ORIGINAL PAPER

Chemiluminescence detection flow cells for flow injectionanalysis and high-performance liquid chromatography

Jessica M. Terry & Stephan Mohr & Peter R. Fielden &

Nick J. Goddard & Neil W. Barnett & Don C. Olson &

Duane K. Wolcott & Paul S. Francis

Received: 2 December 2011 /Revised: 22 February 2012 /Accepted: 24 February 2012 /Published online: 28 March 2012# Springer-Verlag 2012

Abstract We have examined a range of new and previouslydescribed flow cells for chemiluminescence detection. Thereactions of acidic potassium permanganate with morphineand amoxicillin were used as model systems representingthe many fast chemiluminescence reactions between oxidis-ing agents and organic analytes, and the preliminary partialreduction of the reagent was exploited to further increase therates of reaction. The comparison was then extended tohigh-performance liquid chromatography separations of α-and β-adrenergic agonists, with permanganate chemilumi-nescence detection. Flow cells constructed by machiningnovel channel designs into white polymer materials (sealedwith transparent films or plates) have enabled improvementsin mixing efficiency and overall transmission of light to thephotodetector.

Keywords Chemiluminescence . Flow cells . Acidicpotassium permanganate . High-performance liquidchromatography

Introduction

Chemiluminescence is an attractive mode of detection offeringremarkable sensitivity and the ability to detect analytes thatlack a strong chromophore or fluorophore, using very simpleinstrumentation [1–5]. Like electrochemical detection, chemi-luminescence is dependent on oxidation and reduction pro-cesses, but the interactions that initiate the light-producingpathways can be considerably more complex than electrontransfer. However, this can be exploited to rapidly assess thechemical reactivity of an entire sample (using flow injectionanalysis) [6–8] or individual sample components (afterchromatographic separation) [9–11].

The construction of chemiluminescence flow cells forflow injection analysis and liquid chromatography by

Paul S. Francisis a Senior Lecturer in Chemistry atDeakin University (Australia) andcurrently holds an Australian Re-search Council Future Fellowshipto study the chemistry, spectroscopyand analytical applications of chemi-luminescence and electrochemilumi-nescence reactions. For his researchin these areas, he has received aVictoria Young Tall Poppy ScienceAward (Australian Institute ofPolicy and Science), the RobertCattrall Medal (Royal AustralianChemical Institute) and the Gordon

F. Kirkbright Award (Association of British Spectroscopists).

Published in the special issue Young Investigators in Analytical andBioanalytical Science with guest editors S. Daunert, J. Bettmer, T.Hasegawa, Q. Wang and Y. Wei.

Electronic supplementary material The online version of this article(doi:10.1007/s00216-012-5902-1) contains supplementary material,which is available to authorized users.

J. M. Terry :N. W. Barnett : P. S. Francis (*)School of Life and Environmental Sciences, Deakin University,Geelong, Victoria 3216, Australiae-mail: paul.francis@deakin.edu.au

S. Mohr : P. R. Fielden :N. J. GoddardSchool of Chemical Engineering and Analytical Science,The University of Manchester,Manchester M60 1QD, UK

D. C. Olson :D. K. WolcottGlobal FIA,P.O. Box 480, Fox Island, WA 98333, USA

Anal Bioanal Chem (2012) 403:2353–2360DOI 10.1007/s00216-012-5902-1

machining channels into polymer materials has enabledexploration of designs that are not accessible using thetraditional coiled-tubing approach [12–21]. Studies by ourresearch group and others have shown: (1) repeated changesin the direction of solution flow (such as reversing turns)enhances the mixing efficiency and therefore the emissionintensity [12, 19, 20, 22]. (2) Relatively large chambers orwells in the detection zone generally delay the onset of themaximum emission as the solution rapidly moves throughthe path of least resistance, leaving surrounding areas ofpoor solution mixing [19, 21]. (3) Channels machined oretched into an opaque white polymer and sealed with a thinpiece of transparent material minimise the loss of lightthrough surfaces not exposed to the photodetector [17, 20,23]. (4) A mixing tube/channel prior to the entrance of thedetection zone is normally required for the reacting mixtureto reach maximum emission intensity in front of the photo-detector [12, 19, 24], but in the case of very rapid chemilu-minescence systems, merging the solutions within thedetection zone may be superior [21].

