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Droplet microfluidics for post-column reactions in capillary electrophoresis Aemi S. Abdul Keyon, Rosanne M Guijt, Christopher J. Bolch, and Michael C. Breadmore Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/ac5033963 • Publication Date (Web): 13 Oct 2014 Downloaded from http://pubs.acs.org on October 23, 2014

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Analytical Chemistry

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Droplet microfluidics for post-column reactions in capillary electrophoresis

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Aemi S. Abdul Keyon1, 2, 3, 4

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Rosanne M. Guijt2

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Christopher J. Bolch3

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Michael C. Breadmore1*

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Australian Centre for Research on Separation Science, School of Physical Sciences, University of Tasmania, GPO Box 252-75, Hobart, Tasmania 7001, Australia

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Prof Michael Breadmore,

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Australian Centre for Research on Separation Science,

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School of Chemistry,

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University of Tasmania,

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GPO Box 252-75, Hobart, Tasmania 7001, Australia

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Phone: +61-3-6226-2154

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Fax: +61-3-6226-2858

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Email: [email protected]

Pharmacy School of Medicine, Australian Centre for Research on Separation Science, University of Tasmania, GPO Box 252-26, Hobart, Tasmania 7001, Australia National Centre for Marine Conservation and Resource Sustainability, Australian Maritime College, University of Tasmania, 7250, Launceston, Tasmania, Australia Department of Chemistry, Faculty of Science, Universiti Teknologi Malaysia, 81310, UTM Johor Bahru, Johor, Malaysia *Address Correspondence to:

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List of abbreviations

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CE Capillary electrophoresis

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CZE Capillary zone electrophoresis

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C4D Capacitively coupled contactless conductivity detection

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EOF Electroosmotic flow

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µeff Effective mobility

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FLD Fluorescence detection HPLC High performance liquid chromatography

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He-Cd Helium-Cadmium

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LC Liquid chromatography

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LED Light emitting diode

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LOD Limit of detection

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MEKC Micellar electrokinetic chromatography

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MS Mass spectrometry

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NEO Neosaxitoxin

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PEEK Polyetheretherketone

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PSTs Paralytic shellfish toxins UV Ultraviolet

AOAC Association of Official Analytical Chemists BGE Background electrolyte

dcGTX2 decarbamoylgonyautoxin2

Keywords: Droplet microfluidics, capillary electrophoresis, post-column reaction, paralytic shellfish toxins Total number of words including figure and table legends: 5100


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ABSTRACT

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A post-column reaction system based on droplet microfluidics was developed for capillary

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electrophoresis (CE). Analytes were separated using capillary zone electrophoresis (CZE) and

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electrophoretically transferred into droplets. The use of a micro cross for positioning a salt

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bridge-electrode opposite the separation capillary outlet is the key element for maintaining

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the electrical connection during electrophoretic separation. As the first of its kind,

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positioning the droplets in the electric field eliminated the need for electroosmotic flow

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(EOF) or hydrodynamic flow for droplet compartmentalisation. Depending on the total flow

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rate of both aqueous and oil phases, droplets of water-in-oil could be formed having

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frequencies between 0.7-3.7 Hz with a size of approximately 14 nL per droplet.

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Compartmentalised in the droplets, analytes reacted with reagents already present in the

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droplets to facilitate detection. The periodate oxidation of paralytic shellfish toxins (PSTs)

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was demonstrated, overcoming the limitation of pre-column oxidation, which results in

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multiple and sometimes identical oxidation products formed from the different PSTs.

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Compartmentalisation allows the oxidation products for each peak to be contained and to

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contribute to a single fluorescence signal, preserving the selectivity of CZE separation while

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gaining the sensitivity of fluorescence detection.

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74

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INTRODUCTION

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Capillary electrophoresis (CE) is a high-resolution separation technique allowing for the fast

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separation of a wide range of analytes. With many analytes not ultraviolet (UV) absorbing,

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fluorescent or electrochemically active, derivatisation is often required for sufficient

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sensitivity. In pre-column derivatisation, the use of large reagents may compromise the

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selectivity of the separation of small molecules, especially when the targets are smaller than

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the label. This is overcome in liquid chromatography (LC) by performing post-column

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reactions where reagents are introduced to the LC effluent, however this approach leads to

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slight peak broadening due to mixing, dilution and diffusion during the extra time required

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in the reaction coil.

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Despite the experimental complications introduced by the electric field and the smaller

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scale of CE, several post-column reaction schemes exist, including gap reactor1-3, coaxial

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reactor4-6, sheath flow cuvette reactor7-10, membrane reactor11 and a laser drilled capillary

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reactor12. Technically, some of these reactors require non-trivial construction, alignment or

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interfacing procedures. Typically, hydrodynamic flow in the reactor results in zone

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broadening and a loss of resolution, an effect more pronounced than in LC because of the

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much sharper peaks2,4. To minimise the extent of broadening, fast kinetic reagents (i.e.

