Split Injection: A Simple Introduction Of Subnanoliter .

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Microchemical Journal 99 (2011) 180–185Contents lists available at ScienceDirectMicrochemical Journalj o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / m i c r o cSplit injection: A simple introduction of subnanoliter sample volumes forchip electrophoresisA. Gáspár a,⁎, P.I. Koczka a, H. Carmona b, F.A. Gomez babDepartment of Inorganic and Analytical Chemistry, University of Debrecen, H-4010 Debrecen, HungaryDepartment of Chemistry and Biochemistry, California State University, Los Angeles, CA, USAa r t i c l ei n f oArticle history:Received 20 April 2011Accepted 4 May 2011Available online 11 May 2011Keywords:ChipPoly(dimethylsiloxane)Pressure injectionChip electrophoresisa b s t r a c tThe ability to accurately inject small volumes of sample into microfluidic channels is of great importance inelectrophoretic separations. While electrokinetic injection of nanoliter scale volumes is commonly utilized inmicrochip capillary electrophoresis (MCE), mobility and matrix bias makes quantitation difficult. Herein, wedescribe a new injection method based on the simple patterning of the crossing of channels that does notrequire sophisticated instrumentation. The sample volume injected into the separation channel is dependenton the ratio of the widths of the crossing channels. This injection method is capable of introducing, into aseparation channel, multiple plugs of sample on a large scale. This injection technique is tested for zoneelectrophoresis in native and surface modified poly(dimethylsiloxane) (PDMS) chips. 2011 Elsevier B.V. All rights reserved.1. IntroductionThe injection of minute quantities of sample solution into aseparation channel is a prerequisite for effective separations.Electrokinetic injection is the most commonly utilized form of sampleinjection in microchip capillary electrophoresis (MCE) mainly due toits ease of use (no external pumps or valves are required tomanipulate fluids in the chip) [1,2]. While the most commonlyapplied electrokinetic methods are pinched [3] and gated injection [4],both techniques suffer from injection bias resulting in significantproblems for quantitative applications in CE. During electrokineticinjection, variations in the conductivity of samples are due to acombination of matrix effects (matrix bias) and different mobilities ofthe analyzed components (mobility bias), thereby resulting indifferent loaded quantities [5]. Leakage of sample into the separationchannel has also been reported in these techniques. A number ofpapers have detailed how to compensate for biased injections andleakage problems [6,7].Although it is well-known that pressure (hydrodynamic orhydrostatic) injections can provide bias-free injections, only a fewrecent publications (compared to publications dealing with electrokinetic injections) have detailed these types of injection procedures.Pressure injection modes utilize external pressures induced by pumpsor hydrostatic forces and are often combined with voltage-gated⁎ Corresponding author.E-mail address: gaspara@tigris.unideb.hu (A. Gáspár).0026-265X/ – see front matter 2011 Elsevier B.V. All rights reserved.doi:10.1016/j.microc.2011.05.001schemes to prevent the sample solution from entering the separationchannel except during the injection step [8]. Some combined flowinjection capillary electrophoresis (FI-CE) systems have been developed where pressure-driven hydrodynamic flow (e.g., sampling loop,injection valve) is used for the sample injection [9]. Simple yet slightlyreliable methods include the addition of extra sample into the samplereservoir relative to the other reservoirs [10] or the tilting of themicrochip causing the fluid in a sample reservoir to be of a differentheight [11]. Numerous pressure injection techniques have beenpublished which either require complicated instrumentation (e.g., asystem in which the samples are injected by applying a pressure pulseto a membrane on the reservoir using a mechanical actuator [12],dual-syringe pump with switching valve [13]) or integrate sophisticated devices, thereby losing the disposable feature of the microchip(e.g., integrated diaphragm pump [14]). Lin et al. combinedhydrostatic pressure (generated simply by adjusting the liquid levelin different reservoirs) with electrokinetic forces in a cross-formmicrofluidic chip [15]. Attiya et al. presented a method for interfacinglarge-scale flow channels with 10 μm scale channels allowing nearcontinuous sampling of external reservoirs [16].An optimal injection method should be characterized by little to nobias, no voltage application on the sample reservoir (voltage mayresult in a change in sample composition and/or pH due toelectrolysis) and simplicity to make disposability of the microchipeasy. Herein, we describe a new approach to sample introduction inpoly(dimethylsiloxane) (PDMS) microfluidic chips that utilizespressure applied by a peristaltic pump instead of electrokinetic forces.Food dyes were utilized in the injection schemes to visualize sampleflow, plug formation and electrophoresis.