In this study, we compare a range of chemiluminescenceflow cells that incorporate one or more of these advances,for both flow injection analysis and high-performance liquidchromatography, using the fast chemiluminescence reac-tions of acidic potassium permanganate with morphine,amoxicillin, α-adrenergic agonists (from Citrus aurantium[25]) and β-adrenergic agonists.

Experimental methods

Instrumentation

Flow injection analysis

The instrument was constructed as previously described[21]. With each change of flow cell, the light-tight housingwas re-sealed and the instrument was left for 40 min toavoid the temporary increase in baseline signal that wasobserved under the most sensitive settings. All tubing enteringand exiting the detector was black PTFE (0.76 mm, i.d.,Global FIA). The output signal from the detector wasrecorded with an ‘e-corder 410’ data aqcuisition system(eDAQ, NSW, Australia).

High-performance liquid chromatography

Analyses were carried out on an Agilent Technologies 1200series liquid chromatography system, equipped with a qua-ternary pump, solvent degasser system and autosampler(Agilent Technologies, Victoria, Australia), using a SynergiHydro-RP 80A (250×4.6 mm, i.d., 4 μm; for the C. aur-antium compounds) or an Alltech Alltima C18 column

(250×4.6 mm, i.d., 5 μm; for the β-adrenergic agonists)with an injection volume of 20 μL and a flow rate of1 mL min−1. An analogue to digital interface box (AgilentTechnologies) was used to convert the signal from thechemiluminescence detector. Before use in the high-performance liquid chromatography (HPLC) system, allsample solutions and solvents were filtered through a0.45-μm nylon membrane.

For the separation of C. aurantium compounds, isocraticelution was performed with 98% solvent A: deionised wateradjusted to pH 2.15 with trifluoroacetic acid and 2% solventB: methanol [26]. The column eluate (1 mL min−1) and acidicpotassium permanganate reagent (1 mL min−1) merged at aconfluence point located immediately prior or within thedetection zone of the flow cells. For the separation of β-adrenergic agonists, gradient elution was performed withdeionised water adjusted to pH 2.5 with trifluoroacetic acid(solvent A) and methanol (solvent B) as follows: 0–7 min,10% B; 7–9 min, 10–50% B; 9–10 min, 50% B; 10–11 min,50–40% B; 11–12 min, 40–10% B; and 12–13 min, 10% B.

Flow cells

Flow cell A was a coil of polymer tubing, which has beencommonly used in chemiluminescence studies and adopted inseveral commercially available detectors [27]. In our case, theflow cell was constructed bymounting a 3-cm diameter coil oftransparent PTFE-PFA tubing (0.8 mm, i.d.; DKSH, Queens-land, Australia) on a thin metal sheet (3.5×5 cm). The tubingat the centre of the coil passed through a small slit in the sheetand was connected to a plastic barbed T-piece by slippingsilicon tubing over both parts.

Flow cell B was a novel variation on this design in whichthe tubing was glued into a spiral channel machined into apolished aluminium plate (Fig. 1). Acting as reflector, themachined channel had a semi-spherical profile matching theradius of the tubing. Prior to fabrication, a 3D model of thebacking plate (Fig. S1 in the Electronic supplementary

Fig. 1 Construction of flow cell B. Left, a spiral channel machinedinto an aluminium plate. Right, PTFE-PFA tubing (0.8 mm, i.d.) gluedinto the channel

2354 J.M. Terry et al.

material (ESM)) was drawn using AutoDesk Inventor(Autodesk Inc., San Rafael, USA). EdgeCAM software(Pathtrace, Kent, UK) was used to convert the 3D modelsinto machine code for the CNC precision milling machine(Datron CAT3D M6, Datron Technology Ltd., MiltonKeynes, UK).