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naphthalene-2,3-dicarboxyaldehyde, o-phthaldialdehyde or fluorescamine) are required to

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rapidly produce fluorescent derivatives and preserve resolution, eliminating reagents with

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relatively slow kinetics.

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Droplet microfluidics or segmented flow of aqueous droplets in an oil phase within

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microchannels, tubing or capillaries provides a unique way of compartmentalising aqueous

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fractions as the non-miscible boundary restricts dispersion and limits cross-contamination13.

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At the microscale, this has been used to facilitate the preservation of microscale


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separations14-16, whilst in the droplets increased reaction rates can be realised because of

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rapid mixing17,18. Combining these two features in a post-column reactor eliminates the

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need for reagents with fast reaction kinetics. Compartmentalisation of separation effluent

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into droplets has been applied in various separation systems including LC and microchip

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electrophoresis. Ji et al.19 used droplets for post-column enzymatic digestion of proteins, to

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be later subjected to mass spectrometry (MS) analysis. Nie and Kennedy20 demonstrated the

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addition of fluorescent reagent to proteins collected into droplets from capillary LC (cLC).

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Edgar et al.21 used the electroosmotic flow (EOF) to form aqueous droplets from the

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background electrolyte (BGE) in a microfluidic structure filled with oil, compartmentalising

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separated analyte bands. Following on from this, Draper et al.22 demonstrated the

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compartmentalisation of separated bands in micro capillary gel electrophoresis (µCGE). Due

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to the absence of EOF, an aqueous carrier was used to collect the bands and form the

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droplets. In both cases, the electrical field was terminated after separation and prior to

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compartmentalisation.

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Here, we present a new approach where analytes are electrophoresed into the droplets

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without disruption of the electric field, and used for post-column reaction. Off-the-shelf

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components were used for droplet formation and interfacing with fused silica capillaries in a

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commercial CE instrument. The potential application of this approach was demonstrated for

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post-column periodate oxidation of paralytic shellfish toxins (PSTs) in CE, marrying the

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selectivity of capillary zone electrophoresis (CZE) with the sensitivity and selectivity of

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fluorescence detection (FLD).

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MATERIALS AND METHODS



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Chemicals and reagents

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Chemicals were purchased from Sigma Aldrich (St. Louis, MO, USA) unless otherwise

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specified. Decanol was used as the oil phase for all experiments. All aqueous solutions were

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prepared with Milli-Q water (Millipore, Bedford, MA, USA). The BGE for CZE of fluorescein

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and calcein was 3 mM disodium phosphate prepared from disodium hydrogen phosphate

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anhydrous and adjusted to pH 7.8 with phosphoric acid (BDH, Melbourne, Australia).

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Fluorescein and calcein were dissolved in the BGE. The BGE for CZE of PSTs was 20 mM

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formic acid pH 2.8. Standards for PSTs (neosaxitoxin (NEO) and decarbamoylgonyautoxin 2

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(dcGTX2)) were purchased from Institute for Marine Biosciences (National Research Council

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of Canada, Halifax, Canada). Both PSTs standards, each having different concentration, were

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diluted with Milli-Q water to the desired working concentration and used without further

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purification. The PSTs mixture consisted of 10 µg/mL of NEO and 20 µg/mL dcGTX2. The

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oxidant reagent was prepared daily by mixing 5 mM periodic acid and 100 mM phosphoric

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acid, adjusted to pH 7.8 with 5M NaOH.

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Droplet formation

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The schematic outline of droplet formation was presented in Fig.1a. A dual syringe pump

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(Model 33, Harvard Apparatus, United States) was used to infuse the oil and aqueous

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phases into a Labsmith tee connector with 250 µm thru-holes (Labsmith, California, United

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States), with the aqueous phase entering the tee at 90° to the oil flow. The reagents were

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delivered to the tee using disposable syringes (3 mL volume equipped with luer lock,

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Hapool, China) via two separate polyimide coated fused silica capillaries (16 cm long 150 μm

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I.D, Polymicro-Technologies Phoenix, Arizona, United States). During optimisation, flow


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rates for each of the aqueous and oil phases ranged from 0.6 to 8 μL/min. A 40 cm long, 150

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μm I.D polyimide coated fused silica capillary connected the third tee arm with a polyether

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ether ketone (PEEK™) micro cross with 150 μm thru-holes (Upchurch Scientific, United

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States) for interfacing with the separation capillary. A manual 3-way valve (Labsmith,

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California, United State) was connected on the 40 cm long capillary, positioned between the

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tee and micro cross. The droplet valve port was closed during sample injection (droplet

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flowed to waste at the same time) and rapidly opened at the end of injection to allow

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droplet re-flow into the micro cross.