A. Gáspár et al. / Microchemical Journal 99 (2011) 180–1852. Experimental2.1. MaterialsReagents of analytical grade were obtained from various distributors. Sodium dihydrogen phosphate and sodium hydroxide werepurchased from Reanal (Hungary). Sodium dodecyl sulphate (SDS),cetyltrimethyl-ammonium bromide (CTAB) and methyl cellulosewere purchased from Sigma. Stock solutions of food dyes (FD&Cblue#1, and FD&C red#40) were obtained from McCormick & Co., Inc.(MD, USA). The buffer electrolyte for the CZE separation contained50 mM phosphate, pH, 6.8. The microchannels and capillaries werepreconditioned with the buffer electrolyte for 5 min. All solutions(buffer, samples and methanol) were degassed and filtered through a0.45 μm syringe filter.2.2. InstrumentationAll solutions are transported into the chip using a low-rate (0.01–1 μL/min) peristaltic pump (IPC, Ismatec). The movement of the plugswas monitored using an inverted microscope (Axio Observer A1,Zeiss) equipped with a high speed CCD camera (AxioCam ICC3, Zeiss).Videos and images were recorded by AxioVision 4.6.3 (Zeiss)software. The software allows for the measurement of the intensityof colors (RGB) at any part of the channel at any in time yielding anelectropherogram showing the separation of dyes recorded as a video.Although the sensitivity of this type of detection is poor, it is sufficientfor optimizing experimental conditions and is less expensive andtime-consuming than other detection modes.The samples (0.5–2 μL) were intoduced into the peristaltic pumptubing (ID: 0.3 mm) initially filled with electrolyte. The sample tubingis connected to the inlet port and the other tubings are connected tothe outlet ports of the waste channels. For the ΜCE separation, aminiaturized power supply with positive ground was used (0.5–2 kV,Cetox Ltd., Hungary). The Pt electrodes are inserted to the outlet portand the end port of the separation channel.The CE instrument was a HP 3DCE model (Agilent, Waldbronn,Germany). Hydrodynamic sample introduction was used for sampleinjecton (100 mbar .s). Separations were performed using polyimide-ac181coated fused-silica capillaries of 64.5 cm 50 μm i.d. (PolymicroTechnology, Phoenix, AZ, USA). The sample is injected at the detectionend of the capillary, thus the effective length of separation was 8.5 cm.The applied voltage was 25 kV. The detection was carried out by oncolumn photometric measurement at 200 nm.2.3. Microchip fabricationThe PDMS chips were prepared by using a mold created by softphotolithography [17]. The pattern consisted of a crossing of three50-μm-wide channels and one 150–200-μm-wide channel designedand printed as a high resolution (10,000 dpi) photomask (Fig. 1aand b). The separation channel consisted of a straight channelcomponent and an arched segment for a total separation length of8 cm. (Serpentine or folded channels are not recommended forΜCE [18].) Each lithographic mask includes three separate channelpatterns.Negative type photoresist (SU-8 2025, Microchem, Newton, MA)was spin-coated onto a 3″ silicon wafer at 3000 rpm for 30 s to athickness of 30 μm. The photoresist coated wafer was baked for15 min at 95 C. The pattern on the mask was transferred to the waferthrough UV exposure (365 nm) for 2 min. The exposed wafer wasbaked at 95 C for 5 min and the unexposed areas were removed byrinsing with SU-8 developer (Microchem, Newton, MA) (Fig. 1c). ThePDMS chip was fabricated by cast molding of a 10:1 mixture of PDMSoligomer and cross-linking agent (Sylgard 184, Dow Corning,Midland, MI). The PDMS mixture was degassed and baked at 80 Cfor 30 min. The PDMS replicas were peeled off from the mold. Holes(300 μm diameter) for the liquid connections were punched throughthe PDMS chip. The PDMS chip was irreversibly sealed onto a glassslide of 1.2 mm thickness after oxygen plasma treatment (PDC-32 GHarrick) (Fig. 1d).3. Results and discussion3.1. The principle of split injectionThe proposed injection method involves pressure injection ofsample using a peristaltic pump without the use of any special valves.bdFig. 1. Preparation of microchip for split-injection zone electrophoresis using (a) lithographic mask includes 3 channel patterns (the crossing is magnified (b)), (c) mold (includes 2identical chip patterns). (d) PDMS chip sealed to a glass slide.