Flow cell C was constructed by machining channels(0.7×0.7 mm) into both faces of a polycarbonate chip(46×57×6 mm) and sealing the channels with a transparentepoxy-acetate film [20, 28]. The front face contained aserpentine channel configuration, with a central entrancelocated above the confluence point on the back face(Fig. 2). Solution lines were connected to the flow cell usingMinistac fittings (Aviaquip, Victoria, Australia). A mirrorwas attached to the back face of the flow cell to enhance thetransmission of light to the photodetector.

Flow cell D was identical to flow cell C, except that thechannels were machined into a white Acetal chip, and nomirror was used (Fig. S2 in the ESM). This flow cell wasused in our previous investigation into channel size, designand chip material [20].

Flow cell E was a Teflon disk with machined serpentinechannel (0.76 mm width×0.89 mm depth), contained withina commercially available ‘GloCel’ chemiluminescence de-tector (Global FIA, WA, USA) [19]. Solution inlet andoutlet ports in the back plate of the detector were alignedwith holes drilled through the disks. A sapphire windowserved as the transparent top surface of the flow cell. Thesolutions were merged at a Y-piece outside the detector. Thelength between the confluence point and detection zone wasapproximately 4 cm (∼20 μL).

Flow cell F was a modified version of flow cell E,containing two solution inlets at the centre of the Teflondisk and appropriately adjusted back plate [19].

Flow cells G and H were identical to flow cells E and F,except that the disks were constructed from Teflon impreg-nated with 25% glass microspheres to improve the trans-mission of light to the photomultiplier tube.

Flow cell I was a commercially available borosilicatechip (48×57×11 mm) containing a spiral channel (1 mm,i.d.) and sealed by thermal bonding (Hellma, Singapore).The confluence point was located below the central entranceto the spiral (Fig. 3). Inlet and outlet ports had 1/4"-28thread for conventional fittings. The back of the cell wascoated with a mirror surface. Flow cell J was identical toflow cell I, except that all surfaces not exposed to the PMTwere covered with aluminium foil.

Chemicals and reagents

Deionised water (Continental Water Systems, Victoria,Australia) and analytical grade reagents were used unlessotherwise stated. Chemicals were obtained from the followingsources: amoxicillin, epinephrine hydrochloride, fenoterolhydrobromide, isoprenaline hydrochloride, metaproterenolhemisulfate salt, octopamine, sodium polyphosphate, sodiumthiosulfate, syneprhine, trifluoroacetic acid and tyramine fromSigma-Aldrich (NSW, Australia); N-methyltyramine fromCSIRO Animal Health Laboratories (Victoria, Australia); sal-butamol hemisulfate from Fluka (NSW, Australia); morphinefrom GlaxoSmithKline (Victoria, Australia); potassium per-manganate from Chem-Supply (SA, Australia); and methanoland sulfuric acid fromMerck (Victoria, Australia). Hordeninewas synthesised as previously described [26].

The C. aurantium compounds (hordenine, N-methyltyr-amine, octopamine, synephrine and tyramine; 1×10−3 M)were prepared in deionised water and diluted into the HPLCmobile phase (98% solvent A, deionised water adjusted topH 2.15 with trifluoroacetic acid; 2% solvent B, methanol)as required. The β-adrenergic agonists (epinephrine, feno-terol, isoprenaline, metaproterenol and salbutamol; 1×10−3 M) were prepared and diluted in deionised water thathad been adjusted to pH 2.5 with trifluoroacetic acid.