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Interfacing of CE with droplets

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Separations were performed using an Agilent HP3DCE system (Agilent Technologies,

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Waldbronn, Germany), using a PEEK™ micro cross to interface the separation capillary with

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the segmented flow and electrode as described above (Fig.1a). The micro cross was

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positioned inside the capillary cassette. A photograph of the micro cross positioned in a

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capillary cassette is shown in Fig. 1b. A polyimide coated fused silica capillary was used for

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CZE (57 cm long, 25 μm I.D) with the outlet positioned 90° to the segmented flow. High

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voltage was applied to a Pt ground electrode positioned opposite the separation capillary

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outlet with electrical connection ensured using a salt bridge constructed by filling a 0.5 cm

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long, 250 μm I.D capillary with 3% w/w agarose prepared in 3 mM disodium phosphate

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buffer. Droplets containing the separation effluent flowed through another piece of fused

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silica capillary (30 cm long, 150 μm I.D) to the detection window 10.5 cm from the micro

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cross. For experiments with fluorescein and calcein, a blue light emitting diode (LED) at

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wavelength of 470 nm having luminous intensity of 12,000 millicandela (Lucky Light, China)

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was used in combination with 470 nm and 525 nm band pass filters (25 mm diameter,



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Chroma, Vermont, United States) for excitation and emission, respectively. For detection of

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the oxidised PSTs, a Helium-Cadmium laser (He-Cd, Melles Griot, California, United States)

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was used at ƛex = 330 nm, ƛem = 390 nm using a 325 nm He-Cd laser clean-up filter and 390-

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40 nm band pass filters (25 mm diameter, Semrock, New York, United States). The

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fluorescence detector was constructed using a 5-port manifold (Upchurch Scientific, United

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States), the blue LED or He-Cd laser spot was aligned to on-column detection window via

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two fibre optics (400 µm for excitation, 600 µm for emission; Ocean Optics, Florida, United

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States)23. Fluorescence was measured by a photomultiplier tube (Hamamatsu, Japan) and

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the emitted light was collected using an Agilent 35900E analogue-to-digital convertor

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(Agilent Technologies, Waldbronn, Germany). Separation and outlet capillaries were rinsed

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with Milli-Q water and BGE by applying pressure of 2 bar to the inlet vial before and in

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between analyses. Samples were electrokinetically injected at 5 kV for 25 s and separations

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were performed at 25°C, unless otherwise specified. Fluorescein and calcein were separated

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using a voltage of +30 kV (field strength of 530 V/cm), while the PSTs were separated using

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+25 kV (field strength of 440 V/cm).

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Droplet microfluidics post-column oxidation

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The oxidant consisted of 5 mM periodic acid, 100 mM phosphoric acid and adjusted to pH

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7.8 with 5 M NaOH24 was delivered as aqueous phase segmented by oil to the micro cross.

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Droplet formation was initiated directly after electrokinetic injection of sample.

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191

RESULTS AND DISCUSSION


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Characterisations of droplets and CE-droplet microfluidic interface

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The aim of the current study is to electrophorese analytes into preformed droplets for

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compartmentalisation to maintain resolution during post-column reactions. To do this in a

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no or low EOF environment, the electrical connection needs to be maintained across the

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segmented flow. This is fundamentally different from all previous studies in which the

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electric field was terminated after separation and prior to droplet generation21,22. These

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works also required liquid flow through the microchannel or the addition of a make-up flow

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for droplet formation. Here, we choose to terminate the electric field after

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compartmentalisation into the droplets (illustrated in Fig.1a) with electrical connection

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established within the CE-droplet microfluidics interface with a salt bridge connected to a Pt

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electrode positioned directly opposite the separation capillary. This was constructed using a

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commercial micro cross, with droplets formed upstream in a tee connector from flows of

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decanol (the oil phase) and buffer or reagent (aqueous phase). The salt bridge-electrode and

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separation capillary were positioned opposite to each other to provide a direct conducting

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pathway during compartmentalisation. A micro cross with bigger through hole (i.e. 500 µm)

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can also be used, although the larger droplets result in a greater dilution of the effluent.

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Critical to this form of compartmentalisation is the formation of droplets with an

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appropriate frequency and maintenance of the electric current.

