Multistacking from Two Sample Streams in Nonaqueous Microchip

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Multi-stacking from two sample streams in nonaqueous microchip electrophoresis Lee Yien Thang, Hong Heng See, and Joselito P Quirino Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.6b02790 • Publication Date (Web): 26 Sep 2016 Downloaded from http://pubs.acs.org on September 27, 2016

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Analytical Chemistry

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Multi-stacking from two sample streams in nonaqueous microchip electrophoresis

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Lee Yien Thanga,b, Hong Heng Seea,b,*, Joselito P. Quirinoa,b,c,*

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a

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Research, UniversitiTeknologi Malaysia, 81310 UTM Johor Bahru, Johor, Malaysia

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b

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Johor Bahru, Johor, Malaysia

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c

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Chemistry, University of Tasmania, 7001 Hobart, Tasmania, Australia

Centre for Sustainable Nanomaterials, IbnuSina Institute for Scientific and Industrial

Department of Chemistry, Faculty of Science, UniversitiTeknologi Malaysia, 81310 UTM

Australian Centre for Research on Separation Science, School of Physical Sciences –

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Corresponding author,

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* Joselito P. Quirino, E-mail: [email protected]

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Australian Centre for Research on Separation Science, School of Physical Sciences –

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Chemistry, University of Tasmania, 7001 Hobart, Tasmania, Australia

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Tel: +61 448 122 456; Fax: +61 3 6266 2858

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Additional corresponding author,

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* Hong Heng See, E-mail: [email protected]

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Centre for Sustainable Nanomaterials, IbnuSina Institute for Scientific and Industrial

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Research, UniversitiTeknologi Malaysia, 81310 UTM Johor Bahru, Johor, Malaysia

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Tel: +60 7 553 6270; Fax: +60 7 553 6080

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ACS Paragon Plus Environment

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Abstract

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The translation of stacking techniques used in capillary electrophoresis (CE) to

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microchip CE (MCE) in order to improve concentration sensitivity is an important area of

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study. The success in stacking relies on the generation and control of the stacking boundaries

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which is a challenge in MCE because the manipulation of solutions is not as straightforward

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as in CE with a single channel. Here, a simple and rapid on-line sample concentration

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(stacking strategy) in a battery operated nonaqueous MCE device with a commercially

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available double T-junction glass chip is presented. A multi-stacking approach was

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developed in order to circumvent the issues for stacking in nonaqueous MCE. The cationic

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analytes from the two loading channels were injected under field-enhanced conditions and

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were focused by micelle-to-solvent stacking. This was achieved by the application of high

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electric fields along the two loading channels and a low electric field in the separation

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channel, with one ground electrode in the reservoir closest to the junction. At the junction,

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the stacked zones were re-stacked under field-enhanced conditions and then injected into the

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separation channels. The multi-stacking was verified under a fluorescence microscope using

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Rhodamine 6G as the analyte, revealing a sensitivity enhancement factor (SEF) of 110. The

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stacking approach was also implemented in the nonaqueous MCE with contactless

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conductivity detection of the anticancer drug tamoxifen as well as its metabolites. The multi-

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stacking and analysis time was 40 s and 110 s, respectively, the limit of detections was from

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10 to 35 ng/mL, and the SEFs were 20 to 50. The method was able to quantify the target

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analytes from breast cancer patients.

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Introduction

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The ability of capillary electrophoresis (CE) to handle analyses of various analytes at

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relevant levels in real samples or even to trace analyses is due to innovative offline sample

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preparation methods1-4 and more interestingly due to the advancements in stacking.5-8

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Stacking is generally performed by changing the solution chemistry of the sample to affect

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the enrichment of analytes injected into a narrow band inside the separation channel.

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Stacking techniques in electrophoresis using capillaries have remained popular but are not as

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popular when using the microchip format.9 This is primarily due to the difficulty in the

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generation and control of the stacking boundaries in MCE reservoirs and channels. The

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development of stacking strategies in commercially available microchips and MCE devices

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will increase the application of MCE assays to a wide variety of applications.