The ‘standard’ permanganate reagent was prepared by dis-solution of potassium permanganate (1×10−3 M) in 1% (m/v)sodium polyphosphate and adjusting to pH 2.5 with sulfuric

Fig. 2 Flow cell C: a polycarbonate chip with serpentine channel Fig. 3 Flow cell I: a borosilicate chip with spiral channel

Chemiluminescence detection flow cells for FIA and HPLC 2355

acid. The ‘enhanced’ permanganate reagent was prepared bydissolution of potassium permanganate (1.9×10−3 M) in 1%(m/v) sodium polyphosphate and adjusting to pH 2.5 withsulfuric acid and then adding sodium thiosulfate (0.6 or1.0 mM), using a small volume of a 0.1-M solution [29].

Results and discussion

Flow injection analysis

The ten flow cell configurations were initially comparedusing the chemiluminescence reactions of morphine andamoxicillin with the two acidic potassium permanganatereagents. The reaction of morphine with permanganate wasselected because it has been used extensively in flow anal-ysis and HPLC applications [30, 31] and is an exemplar ofthe many fast chemiluminescence reactions between oxidis-ing agents and organic analytes [32]. Amoxicillin is a β-lactam antibiotic that has been detected with various per-manganate chemiluminescence reagent systems [33–35].Our previous investigations involving these and other ana-lytes have shown that considerable increases in reaction rateand chemiluminescence intensity can be obtained by a pre-liminary partial reduction of permanganate to create a stable,relatively high concentration of Mn(III) in the reagent solu-tion [29, 36]. The flow cells were therefore also comparedwith the ‘enhanced’ permanganate reactions as examples ofextremely rapid chemiluminescence systems.

Using FIA methodology, the analytes were injected (tenreplicates) into a water carrier stream, which merged withthe permanganate reagent at a flow rate of 3.5 mL min−1 perline. In agreement with our previous work [29], the en-hanced reagent provided a considerable increase in chemi-luminescence intensity (4- to 5-fold for morphine and 32- to38-fold for amoxicillin for most flow cells; Table S1 in theESM). Greater enhancement was observed for flow cells Fand H (7-fold for morphine and 50- to 58-fold for amoxi-cillin), where the merging of reactants within the detectionzone enabled a greater proportion of the light emitted fromthese reactions to be captured. As shown in Fig. 4, severalflow cells provided greater chemiluminescence signals thanthe traditional coiled tubing approach (flow cell A). Flowcell B enabled the tubing to be tightly coiled on a reflectivebacking plate (Fig. 1), the advantage of which was mostevident using the faster (enhanced) permanganate reactions.The ‘GloCel’ detector (flow cells E–H) provided a relativelyinexpensive commercial option with a purpose-built hous-ing. Machining the reaction channels into disks constructedfrom Teflon impregnated with glass microspheres (flowcells F and H) increased the signal intensities by up to 23%.

Although flow cell D was also constructed by machiningchannels into a white polymer material, it produced

significantly larger chemiluminescence signals (between36 and 210% greater than the signals for flow cells E–H;Fig. 4). We attribute these differences to the thin transparentseal and ability to place the photomultiplier tube flushagainst the chip, which enables a greater transfer of lightto the photodetector. Figure 5 shows the distribution ofemitted light (the characteristic red emission (λmax0689±5 nm) emanating from an electronically excited manganese(II) species [37]) for the three flow cells providing thegreatest chemiluminescence intensities (B, D and H) incomparison with flow cell A. In agreement with our previ-ous investigation [20], flow cells I and C, which were bothconstructed from transparent materials and backed with amirror, produced much lower signals due to the loss of lightthrough surfaces not exposed to the photodetector (Fig. 6).The addition of a layer of aluminium foil around the surfa-ces of flow cell J enhanced the transmission of light to thedetector by 25–44%. However, the absolute signals werestill lower than most of the other flow cells.