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To test this idea, the formation of droplets was examined at different flow rates and with

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the separation capillary filled with BGE (3 mM phosphate buffer pH 7.8) whilst the droplets

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were formed from BGE spiked with 1 µg/mL fluorescein. Fig.2a shows the fluorescence

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signal when an electric field (field strength of 530 V/cm) was applied over the separation

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capillary, with each peak corresponding to a droplet passing the detection window. The flow



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rate for the oil was varied from 2.4-8 µL/min, for the aqueous phase from 0.6-2 µL/min,

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resulting in a total flow rate of 3-10 µL/min, at an oil : aqueous flow rate ratio of 4. These

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translate into droplet frequencies of approximately 0.7-3.7 Hz. At a flow rate of 3 µL/min,

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formation of droplets was inconsistent as evidenced by irregular peak width and spacing. At

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flow rates from 4-7µL/min, regularly spaced consistent peaks were observed. At flow rates

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from 10 µL/min, droplet formation at this large scale was compromised, with droplets

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partially merging, as evidenced by the irregular fluorescence signal. Consistent droplet

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formation is desired to ensure efficient compartmentalisation, thus only droplets formed at

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4-7 µL/min should be used. The repeatability of droplet formation at 4-7 µL/min

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(percentage relative standard deviations (%RSD), n = 3) for intra-day variations in droplet

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frequency were < 8.1%. The physical length of the droplets ranged from 730-810 µm (n =3,

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< 2.7% RSD for intra-day variation) by digital microscope imaging; thus each droplet had a

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volume of approximately 14 nL.

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As can be seen in Fig.2b during the CE-droplet experiments using 3 mM disodium phosphate

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(pH 7.8), a current of 2.0 µA was generated when applying a voltage of 30 kV. The current

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was maintained at 2.0 µA throughout the experiments, indicating stable electrical

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connection when droplets were delivered at both low and high flow rates. The reliability of

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electrical connection at 4 µL/min calculated by %RSD (n = 5) for inter-day variation was

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2.5%. While stable electrical connection was obtained using low conductive BGE

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(conductivity value of 0.030 S/m), it was unstable or absent when using relatively high

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conductivity BGEs (conductivity values > 0.10 S/m) at the same field strength. To investigate

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this occurrence, experiments were conducted using a fluorescence microscope to visualise

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the interface using a low and high conductivity BGE. When using a high conductivity BGE,


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and hence high current, bubbles were generated in the CE-droplet interface once the

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voltage was applied, followed by visual observation of liquid leakage from the micro cross.

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We believe that the salt bridge ruptured due to the aggressive bubble formation, exposing

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the Pt electrode to a non-conductive oil film, eventually terminating the electrical

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connection and triggering liquid leakage. This limitation may be overcome by downscaling

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and purpose-engineering the droplet generation and interface.

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CE-droplet compartmentalisation

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The performance of the CE-droplet microfluidics system to compartmentalise/collect the

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electrophoresed analytes was examined with the CZE separation of fluorescein and calcein.

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Droplets were delivered at 4 µL/min, giving droplet frequency of 1 Hz for

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compartmentalisation. From the detection trace in Fig. 3a, the droplets containing just BGE

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had a lower background signal than the oil phase, which we consider is due to the difference

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in refractive index of the water and decanol resulting in more excitation light reaching the

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emission fibre with decanol. The fluorescence signals for both fluorescent dyes were higher

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than the background and resulted in positive peaks. To compare the data with on-capillary

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detection (detection window on the separation capillary), an electropherogram was

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reconstructed

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electropherogram of Fig.3a is given in Fig.3b, while Fig.3c is with on-capillary detection. To

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compensate for the different detector position between the two setups, a mobility scale

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was used instead of a timescale25. The insert in Fig. 3a shows that the fluorescein peak was

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compartmentalised into approximately 50 droplets, which was well over the minimum 8-10

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droplets usually needed to preserve peak shape20. The variance in effective mobility

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between CE-droplet microfluidics and on-capillary detection setups was less than 6.5%,

using

the

signal

obtained

in

the

droplets.


The

reconstructed


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indicating stable pressure and electrical connection during injection, and successful

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separation and compartmentalisation when compared with on-capillary detection. Peak

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widths of the compartmentalised fluorescein and calcein increased 4 and 1.2-times,

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respectively, when compared to the same analyte peaks with on-capillary detection,

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indicating zone broadening. The LODs (calculated at S/N = 3) for fluorescein and calcein

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using CE-droplet compartmentalisation were 2.2 and 45 µg/mL compared with 0.22 and 5.1

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µg/mL using on-capillary detection. These are 10 and 9-times higher, although are lower

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than anticipated based purely on dilution in the droplet and is due to the longer detection

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pathlength in the droplet system (150 µm) than the on-capillary detection system (25 µm).