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Stacking in microchip CE (MCE) was first demonstrated by Jacobson and Ramsey10

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using field enhancement in capillary zone electrophoresis in 1995 and a year later by

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Thormann and co-workers11 in micellar electrokinetic chromatography. Field-enhanced

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stacking is achieved by using a sample of a much lower conductivity than the background

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electrolyte (BGE).12,13 Recent work on the use of field-enhanced stacking in MCE included

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the analysis of amino acids and vitamin B3 in functional drinks,14 bacteria in surface water,15

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inorganic anions and oxalate in atmospheric aerosols16 and oligosaccharides from bovine

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ribonuclease B.17 Other stacking mechanisms such as sweeping,18 isotachophoresis (ITP)19

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and dynamic pH junction20 have been employed in MCE. For example, sweeping for the

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analysis of oestrogen and oestrogen binding21 and dyes,22 ITP for increasing the sensitivity of

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DNA microarray hybridization23 and of the cardiac biomarker troponin I,24 and dynamic pH

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junction for the analysis of mono- and di-saccharides.25 An advantage of using planar


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microchips is the visualization of stacking along the channels with the aid of a microscope

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and fluorescence. This has provided many insights on the focusing mechanism involved.22,26-

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28

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limited number of electrolytes and additives that can be used and the low conductivity of the

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BGE since the diluent is an organic solvent.

The implementation of stacking in nonaqueous MCE is however difficult because of the

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In this work, we demonstrate for the first time multi-stacking by field-enhanced

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stacking and micelle-to-solvent stacking (MSS) in nonaqueous MCE using a commercial

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microchip and MCE device. This multi-stacking resulted to significant improvement in

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sensitivity compared to standard gated injection.

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conducted by the appropriate positioning of sodium dodecyl sulfate (SDS) micellar solution,

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nonaqeous BGE, and sample solution into the channels and reservoirs in a standard double T

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microchip. This allowed the generation and control of the stacking boundaries when the

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voltages were applied. The field-enhanced sample injection (FESI)29 or electrokinetic

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injection of analytes in low conductivity sample diluent (acidified methanol (MeOH)) from

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two sample reservoirs was achieved by the proper control of the electric field strengths along

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the three channels of a double T-junction microchip. MSS30,31 which was based on the

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effective electrophoretic mobility reversal of a charged analyte was invoked by filling the

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microchip channels with the SDS micellar solution. Stacking was clearly confirmed using

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fluorescence microscopy in the microchip and was applied to real sample analysis using

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nonaqueous MCE with a contactless conductivity detector (C4D).

Multi-stacking and separation was

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Experimental section

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MCE device set-up

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Fig. 1 illustrates the experimental set-up. The high voltage sequencer and C4D were

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powered by a new in-house 12-volt DC output NiMH rechargeable battery pack (RS Pro,

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Corby, UK). The microchip, MCE platform, C4D elements and high voltage sequencer were

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obtained from eDAQ (Denistone East, NSW, Australia). The sequence and data acquisition

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were controlled by Sequencer and PowerChrom® software from eDAQ. The microchip had

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channels with a cross-section of 100 µm width and 10 µm depth. The microchip consists of

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separation and loading channels (L1 and L2), reservoirs (R1, R2, R3, R4) and embedded

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electrodes for C4D. The embedded electrodes (measuring and reference) measured 200 µm ×

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500 µm and were 200 nm thick. They lay parallel to each other in the microchip, separated by

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a distance of 2.9 mm. The microchip was placed on the MCE platform and connected to the

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high voltage sequencer and C4D data system. The electrodes for stacking and separation were

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attached to the reservoirs.

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Reagents and materials

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Ethyl acetate (EtOAc), glacial acetic acid, MeOH, and 2-propanol (IPA) were

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purchased from Merck (Darmstadt, Germany). Deoxycholic acid sodium salt (NaDCHA),

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Rhodamine 6G, SDS, and sodium hydroxide (NaOH) were obtained from Sigma-Aldrich

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(Buchs, Switzerland). 4-(dimethylamino)pyridine and 18-crown-6 were purchased from

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Fluka (Buchs, Switzerland). Standard drugs tamoxifen, N-desmethyltamoxifen, 4-hydroxy-N-

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desmethyltamoxifen (1:1 E/Z mixture), and (Z)-4-hydroxytamoxifen were purchased from


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Toronto Research Chemicals (Toronto, Canada). Stock solution of 1 µg/mL Rhodamine 6G

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was prepared in MeOH. Stocks of solutions of each drug at a concentration of 500 µg/mL

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were prepared in MeOH and kept at 4°C. Working standard solutions at lower concentrations

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were prepared by dilution in acidified MeOH (0.1mM acetic acid in MeOH) in order to

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ionize the analytes.

