FTC-Spn were negative due to theirmigration behind the micelle phase as a consequence ofthe electrophoretic retardation towards the anode. FTC-His and FTC-Tyr, due to the presence of hydroxylicgroups, were less solubilized by the micelles and showedlower capacity factors.

3.2 Sample injection and separation

High electric fields of 600 V/cm and a relatively shortseparation channel allowed for a rapid separation of theeight FTC-amines and the internal standard. An electro-pherogram of the biogenic amines and internal standardcan be seen in Fig. 5. Complete separation was achievedin 75 s. Injection was performed by applying 600 v/cm inthe injection channel for 5 s. This drove the sample fromreservoir 1 to reservoir 2 across the intersection point. Byswitching the potential to the separation channel, thesample plug created at the intersection was injected andseparated. In principle the sample plug should have beendetermined by the geometry of the intersection and beindependent of injection time. However, it was noticedthat the peak area increased with injection time. Theseresults are consistent with previously reported diffusion ofsample at channel intersections during injection andduring delayed periods between termination of sampleinjection and the start of the separation [6, 35]. Injectiontimes needed to be optimized in order to avoid dispersion

122 I. Rodríguez, H. K. Lee and S. F. Y. Li Electrophoresis 1999, 20, 118±126

Table 1. Partition properties of the FITC-derivatized biogenic amines in MEKC on amicrofabricated glass chip for the buffer electrolyte of 120 mM borate, pH 9.4,modified with 40 mM SDS

Detection time of bulk flow, to, (min) in capillary 5.63Detection time of micelle, tm, (min) in capillary 23.87Migration time window, to/tm 0.24Detection time of bulk flow, to, (min) in microchannel 0.16Detection time of micelle, tm, (min) in microchannela) 0.67Capacity factorsb)

k ¢ FTC-Put 0.98k ¢ FTC-His 0.66k ¢ FTC-Tyr 0.56k ¢ FTC-Cad 2.49k ¢ FTC-Phe 0.81k ¢ FTC-Tryp 1.10k ¢ FTC-Spd ±5.25k ¢ FTC-Spn ±9.61

a) Estimated value (see Section 3.1)b) Calculated according to k ¢ = (tr±to)/to (l±tr±tm), where tr is the migration time of the FTC-

amines [34]

of the sample plug and to achieve the highest separationefficiency. Figure 6 shows the height equivalent theore-tical plates (HETP) obtained at various injection times foran injection voltage of 300 V between reservoirs 1 and 3.Plate heights increased with the injection time. Efficien-cies up to 3.2 mm plate heights were obtained for a 3000 Vseparation potential with a 3 s injection time. However, 5 swas the minimum injection time for unbiased, representa-tive injection of all the sample components. At injectiontimes below 5 s, FTC-Spn was not present in the injectionplug, probably due to its slower mobility. The level ofsample leakage from the injection channel to theseparation channel was measured by the increase ofsignal due to the presence of sample in the injectionchannel. The increase in signal was 2.5% of the totalsignal. This level of leakage, although it increased thebaseline noise, was acceptable for the present separationand application. Leakage of sample from the injectionchannel at the channel intersection has been ascribed [2,35] to the induced convective flow in the injection channelby the electroosmotic flow in the separation channel.

3.3 Efficiency, reproducibility and detectionlimits

The efficiency of the separation in the microchanneldevice for the injection and separation conditions de-scribed above was measured in terms of theoreticalplates. Efficiencies obtained were in the range of120 000±45 000 theoretical plates for the later elutingpeaks corresponding to plate heights in the micrometerrange. Table 2 shows the data obtained for thedifferent FTC-amines. Better efficiencies should be read-ily achieved with an optimized system allowing control ofthe sample diffusion at the intersection of channels and, in

Electrophoresis 1999, 20, 118±126 Microchannel MEKC of biogenic amines 123

Figure 5. Electropherogram of fluorescently labeledbiogenic amines obtained by microchip-CE. Buffer: 120mM borate, 40 mM SDS, pH 9.4; separation channel, 50mm � 8 mm � 4.5 cm (effective length 2.8 cm); injectionvoltage: 300 V between reservoirs 1 and 3, 5 s;separation voltage: 3000 V between reservoirs 2 and 4;detection: excitation wavelength, 470±490 nm; emissionwavelength, 520 nm; Peak identities: 1, Put; 2, His; 3, Tyr;4, Cad; 5, Phe; 6, Tryp; 7, Spd; 8, Spn; IS, internalstandard (1,6-diaminohexane). Concentration of eachpeak is ca. 10±5 mol/L.