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The commercially available tee connector with 250 µm thru-holes used in our droplet setup

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formed droplets with a volume of approximately 14 nL – 20-47 times larger than would be

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encountered in microchip formats21,22. It is envisaged that considerable improvement in

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performance would be achieved by using smaller droplets as the effect of sample dilution

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would be considerably smaller.

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Droplet volume and droplet flow contribute to the dilution effect, therefore we examined

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the relationship of total flow rate and dilution with the results shown in Fig. 4a. When the

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total flow rate was reduced from 10 to 3 µL/min, droplet frequency decreased from 3.7 to

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0.7 Hz, resulting in a higher proportion of the electrophoresed components in each droplet,

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and a corresponding significant increase in detector response. Higher droplet frequencies

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allow for maintenance of resolution: for a minimum of 10 droplets per peak, 2.7 s wide

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peaks could be analysed at 3.7 Hz, whereas the minimal peak width increases to 14 s wide

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when using 0.7 Hz. It can also be seen from the figure that as the flow rate was increased

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that the effective mobility also increased. This is due to an increase in pressure from the


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flowing liquid which counters the EOF from the capillary thus reducing the overall liquid

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flow through the capillary and increasing the effective mobility. Comparison of the signal

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response (Fig. 4b) indicates an increase by 14-times when changing from 10 to 3 µL/min,

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thus the lower droplet frequency will be better for preserving analytical sensitivity.

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To understand the broadening observed in Fig.3, experiments were performed using a

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fluorescence microscope equipped with colour charge-coupled device camera. Images were

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acquired during the experiment to observe the movement of droplets into the micro cross

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during application of high voltage. Fig. 5 shows a series of screen shots of fluorescein-doped

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droplets through the micro cross interface with an electric field strength of 530 V/cm. As

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can be seen from Fig.5a, a fraction of the fluorescent aqueous droplet remained stationary

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in front of the salt bridge-electrode to coalesce with a subsequent droplet (Fig.5b-c). The

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fluorescent droplet facilitated the conducting pathway from the separation capillary to the

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salt bridge and Pt electrode to close the electric circuit. Due to differences in wettability, the

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aqueous phase wets the salt bridge, preventing the salt bridge from being covered with a

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film of oil that would disrupt the electrical circuit due to oil’s insulating properties.

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Experiments using the Pt electrode directly and no salt bridge did not produce a stable

300

current which we believe is due to the formation of an oil coating on the electrode surface.

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Thus, the hydrophilicity of the salt bridge allows for preferential wetting with aqueous

302

phase and as such provides a continuous electrical pathway. While this assisted in achieving

303

the electrical connection required for electrophoresis, the capture and coalescence of

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droplets visualised in Fig.5 is undesirable and attributes to the loss in resolution as part of

305

the electrophoresed analyte remains behind in the interface to leech back into the

306

subsequent droplet(s), compromising compartmentalisation. While we consider this



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accounted for a major component of the decreased efficiency, there is also likely to be a

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small hydrodynamic flow from the separation capillary outlet that will also induce

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broadening, although this is minimised by using a 25 µm I.D capillary. Fabrication of a salt

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bridge in a microfluidic device positioned at the end of the flow-through channel should

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overcome the droplet capture and mixing issue and will preserve the efficiency better.

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CE with droplet-based microfluidics post-column reaction: Paralytic shellfish

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toxins as model analytes

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To demonstrate the droplet approach for post-column reaction in CE, the detection of

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paralytic shellfish toxins (PSTs) is chosen as a challenging application. PSTs are natural

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marine toxins produced by dinoflagellates during algal blooms that accumulate in filter-

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feeding shellfish, fish or plankton. Consumption of PSTs-contaminated shellfish may result in

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respiratory arrest and cardiovascular shock that may lead to death26. PSTs are natively non-

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fluorescent but yield fluorescent products upon oxidation24,27. Pre-column oxidation with

321

HPLC-FLD was the first official method used for PSTs detection, however, some toxins

322

produced more than one fluorescent oxidation product and identical fluorescent oxidation

323

products are formed from some of the toxins27, compromising selectivity and specificity. To

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maintain selectivity, the alternative official method uses post-column oxidation and is

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preferred24, although a post-column reactor setup is needed. Both methods offer detection

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limits in the low ng/mL range for the analysis of PSTs. Recently, we evaluated four different

327

detection methods for PSTs analysis by CE28. FLD with pre-column oxidation was the most

328

sensitive, although micellar electrokinetic chromatography (MEKC) separation was required

329

to separate the neutral oxidation products. The CE method suffered from the same loss in


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330

selectivity due to the multiple, overlapping oxidation products reported for the HPLC

331

method with precolumn oxidation. In this work, droplets containing periodic acid were used

332

for compartmentalisation of the electrophoretically separated analytes for oxidisation, thus

333

providing the sensitivity of FLD with the selectivity of CZE. It was however not possible to

334

use our previously developed CZE BGE because it had too high ionic strength to provide a

335

stable current.