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The BGE for visualization using Rhodamine 6G as probe was 150 mM acetic acid in

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MeOH. Acetic acid was widely employed as a BGE in CE analyses with C4D especially

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when the low pH buffer range was required.32-34 The BGE for the anti-cancer drugs analysis

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was prepared by mixing respective amount of sodium deoxycholate with acetic acid and 18-

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crown-6 in methanol.35 The micellar solution was 10 mM SDS in 50 mM acetic acid.

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Ultrapure water from Milipore (Bedford, MA) was used for the rinsing of microchannels. All

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solutions were filtered through the 0.2µm PTFE syringe filters prior to introduction into the

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microchannels.

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General and multi-stacking MCE procedures

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A new microchip was activated by manually flushing with 0.5 M NaOH solution for 2

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min, followed by water for 5 min, and separation media for 5 min. The microchip was

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flushed with separation media for 2 min in between runs.

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The typical injection of anti-cancer drugs was carried out with 50 µL of BGE in all

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reservoirs except in R2, which received 50 µL of sample solution. A constant flow of sample

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solution from R2 to R1 was achieved by application of 900 V at R2, grounded at R1. The

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BGE in the separation channel were maintained by application of 1000 V at R3, grounded at


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R4. The sample at the junction was injected by stopping the voltage applied at R3 for 1 s.

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Separation was achieved by application of positive voltage at R2, grounded at R4. For multi-

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stacking, the separation channel was filled with micellar solution and R2 and R4 with 50 µL

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BGE. The sample solution (50 µL) was then loaded into R1 and R3. The stacking occurred

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when a positive voltage of 250 V was applied at R1 and R3 while 70 V was applied at R4.

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The ground was at R2. After a certain focusing time, a positive separation voltage of 1000 V

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was then applied at R2 and ground at R4.

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The visualization of multi-stacking was done using an inverted fluorescence

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microscope AF4115-GFBW (Dino-Lite, Hsinchu, Taiwan) that was focused into the inlet

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section of the microchip with excitation and emission wavelength of 480 nm and 510 nm,

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respectively. The separation channels were filled with micellar solution and R2 and R4 with

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50 µL BGE. The sample solution (50 µL) was then loaded into R1 and R3. A positive voltage

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of 250 V was applied from time = 0 s at R1 and R3 while 70 V was applied at R4. The

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ground was at R2. A positive voltage of 1000 V was then applied at time = 20 s at R2 and

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ground at R4.

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Results and discussion

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Fig. 2 shows snapshots taken at 15 s (a), 18 s (b), 19 s (c), 20.2 s (d), 20.4 s (e) and

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20.8 s (f) during the multi-stacking and injection of cationic Rhodamine 6G.

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Experimental section for the starting situation. After time = 0 s, the micelles from the

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separation channel between the double-T junction and R2 migrated towards L1 or L2 while

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those between the junction and R4 migrated towards R4. Rhodamine 6G was

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electrokinetically introduced under field-enhanced conditions (FESI) from two sample


Please see

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streams R1 and R3 and was stacked on the MeOH-rich side of the solvent and concentration

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boundaries (SCB) via MSS. The SCBs migrated with the EOF that moved towards the

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junction from R1 or R3. The FESI were achieved by dissolving the analytes in acidic MeOH.

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The MeOH-rich matrix entered the L1 and L2 channels from R1 and R3, respectively. The

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electrophoretic velocity of the cationic dye (v) in the MeOH zone is equal to equation 1.

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= .

(1)

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µ is the electrophoretic mobility of the dye and E(LMeOH) is the electric field strength in the

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MeOH zone inside L1 or L2. The high E(LMeOH) allowed a large number of dye molecules to

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be injected. The SDS micelles from L1 and L2 electrophoretically migrated towards R1 and

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R3, respectively. The injected molecules entered the micellar zones where the velocity was

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approximated by the effective electrophoretic velocity, veff, given by equation 2.