Fig. 4 Comparison of chemiluminescence detection flow cells for thereactions of (a) 1×10−7 M morphine and (b) 1×10−5 M amoxicillinwith the standard permanganate reagents (white columns, left scale)and the enhanced permanganate reagent (grey columns, right scale),using flow injection analysis

2356 J.M. Terry et al.

The greater chemiluminescence intensity obtained usingflow cell D also translated to a slightly lower limit ofdetection for amoxicillin (9×10−8 M) compared with theconventional coiled-tubing approach (2×10−7 M). Usingthe enhanced reagent, the detection limit was furtherimproved to 3×10−9 M, which is superior to all previouslyreported values for this analyte with permanganatechemiluminescence [33, 34, 38].

High-performance liquid chromatography

The flow cells that provided the greatest chemiluminescenceintensities with the enhanced permanganate system (D and H)were evaluated for post-column HPLC detection, in compari-son with the conventional coiled-tubing approach. We initiallycompared these flow cells for the detection of a mixture of α-adrenergic agonists from C. aurantium, due to the recentinterest in their potentially adverse health effects as ingredientsin weight-loss supplements [39]. Our previous work has dem-onstrated the rapid determination of these analytes with mono-lithic column chromatography and permanganatechemiluminescence detection [26, 40]. More recently, we haveshown that the emission intensities with these analytes can beimproved by up to two orders of magnitude with enhancedpermanganate reagents, largely due to increased reaction ratesthat were more compatible with HPLC-based detection [36].

In this study, we have employed a reverse-phase particle-packed column designed for highly aqueous mobile phases

(98% solvent A, deionised water adjusted to pH 2.15 withtrifluoroacetic acid; 2% solvent B, methanol). Separationtimes with this column were much longer than those in ourprevious reports [26, 40], providing greater peak resolution.Using flow cell A, the enhanced reagent provided 15- to 49-fold larger signals than the standard reagent (Fig. 7). The useof flow cells D and H, which both contained channelsmachined into white polymer materials, further increasedsignal intensities by 55% and 10%, respectively. Very littledifference in peak widths was observed using these threeflow cells (Fig. S3 in the ESM), as the time required for thereacting mixture to pass through the flow cell (largely de-pendent on the combined flow rates of the column eluateand chemiluminescence reagent, in conjunction with theflow cell volume) was significantly less than the analyteband width after chromatographic separation.

We also examined the separation and detection of β-adrenergic agonist pharmaceuticals, which required higherproportions of organic modifier in the chromatographicmobile phase (up to 50% solvent B, methanol). Poor com-patibility between the epoxy resin (used to attach the trans-parent seal to flow cell D) and the HPLC mobile phasemeant that it was not suitable for use in this analysis.

As only fenoterol had previously been detected with theenhanced reagent [29], we optimised the concentrations ofpermanganate and thiosulfate required to generate the great-est chemiluminescence intensities with each analyte usingFIA (Fig. S4 in the ESM), prior to implementing this

Fig. 5 Photographs ofchemiluminescence from thereaction of morphine with theenhanced permanganate reagentin flow cells A, B, D and H.The reactant solutions werecontinuously merged at a flowrate of 3.5 mL min-1 per line. A20-s exposure time was used foreach photograph

Chemiluminescence detection flow cells for FIA and HPLC 2357

detection system in HPLC. Whilst the degree of enhance-ment was somewhat analyte dependant, the greatest intensi-ties were generally obtained using a reagent containing 1.9×10−3 M permanganate and 1.0×10−3 M thiosulfate. The use

of the enhanced reagent resulted in significant increases inchemiluminescence intensity for both salbutamol and feno-terol (Table S2 in the ESM). Moderate enhancement wasobtained for metaproterenol (11-fold), and as a consequencethe extremely low response obtained with the standardreagent became comparable with that of epinephrine andisoprenaline. Similar trends were observed in the HPLCexperiments (Fig. 7; Table S3 in the ESM), with discrep-ancies between the two experimental approaches attribut-able to differences in flow rates and the proportion oforganic modifier in the carrier/eluent stream. The limits ofdetection for the five analytes using FIA under these con-ditions (with flow cell H) ranged from 1×10−10 to 5×10−9 M (compared with 3×10−8 to 1×10−7 M using thestandard permanganate reagent; Table S4 in the ESM),which were better than all previously reported values forepinephrine, isoprenaline, salbutamol and fenoterol usingpermanganate chemiluminescence systems [29, 41–45].Metaproterenol had not previously been detected with thesereagents.