336

In HPLC with post-column oxidation, the separation of PSTs usually takes ~30 min and

337

reaction times typically between 1 to 2 min were used depending on the flow rate of

338

oxidant and the length of the reaction coil24. Given that enhanced mixing in droplets

339

improves reaction rates18, we anticipated shorter reaction time in the droplet oxidation

340

system. We examined the effect of the reaction time on the fluorescence intensity by

341

changing the distance of the detection window from the micro cross. Distance of 10.5, 20.5,

342

25.5and 30.5 cm were used, corresponding to oxidation times of approximately 14, 16, 18

343

or 20 s when the droplets were delivered at total flow rate of 4 µL/min (1 Hz). No significant

344

difference in fluorescence was recorded, therefore the shortest time of 14 s was selected as

345

the reaction time. Shorter or longer times were not examined due to the practical difficulty

346

of positioning the detector closer or further to the micro cross. Fig.6a-b show the droplet

347

trace and reconstructed electropherograms for NEO and dcGTX2 with the detection window

348

positioned 10.5 cm from the cross. NEO and dcGTX2 have an effective mobility of 41 and

349

21 x 10-9 m2/Vs, respectively, and were separated and detected within 10 min. The

350

performance of CZE-droplet post-column oxidation method was evaluated in terms of

351

linearity of the detector response, LODs, intra-day and inter-day precisions (Table 1). The

352

linear ranges for NEO and dcGTX2 were between 670-10,000 ng/mL (regression coefficient



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Page 16 of 27

353

(R2) > 0.982) and between 430-20,000 ng/mL (R2> 0.980), respectively. The LODs for NEO

354

and dcGTX2 were 670 ng/mL and 430 ng/mL, respectively. These values are approximately

355

11 and 7-times higher than previously reported with pre-column oxidation with on-capillary

356

detection28 (detection window on the separation capillary), and consistent with the 9-10

357

times drop in sensitivity for calcein and fluorescein as a consequence of dilution in the

358

droplets. This suggests that comparable oxidation (in term of fluorescent yield) between our

359

previous pre-column oxidation (3 min) and our current post-column oxidation (14 s) was

360

achieved, with droplet post-column oxidation oxidising only one toxin at a time, avoiding

361

the issues around multiple, overlapping and identical oxidation products complicating

362

identification following pre-column oxidation. While the detection limits were only

363

marginally improved when compared to UV detection (Fig. 6c), oxidation and fluorescence

364

increases specificity of detection allowing more reliable and accurate identification of toxin

365

peaks. To be applicable to the analysis of PST content in shellfish samples, it is necessary to

366

be able to detect toxins lower than the regulatory limit of 800 ng/mL27, thus the sensitivity

367

of the methods needs to be further improved. This could be achieved by various stacking

368

approaches, such as the transient isotachophoresis (tITP) method we have previously

369

developed for capacitively coupled contactless conductivity detection (C4D)29, and/or

370

through reduction of

371

compartmentalisation.

the

droplet

size

to reduce

the

dilution factor

upon

372

373

CONCLUSIONS

374

We demonstrate that droplet microfluidics can be used to compartmentalise

375

electrophoretically transferred analytes for post-column reaction in CE. The use of a PEEK™


Page 17 of 27

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376

micro cross for positioning a salt bridge across the droplet flow from the separation capillary

377

outlet enables the compartmentalisation/collection of the electrophoretically transferred

378

analytes while maintaining the electrical connection. Positioning the droplets in the electric

379

field eliminated the need for EOF or hydrodynamic flow and allowed electrophoresing

380

effluents into the droplets. We show that the system could be successfully applied to the

381

post-column oxidation of PSTs, allowing for the combination of the selectivity of CZE with

382

the sensitivity of FLD. We believe this new approach for post-column droplet collection in CE

383

can be very powerful when miniaturised further to reduce the droplet size which will reduce

384

the dilution factor and enable a range of post-separation processing to be performed.

385

386

ACKNOWLEDGEMENTS

387

The authors would like to thank the Australian Research Council, ARC QEII Fellowship

388

(DP0984745) and Future Fellowship (FT130100101) awarded to M.C.B and University of

389

Tasmania for funding support. We acknowledge scholarships from the Ministry of Education

390

Malaysia (MOE) and Universiti Teknologi Malaysia for A.S.A.K. The authors would like to

391

acknowledge the expertise and support from Mr. John Davis, Mr. Paul Waller and Mr. Chris

392

Young of the Central Science Laboratory, University of Tasmania. A.S.A.K would like to

393

acknowledge Mr. Burhanuddin Mohamad for his help on post-processing fluorescence

394

images using Adobe® Photoshop® and Adobe® Lightroom® softwares.