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= +

(2)

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k is the retention factor, µmc is the electrophoretic mobility of SDS micelles and E(Lmc) is the

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electric field strength in the micellar zone inside L1 or L2. The νeff is anodic and thus the

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dyes in the micellar zone electrophoretically migrated to the MeOH-rich zone but at a low

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velocity due to the low E(Lmc). The micelles that carried the analytes collapsed due to a

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sudden increase in the critical micelle concentration in the MeOH-rich zone adjacent to the

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SCB. The k value in equation (2) became zero and the electrophoretic velocity of the dye

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reverted to equation (1). The dyes were released and eventually stacked in the MeOH-rich

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zone of the SCB and this stacking by MSS is shown in Fig. 2a.

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The total electric field across L1 was higher than in L2 because the distance of R1 to

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R2 (ground) was smaller than R3 to R2 due to the 1 mm junction in the separation channel.

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Therefore, the SCB and the stacked zone from R1 entered the junction before the SCB from

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R3, as shown in Fig. 2b. The SCB from R3 then entered the junction while the stacked zone

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from R1 was already in the junction, as shown in Fig. 2c. The sample diluent (MeOH) from

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L1 and the aqueous solution from L2 were also in this filled channel. The concentration of

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MeOH in the junction was approximately 50%, as calculated based on the EOF velocities in

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the channels. There was lowering of the fluorescent intensity of the stacked zone from L2 due

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to dilution at the junction and the depletion of micelles for MSS. At this point, all the

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channels were devoid of micelles as they migrated into the respective reservoirs due to the

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fast electrophoretic mobility of ionised SDS micelles (-4.147 cm2/V.s 36).

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After time = 20 s, another SCB was formed on the cathodic side of the junction, as

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shown in Fig. 2d. The fluorescent zone inside the separation channel was of low conductivity

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due to the increased concentration of MeOH in this channel (approximately 95% MeOH).

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The concentrated analytes that entered the junction and separation channel were re-stacked

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(second step stacking) at this SCB by field-enhanced sample stacking. The electrophoretic

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velocity of Rhodamine 6G was faster on the right side (methanol rich) than the left side (high

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conductivity aqueous zone) of the SCB. The re-stacked analytes then migrated to the detector

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side (left side of the junction), as shown in Fig. 2e and 2f. The analytes introduced through

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the junction that were not re-stacked migrated into L1 and L2.

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The sensitivity enhancement factor (SEF) was calculated by obtaining the fluorescent

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intensity of the initial and stacked zone from the standard calibration of the Rhodamine 6G

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dye. The standard calibration was established by measuring the fluorescent intensity at the


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junction of the separation channel at concentrations ranging from 0.5 to 100 ng/mL. The

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concentration of the final stacked zone was 3.32 ng/mL, which corresponded to a SEF of 110.

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When Rhodamine 6G was prepared in water with 0.1 mM acetic acid, the fluorescence

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intensity of the stacked zones that entered the L1 and L2 was weak. Also, if the BGE was

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used instead of the micellar solution to fill the channels before electrokinetic injection of

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sample, the intensities were also weak. The synergistic effect of MSS on field-enhanced

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sample injection using MeOH was clearly demonstrated in a commercial microchip format.

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Application to analysis of tamoxifen and its metabolites in plasma

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Tamoxifen is a nonsteroidal triphenylethylene anti-oestrogen drug approved by the

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Food and Drug Administration (FDA) for adjuvant treatment against metastatic breast cancer

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that can be either metabolically activated to 4-hydroxytamoxifen (HTF) and 4-hydroxy-N-

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desmethyltamoxifen (endoxifen) by the cytochrome P4502D6 or routed via N-

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desmethyltamoxifen (NDT) to endoxifen racemate E/Z.37-40 The nonaqueous CE BGE with

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sodium deoxycholate in acetic acid and 18-crown-6 in MeOH35 was re-optimized for MCE

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with a much shorter effective separation length. Separation was performed at 1000 V and the

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typical injection (not for stacking) with C4D gave LODs (signal-to-noise ratio of 3) of 0.5,

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0.8, 0.8, 0.3 and 0.7 µg/mL for NDT, endoxifen-i, endoxifen-ii, tamoxifen and HTF,

20

respectively.

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The channels and reservoirs were loaded as in Fig. 2. In order to achieve high SEF,

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the focusing voltage needs to be comprehensively optimized. The low focusing time were

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inadequate for the stacked zone to meet at the junction while the high focusing time may

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cause the stacked zone to move in opposite direction to sample reservoirs. Thus, a series of


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experiments were conducted by examine the focusing voltage of in the range of 250 V, 500 V,

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1000 V and 1500 V at sample reservoirs R1 and R3. There was a change of voltage at R4 in

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accordance with R1 and R3 at 70 V, 150 V, 300 V and 450 V, respectively. R2 was ground.