Figure 6. Variation of HETP ofthe FTC-amines as a function ofinjection time. The injectionpotential was 300 V betweenreservoirs 1 and 3; separationpotential was 3000 V betweenreservoirs 2 and 4.

consequence, decrease of the effective injected sampleplug size [4, 8]. Further improvements in efficiency couldbe achievable by using narrower injection channels andsmaller detector view points. The reproducibility of thesystem was studied in terms of relative standard devia-tions (% RSD) of the migration times and peak areas (forresults, see Table 2). Good reproducibilities of themigration times were observed from run-to-run and day-to-day. FTC-Spd and FTC-Spn showed higher values of% RSD, probably due to wall interactions (as discussedabove). The values of % RSD for peak area wereexcessively high. This low reproducibility may be due tothe temporal instability of the sample plug shape due todiffusion of sample during injection (as described above).Injection amount was a function of the injection time anddelayed periods between injections and separations and,therefore, the reproducibilities of the peak areas weredependent on the manual control of these times.Reproducibility of the areas is expected to improve withan optimized system that allows for control of the diffusionof sample during injections [6, 24].

Detection limits were measured using serial dilutions ofthe stock solutions of the FTC-amines. For a signal-to-noise ratio (S/N) of 2, detectable concentrations were inthe range of 10±6

M. Detection limits for the FTC-aminesare listed in Table 2. For most applications in the area offood analysis these detection limits are sufficient. How-ever, a further decrease in detection limits could beachieved by optimizing the detector, i.e., by using abrighter source and reducing or eliminating factors thatcontributed to the increase of background noise, such asscattered light and leakage of sample into the separationchannel.

3.4 Application to real sample

To demonstrate the feasibility of microchip separations ofreal samples, determination of biogenic amines was

performed in soy sauce. For this purpose, soy saucesamples were first extracted to eliminate other aminatedcompounds that would react with FITC and interfere withthe identification and detection of the amines. Biogenicamines were extracted by solid-phase ion-pair extraction

124 I. Rodríguez, H. K. Lee and S. F. Y. Li Electrophoresis 1999, 20, 118±126

Table 2. Efficiencies, reproducibilities and detection limits of the FITC-derivatized biogenic aminesa)

Theoretical HETP %RSD for %RSD for %%SD for Detectionplates (mm) migration times migration times peak area limits(N/m) (run-to-run) (day-to-day) (mM)

Put 120 000 8.1 0.35 0.48 1.27 2.95His 110 000 8.8 0.40 0.57 4.90 6.25Tyr 100 000 9.3 0.37 0.52 1.18 5.06Phe 84 000 11.8 0.34 0.56 1.77 5.58Cad 72 000 13.7 0.16 0.43 3.65 4.81Tryp 43 000 23.1 0.15 0.40 10.62 4.27Spd 90 000 11.0 0.25 0.60 5.41 6.54Spn 46 000 21.5 0.54 0.80 10.52 6.57

a) Data obtained from the average of five analyses

Figure 7. Electropherogram of biogenic amines influorescently labeled soy sauce obtained by microchip-CE. Buffer: 120 mM borate, 40 mM SDS, pH 9.4;separation channel: 50 mm � 8 mm � 4.5 cm (effectivelength 2.8 cm); injection voltage: 300 V betweenreservoirs 1 and 3, 5 s; separation voltage: 3000 Vbetween reservoirs 2 and 4; Peak identities; 1, Put; 2, His;8, Spn; IS, internal standard.

and derivatized according to the protocols described inSection 2.4. FTC-amines presented in soy sauce sampleswere analyzed and identified according to their electro-phoretic mobilities and by spiking with FTC-amines athigh concentration. A typical electropherogram of FTC-amines in soy sauce is shown in Fig. 7. Put, His and Spnwere identified. These amines have been identified in soysauce before by other methods [32, 36]. It can be seen(Fig. 7) that numerous other peaks due to excess reagentor other aminated compounds could be resolved, provingthe high resolving power of the microchip CE separationsfor real samples. However, the application of this methodfor the quantification of biogenic amines in real samplespresents some difficulties, derived from the inaccurateinjection amount of the present setup and the depend-ence of the fluorescence signal upon the amount of FITCused in the derivatization reaction [32].

4 Concluding remarks

The results obtained in this study demonstrate that CEanalysis of biogenic amines can now be performed withinan approximately 15-fold shorter separation time com-pared to that in fused-silica capillaries [32]. Satisfactoryseparation of eight biogenic amines plus an internalstandard with high separation efficiencies could beattained on a time scale of seconds. Plate numbers upto 120 000 have been obtained for FTC-Put in a migrationtime of 20 s, with an associated plate height of 8.1 mm.Integration of a sample injector allows the introduction ofminute sample volumes and very reproducible migrationtimes of nonbiased sample components. Better reprodu-cibility of peak areas could be achieved with effectivecontrol of the diffusion of sample during injection anddelayed periods by controlling the potentials of the fourchannels independently. On-chip separation of realsamples proves the high resolving power of the micro-chip-CE system. Detection limits obtained are in the rangerequired to conduct measurements of biogenic amines atthe levels present in food samples. Nevertheless, it isexpected that detection sensitivity could be enhanced bycontrolling the injection channel potentials during separa-tion to eliminate leakage, or by using better optics, deeperchannels, a brighter source and a more sensitive photondetector.

The authors thank the National University of Singapore forfinancial support.

Received June 20, 1998

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