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REFERENCES

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(1) Albin, M.; Weinberger, R.; Sapp, E.; Moring, S. Anal. Chem.1991, 63, 417-422.

398

(2) Zhu, R.; Kok, W. T. J. Chromatogr. A1995, 716, 123-133.

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(3) Rezenom, Y. H.; Lancaster Iii, J. M.; Pittman, J. L.; Gilman, S. D. Anal. Chem.2002, 74, 1572-1577.

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(4) Nickerson, B.; Jorgenson, J. W. J. Chromatogr. A1989, 480, 157-168.

401

(5) Emmer, Å.; Roeraade, J. J. Chromatogr. A1994, 662, 375-381.

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(6) Feltus, A.; Hentz, N. G.; Daunert, S. J. Chromatogr. A2001, 918, 381-392.

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(7) Oldenburg, K. E.; Xi, X.; Sweedler, J. V. Analyst1997, 122, 1581-1585.

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(8) Nirode, W. F.; Staller, T. D.; Cole, R. O.; Sepaniak, M. J. Anal. Chem.1998, 70, 182-186.

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(9) Ye, M.; Hu, S.; Quigley, W. W. C.; Dovichi, N. J. J. Chromatogr. A2004, 1022, 201-206.

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(10) Dada, O. O.; Huge, B. J.; Dovichi, N. J. Analyst2012, 137, 3099-3101.

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(11) Liu, F.; Zhang, L.; Qian, J.; Ren, J.; Gao, F.; Zhang, W. Analyst2013, 138, 6429-6436.

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(12) Yamamoto, D.; Kaneta, T.; Imasaka, T. Electrophoresis2007, 28, 4143-4149.

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(13) Xiao, Z.; Niu, M.; Zhang, B. J. Sep. Sci.2012, 35, 1284-1293.

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(14) Edgar, J. S.; Pabbati, C. P.; Lorenz, R. M.; He, M.; Fiorini, G. S.; Chiu, D. T. Anal. Chem.2006, 78,

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6948-6954.

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(15) Niu, X.; Pereira, F.; Edel, J. B.; de Mello, A. J. Anal. Chem.2013, 85, 8654-8660.

413

(16) Zhao, Y.; Pereira, F.; deMello, A. J.; Morgan, H.; Niu, X. Lab Chip2014, 14, 555-561.

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(17) Song, H.; Tice, J. D.; Ismagilov, R. F. Angew. Chem. Int. Ed.2003, 42, 768-772.

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(18) Song, H.; Chen, D. L.; Ismagilov, R. F. Angew. Chem. Int. Ed.2006, 45, 7336-7356.

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(19) Ji, J.; Nie, L.; Qiao, L.; Li, Y.; Guo, L.; Liu, B.; Yang, P.; Girault, H. H. Lab Chip2012, 12, 2625-2629.

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(20) Nie, J.; Kennedy, R. T. J. Sep. Sci.2013, 36, 3471-3477.

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(21) Edgar, J. S.; Milne, G.; Zhao, Y.; Pabbati, C. P.; Lim, D. S. W.; Chiu, D. T. Angew. Chem. Int.

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Ed.2009, 48, 2719-2722.

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(22) Draper, M. C.; Niu, X.; Cho, S.; James, D. I.; Edel, J. B. Anal. Chem.2012, 84, 5801-5808.


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(23) Huo, F.; Guijt, R.; Xiao, D.; Breadmore, M. C. Analyst2011, 136, 2234-2241.

422

(24) AOAC. Ofﬁcial Method 2011.02 First Action 2011: Paralytic shellﬁsh toxins in mussels, clams,

423

scallops and oysters by liquid chromatography post-column oxidation; AOAC International:

424

Gaithersburg,MD, 2012.

425

(25) Schmitt-Kopplin, P.; Garmash, A. V.; Kudryavtsev, A. V.; Menzinger, F.; Perminova, I. V.;

426

Hertkorn, N.; Freitag, D.; Petrosyan, V. S.; Kettrup, A. Electrophoresis2001, 22, 77-87.

427

(26) Rossini, G. P.; Hess, P. EXS2010, 100, 65-122.

428

(27) AOAC. Official Method 2005.06 Paralytic shellfish poisoning toxins in shellfish.Pre-

429

chromatographic oxidation and liquid chromatography with fluorescence detection; AOAC

430

International: Gaithersburg, MD, 2005.