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The optimum stacking was achieved using a positive voltage of 1000 V at R1 and R3, 300 V

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at R4 and ground at R2 for 40 s. Separation took place at 1000 V at R2 and ground at R4. The

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LODs were 10, 35, 35, 15 and 20 ng/mL for NDT, endoxifen-i, endoxifen-ii, tamoxifen and

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HTF, respectively. The SEF was calculated by dividing the LODs from stacking by the LODs

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from the typical injection. The corresponding SEF was 50, 23, 23, 20 and 35, respectively.

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This optimization was suitable for Rhodamine 6G and other analytes with similar mobilities.

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However, for analytes with different mobilities it is necessary to re-optimize the focusing

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time. The analysis was linear from 0.03–3.5 µg/mL, and the correlation coefficient was above

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0.992 in all cases. The internal standard (IS), 4-(dimethylamino)pyridine was used for

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quantification purpose when plotting a calibration curve by normalization of the peak heights

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obtained for tamoxifen and its metabolites with the peak height of IS versus analyte

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concentration (ng/mL). The repeatability was evaluated according to the variation of peak

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height at a concentration of 0.5 µg/mL. The intraday repeatability %RSD from six

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determinations taken during one day based on peak height and migration time was in the

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range of 1.0−3.4% and 0.5 – 1.4%, respectively. The interday %RSD from six determinations

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each day for 3 days was in the range of 8.3–9.5% and 2.3 – 3.6%, respectively.

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The analysis of tamoxifen and its metabolites in plasma by the proposed method is

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shown in Fig. 3a. Briefly, 300 µL of pooled human plasma spiked with 20 ng/mL of analytes

23

was subjected to liquid-liquid extraction (LLE)35: 300 µL of the plasma sample was placed in

24

a clean tube and 300 µL of 1 % formic acid was added. The tube was vortexed for 30 s. The

25

mixture was then extracted with 900 µL of EtOAc:IPA (95:5; v/v) solvent mixture and


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vortexed for 5 min. The mixture was then centrifuged at 5000 × g for 5 min at 4°C. The

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organic phase was transferred to a clean tube and dried gently under nitrogen flow. The

3

extract was reconstituted with 50 µL of MeOH plus 1 mM acetic acid to make the sample

4

solution. The standard gated injection of 1 µg/mL drug and metabolite standards in MeOH

5

with the reconstitution diluent is shown in Fig. 3b for comparison. The human plasma

6

samples were collected from three women volunteers participating in a clinical study after

7

oral administration of a 20 mg dosage of tamoxifen. The informed consents in written form

8

were obtained from the volunteers prior to sample collection. All plasma samples were kept

9

at –20°C in a freezer and thawed at room temperature prior to pretreatment. The analysis of

10

patient plasma samples using the multi-stacking nonaqueous MCE method revealed 1.47–

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2.50, 0.44–0.89, 0.55–0.68, 1.21–2.40 and 0.56–0.88 µg/mL of NDT, endoxifen-i, endoxifen-

12

ii, tamoxifen and HTF, respectively in three breast cancer patients receiving tamoxifen (see

13

Table S1 in the Supporting Information). The stacking strategy can be directly used with

14

other nonaqueous MCE methods especially for cationic hydrophobic target analytes that are

15

insoluble in water.

16 17

Conclusions

18 19

A novel multi-stacking strategy using two sample streams in nonaqueous MCE with a

20

standard double T-junction microchip was presented. The use of multi-stacking solved the

21

limitations in conducting on-line sample concentration in nonaqueous MCE. Fluorescence

22

microscopy revealed the FESI and focusing by MSS of a cationic dye from two sample

23

reservoirs, the re-stacking of the injected analytes at the junction, and the injection of the

24

final focused zone into the separation channel. Compared to standard gated injection, SEFs of

25

up to 110 were obtained without complicated procedures or modification of a commercially


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available microchip and microchip platform. The multi-stacking in nonaqueous MCE with

2

C4D was then demonstrated for the analysis of anticancer drugs found in plasma at relevant

3

therapeutic levels. Injection from two sample streams may also be re-focused inside the

4

microchip using other stacking mechanisms (i.e., sweeping) and this is currently under study.