The use of flow cell F (dual-inlet serpentine channelmachined into a Teflon disk) for post-column chemilumi-nescence detection provided an increase in signal of 34–69% across the five analytes, compared with flow cell Awith the same reagent (Fig. 8). Flow cell H, in which theTeflon disk was impregnated with glass microspheres, wason average 45 and 129% superior to flow cells F and A,respectively.

Conclusions

The construction of chemiluminescence detection flow cellsbased on a coil of polymer tubing is an effective approach

Fig. 7 HPLC separation of C. aurantium compounds: 1, octopamine;2, synephrine; 3, tyramine; 4, N-methyltyramine; and 5, hordenine.Detection conditions: flow cell A using the standard permanganatereagent (black trace) and flow cells A (blue trace), H (pink trace)and D (yellow trace), using the enhanced permanganate reagent

Fig. 8 HPLC separation of β-adrenergic agonist pharmaceuticals: 1,epinephrine; 2, isoprenaline; 3, metaproterenol; 4, salbutamol; and 5,fenoterol. Detection conditions: flow cell A using the standard per-manganate reagent (black trace) and flow cells A (blue trace), F (pinktrace) and H (yellow trace), using the enhanced permanganate reagent

Fig. 6 Photographs of chemiluminescence from the reaction of morphinewith the enhanced permanganate reagent in flow cells I and C. The reactantsolutions were continuously merged at a flow rate of 3.5 mLmin-1 per line.A 30-s exposure time was used for each photograph

2358 J.M. Terry et al.

that has been used in both FIA and HPLC over decades ofresearch. In flow cell B, we have improved the signals fromthis simple design, using a reflective backing plate withgrooves to ensure a tight coil of the transparent PTFE-PFAtubing. Alternatively, the machining of channels into poly-mer materials enables new configurations to be explored, tomaximise mixing efficiency and the transfer of light to thephotodetector. Comparison of several previously describedflow cells revealed the importance of surface reflectance.The introduction of glass microspheres in the Teflon disks ofthe GloCel detector (flow cells G and H) improved theresponse. Even greater signals were observed from the Ac-etal chip with a similar channel configuration (flow cell D),which may in part arise due the shorter distance from thereaction channel to the photomultiplier window. Despiteincorporating highly reflective back surfaces, the two flowcells constructed from transparent materials (polycarbonateand glass) produced inferior signals due to the loss of lightfrom the sides of the chips. Machining into polymer materi-als also enables the inclusion of threaded inlet/outlet portsfor standard fittings (which overcomes problems associatedwith pressure at high flow rates) and custom designs forincorporation in purpose-built light-tight housings. Thecombination of these flow cells (that provide efficient mix-ing of fast chemiluminescence reactions close to or at thepoint of detection) with the enhanced permanganate reagenthas provided considerable improvements in the limits ofdetection for a range of analytes.

Acknowledgements The authors thank the Faculty of Science andTechnology (Deakin University) and the Australian Research Council(Future Fellowship FT100100646) for funding this research; GeoffreyP. McDermott and Kerryn F. Saltmarsh (School of Life and Environ-mental Sciences, Deakin University) for assistance with the HPLCexperiments; and Donna Squire (Knowledge Media Division, DeakinUniversity) for the photography. J.M.T acknowledges the receipt of anAustralian Postgraduate Award.

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