431

(28) Keyon, A. S. A.; Guijt, R. M.; Gaspar, A.; Kazarian, A. A.; Nesterenko, P. N.; Bolch, C. J.;

432

Breadmore, M. C. Electrophoresis2014, 35, 1496-1503.

433

(29) Keyon, A. S. A.; Guijt, R. M.; Bolch, C. J.; Breadmore, M. C. J. Chromatogr. A.

434 435

436

437

438

439



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440

FIGURES

441

442

CE-droplet microfluidics system (A) Schematic outline of the setup used for post-

443

Fig.1

444

column compartmentalisation incorporated in a CE instrument. The aqueous phase was

445

segmented by oil in the tee connector (outside CE instrument) and passed the separation

446

capillary outlet to collect the electrophoresed effluent in a compartmentalised manner

447

(within CE instrument). (B) Photograph of the micro cross as the droplet microfluidics

448

interface positioned in a capillary cassette. Photo label: connection for (1) droplet capillary

449

(2) separation capillary (3) detection capillary and (4) salt bridge electrode.


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450

451 452

Characterisations of droplets: (A) Fluorescence signal of a selected 6 s window for

453

Fig.2

454

aqueous droplets doped with1µg/mL fluorescein in the presence of electric field (30 kV,

455

field strength of 530 V/cm). Each peak corresponds to an individual droplet. Total flow rate

456

of droplet formation was varied from 4-10 µL/min. (B) Current readout during experiments

457

conducted in Fig.2a.



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Page 22 of 27

458

Separation of fluorescein and calcein mixture; (A) Electropherogram from CE-droplet

459

Fig.3

460

microfluidics

461

electrophoresed analytes into droplets. The insert showed the zoomed in portion of

462

fluorescein peak. Mixture of 230 µg/mL fluorescein and 1900 µg/mL calcein was injected at

463

5 kV for 25 s, while droplets were delivered at4 µL/min (1 Hz). (B) Reconstructed

464

electropherogram from Fig. 3a. (C) Electropherogram from on-capillary detection. Mixture

465

of 19 µg/mL fluorescein and 190 µg/mL calcein was injected at 5 kV for 25 s. Peak

466

identification: (1) fluorescein and (2) calcein. In both experiments, BGE 3 mM disodium

467

phosphate (pH 7.8) and electric field strength of 530 V/cm were used.

system

demonstrating

separation

and


compartmentalisation

of

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468

469 470

Fig. 4 The relationship of droplet flow and dilution; (A) the electropherograms showing

471

fluorescein signal change as a response to droplet frequency. Insert in Fig.4a is the diluted

472

fluorescein (dilution factor of 5) compartmentalised at 3 µL/min, shown to compensate the

473

overloaded signal in main figure. (B) Plot of signal and droplet frequency as functions for

474

total flow rate of oil and aqueous phases. Experiment was conducted by injecting230 µg/mL

475

fluorescein at 5 kV for 25 s. The BGE and field strength used as indicated in Fig. 3.



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

476

Fluorescence images of droplets in the micro cross in electric field strength of 530

477

Fig.5

478

V/cm. The droplets contained BGE doped with fluorescein and were delivered at a total flow

479

rate 4 µL/min (1 Hz); (A) A fraction from the droplet emanating from the droplet capillary (1)

480

was trapped in front of salt bridge-electrode (4). (B-C) The subsequent droplet coalesced

481

with the trapped droplet when passing through the cross. (D) The droplet left the cross

482

flowing into the outlet capillary (3).

483

484

485

486


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487

488

489

490

491

492

493

494

495

Separation of PSTs mixture; (A) Electropherogram from CZE-droplets microfluidics

496

Fig.6

497

system demonstrating separation and compartmentalisation of electrophoresed analytes

498

into preformed droplets for oxidation. (B) Reconstructed electropherogram from Fig. 6a. (C)

499

Electropherogram from CZE-UV. Peak identification: (1) NEO and (2) dcGTX2. In both

500

experiments, BGE 20 mM formic acid pH 2.8 and electric field strength of 440 V/cm were

501

used. Mixture of 10,000 ng/mL NEO and 20,000 ng/mL dcGTX2 was injected at 5 kV for 25 s.



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Page 26 of 27

502

TABLES

503

Table 1 Performance of CZE-droplet post-column oxidation method for PSTs. Toxin LOD (ng/mL) NEO dcGTX2

670 430

Intra-day, (%RSD, n = 3) µeff a Height

Inter-day, (%RSD, n = 3) µeff a Height

8.0 2.5

8.8 5.5

2.9 8.6

504

505

a)

µeff is effective mobility

506


11 5.2

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507

508

509

Graphical Abstract

510 511


Droplet Microfluidics for Postcolumn Reactions in ... - ACS Publications

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