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Acknowledgements

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LYT thanks the Universiti Teknologi Malaysia (UTM) for Zamalah scholarship. HHS thanks

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the Ministry of Science, Technology & Innovation Malaysia (MOSTI) (eScience Fund no.:

10

06-01-06-SF1253), Ministry of Higher Education Malaysia (MOHE) (Fundamental Research

11

Grant Scheme (FRGS) no.: R.J130000.7826.4F705), and the Swiss National Science

12

Foundation (Advanced Mobility Grant P300P2_147771). JPQ was supported by an

13

Australian Research Council Future Fellowships (FT100100213). JPQ also thanks the UTM

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for the visiting researcher grant (GUP Tier 1 no.: Q.J130000.2509.09H91).

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References (1) Chen, Y.; Guo, Z.; Wang, X.; Qiu, C. J. Chromatogr. A 2008, 1184, 191-219. (2) Kuehnbaum, N. L.; Britz-McKibbin, P. Chem. Rev. 2013, 113, 2437-2468. (3) Santos, B.; Simonet, B. M.; Rios, A.; Valcarcel, M. Trac-Trends in Analytical Chemistry 2006, 25, 968-976. (4) Alothman, Z. A.; Dawod, M.; Kim, J.; Chung, D. S. Anal. Chim. Acta 2012, 739, 14-24. (5) Simpson, S. L.; Quirino, J. P.; Terabe, S. J. Chromatogr. A 2008, 1184, 504-541. (6) Kitagawa, F.; Otsuka, K. J. Chromatogr. A 2014, 1335, 43-60. (7) Breadmore, M. C.; Tubaon, R. M.; Shallan, A. I.; Phung, S. C.; Keyon, A. S. A.; Gstoettenmayr, D.; Prapatpong, P.; Alhusban, A. A.; Ranjbar, L.; See, H. H.; Dawod, M.; Quirino, J. P. Electrophoresis 2015, 36, 36-61. (8) Šlampová, A.; Malá, Z.; Pantůčková, P.; Gebauer, P.; Boček, P. Electrophoresis 2013, 34, 3-18. (9) Yang, H.; Chien, R. L. J. Chromatogr. A 2001, 924, 155-163. (10) Jacobson, S. C.; Ramsey, J. M. Electrophoresis 1995, 16, 481-486. (11) von Heeren, F.; Verpoorte, E.; Manz, A.; Thormann, W. Anal. Chem. 1996, 68, 20442053. (12) Chien, R. L.; Burgi, D. S. Anal. Chem. 1992, 64, 489A-496A. (13) Mikkers, F. E. P.; Everaerts, F. M.; Verheggen, T. P. E. M. J. Chromatogr. A 1979, 169, 1-10. (14) Wu, M. L.; Gao, F.; Zhang, Y.; Wang, Q. J.; Li, H. Talanta 2015, 131, 624-631. (15) Wang, Z. F.; Cheng, S.; Ge, S. L.; Wang, H.; Wang, Q. J.; He, P. G.; Fang, Y. Z. Anal. Chem. 2012, 84, 1687-1694. (16) Noblitt, S. D.; Schwandner, F. M.; Hering, S. V.; Collett, J. L.; Henry, C. S. J. Chromatogr. A 2009, 1216, 1503-1510. (17) Kawai, T.; Sueyoshi, K.; Kitagawa, F.; Otsuka, K. Anal. Chem. 2010, 82, 6504-6511. (18) Quirino, J. P.; Terabe, S. Science 1998, 282, 465-468. (19) Foret, F.; Szoko, E.; Karger, B. L. J. Chromatogr. A 1992, 608, 3-12. (20) Britz-McKibbin, P.; Bebault, G. M.; Chen, D. D. Y. Anal. Chem. 2000, 72, 1729-1735. (21) Chen, C. C.; Yen, S. F.; Makamba, H.; Li, C. W.; Tsai, M. L.; Chen, S. H. Anal. Chem. 2007, 79, 195-201. (22) Sueyoshi, K.; Kitagawa, F.; Otsuka, K. Anal. Chem. 2008, 80, 1255-1262. (23) Han, C. M.; Katilius, E.; Santiago, J. G. Lab Chip 2014, 14, 2958-2967. (24) Bottenus, D.; Jubery, T. Z.; Ouyang, Y.; Dong, W. J.; Dutta, P.; Ivory, C. F. Lab Chip 2011, 11, 890-898. (25) Kazarian, A. A.; Hilder, E. F.; Breadmore, M. C. Analyst 2010, 135, 1970-1978. (26) Liu, Y.; Foote, R. S.; Jacobson, S. C.; Ramsey, J. M. Lab Chip 2005, 5, 457-465. (27) Jia, Z.; Lee, Y. K.; Fang, Q.; Huie, C. W. Electrophoresis 2006, 27, 1104-1111. (28) Kirby, B. J.; Wheeler, A. R.; Zare, R. N.; Fruetel, J. A.; Shepodd, T. J. Lab Chip 2003, 3, 5-10. (29) Chien, R. L. Anal. Chem. 1991, 63, 2866-2869. (30) Quirino, J. P. J. Chromatogr. A 2009, 1216, 294-299. (31) Rabanes, H. R.; Aranas, A. T.; Benbow, N. L.; Quirino, J. P. J. Chromatogr. A 2012, 1267, 74-79. (32) See, H. H.; Hauser, P. C. Journal of Membrane Science 2014, 450, 147-152. (33) See, H. H.; Schmidt-Marzinkowski, J.; Pormsila, W.; Morand, R.; Krahenbuhl, S.; Hauser, P. C. Anal. Chim. Acta 2012, 727, 78-82. (34) Mamat, N. A.; See, H. H. J. Chromatogr. A 2015, 1406, 34-39. (35) Thang, L. Y.; Shahir, S.; See, H. H. Electrophoresis 2015, 36, 2713-2719.


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1

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Figure captions

2 3

Fig. 1. Schematic of the battery operated MCE device with double-T geometry fabricated

4

borosilicate glass microchip. The device consists of a MCE platform to hold the microchip,

5

C4D, high voltage sequencer and in-house 12-volt DC output NiMH rechargeable battery

6

pack. The chip consists of separation and loading channels (L1 and L2), reservoirs (R1, R2,

7

R3, R4) and embedded electrodes for C4D. The total separation channel length was 40 mm

8

and the length from the junction to the embedded electrodes was 33 mm. The length of L1

9

and L2 was 5 mm. The length of the junction (1 mm) plus the length of the channel from R2

10

to the junction was 6 mm.

11 12

Fig. 2. Snapshots of multi-stacking in a double T-junction microchip. Sample: 0.04 ng/mL

13

Rhodamine 6G in MeOH with 0.1 mM acetic acid. (a) to (c) show stacking injections from

14

R1 and R3. (d) to (e) show re-stacking and injection of the final stacked zone into the

15

separation channel. Explanations and other conditions are given in the experimental section

16

and main text.

17 18

Fig. 3. Electropherograms for (a) multi-stacking in nonaqueous MCE of 30 ng/mL spiked

19

plasma after LLE and (b) standard injection of 1 µg/mL of standard. Peak identification: 4-

20

(dimethylamino)pyridine (IS or internal standard); NDT (1); endoxifen-i (2); endoxifen-ii (3);

21

tamoxifen (4); HTF (5).

22 23 24 25


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1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16


17


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

R3 MeOH +250V

Page 18 of 21

EOF SCB EOF 18s

15s R4 +70V

19s

R2 0V SCB

MeOH +250V R1

from R1

EOF

(a)

20.2s R4 0V

(b)

(c)

20.8s

20.4s R2 +1000V SCB

(d)

(e)

(f)

Figure 2

1 2 3 4 5 6 7 8 9 10 11 12 13


18

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3 1 IS

4

2

5

A

1 mV

1 2 3 4

IS

5

B

0.0

1

0.2

0.4

0.6

0.8

1.0

1.2

1.4

1.6

Time (min) Figure 3

2 3 4 5 6 7 8 9 10 11 12 13


19


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two sample streams multi-stacking sample reservoir 1 + + ++ + +

stacked + zone

stacking buffer reservoir

detector

enhancement 110-fold stacked zone

+ + + ++ + +

sample reservoir 2

injection


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R3 MeOH +250V

EOF SCB EOF 18s

15s R4 +70V

19s

R2 0V SCB

MeOH +250V R1

from R1

EOF

(a)

20.2s R4 0V

(b)

(c)

20.8s

20.4s R2 +1000V SCB

(d)

(e)

(f)

Figure 2


Multistacking from Two Sample Streams in Nonaqueous Microchip

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