182A. Gáspár et al. / Microchemical Journal 99 (2011) 180–185inletv0per unit time (t) through a capillary tube is directly proportional tothe pressure difference between its ends (ΔP) and to the fourth powerof its internal diameter (d), and inversely proportional to its length (L)and to the viscosity of the fluid (η).LLoutlet 14dV v110Lv3outlet 3(separation channel)v2 Loutlet 2Fig. 2. The design of crossing of channels and illustration of the flow conditions.The principle behind the injection technique resembles the splitinjection often applied in gas chromatography (GC). In GC, a sample isintroduced into a heated small chamber to volatilize the sample andsample matrix. The carrier gas then either sweeps the entire sample(splitless mode) or a portion (split mode) of the sample into thecolumn. In split mode, a part of the sample/carrier gas mixture in theinjection chamber is exhausted through the split vent [19,20]. In thesplit mode the split rate ranges between 1:10 and 1:1000 [19]. Thistype of injection is not used in liquid chromatography.The proposed injection method follows from the the Hagen–Poiseuille law (Eq. 1). The volume of a homogeneous fluid (V) passingaΔPd4 πt128ηLð1ÞSince the transported (injected) volume of liquid depends on thediameter of the capillary (channel), by adjusting the channel widthsof the crossed channels, different liquid volumes can be distributedinto the channels, and a small fraction of the original liquid volumecan be split into the narrowest of channels.Fig. 2 depicts the design of the crossing of channels. The givenvolume of sample is pumped from the inlet port and is split towardthree outlet ports. The ratio of the liquid volume split into theseparation channel (V3) and the volume originally injected into thechip (V0) are expressed in Eq. (2):V3 V0d43L3d43L3 d42L2 ð2Þd41L1Because the width of the broad-channel is four times larger thanthat of the other channels (d) and the length of the separation channelis 10 times longer than that of all the other channels (L),d1 4 d and d0 d2 d3 d;ð3ÞL3 10 L and L0 L1 L2 L:ð4Þbacd50 µmbefFig. 3. Hydrodynamic split-injection (a–d) and zone electrophoresis (e–f). (Sample:mixture of blue and yellow food dyes, separation conditions: 25 mM phosphate, pH:6.8, U 400 V.)Fig. 4. Split injection of cells (Microcystis aeruginosa) into the separation channel(a) and the obtained plug of cells (b).

A. Gáspár et al. / Microchemical Journal 99 (2011) 180–185By substituting Eqs. (3) and (4) into (2), a large difference involumes of the sample originally injected into the chip and the sampleplug split into the separation channel was obtained (Eq. 5):V3 V02560ð5ÞFor example, if 1 μL volume of sample is pumped into the chip (V0),theoretically, only 390 pL is introduced into the beginning of theseparation channel (the length of the plug and the depth of the channelare 260 μm and 30 μm, respectively). This sample plug is 0.26% of thevolume of a 10 cm length separation channel which is expected to beoptimal based on past capillary electrophoretic separations. With thisinjection, it is possible to inject even a 10–50 μm length plug of samplewhich equates to less than 0.1% of the total volume of the separationchannel. However, even larger sample volumes can be injected byincreasing the sample volume. (In conventional CE, a typical samplevolume is 1–2% of the capillary volume.)The proposed channel pattern was designed to create a μL to pL/nLrange sample plug in the separation channel. Such μL volumes can beinjected into channels using other tools which already can be handledwith introduction/injection techniques. Typically, about 1 μL samplevolume is introduced into the chip, from which less then 0.1% is usedup for ΜCE. On initial inspection, it might appear as if a large volumeof sample is wasted, yet, typical delivery of sample by micropipette orsyringe is on the microliter scale. However, a large portion of thesample injected into the inlet port can be regained from the exit portof the broad (waste) channel (outlet 1, see Fig. 2). The split injection of183a dye can be followed in Fig. 3a–d. The duration of the injection isdefined by the pumping rate and is typically less than 2 s.By changing the ratio of the widths of the crossing channels(especially the channels of inlet and outlet 1 in Fig. 2), greater samplevolumes can be formed in the separation channel. The crossingpattern shown in Fig. 2 is applicable in the delivery of pL quantities ofsolution which is useful for CZE or MEKC on chip. Reducing the ratio ofthe widths of the crossing channels will allow for larger sample plugs(in the nL range) to be injected preferred in isotachoresis (ITP) andpreconcentration procedures. Split injection is capable of injecting alltypes of components or liquids into microfluidic channels. In Fig. 4 asmall volume (1 nL) of cells (Microcystis aeruginosa) is injected intoa channel. Given the volume of injected sample is on the order asthat required in CZE, current work is focused on applying this newtechnique to electrophoretic separations.The volume of solution injected into the separation channel doesnot depend on the pumping rate but is dependent on the ratio of thewidth and length of the crossing channels and the volume of theoriginal sample introduced into the inlet port. The viability of the splitinjection technique is dependent on the reproducibility of theintroduction of the original sample into the inlet port. Althoughmicroinjectors for reproducible sampling (b2 RSD%) in the 0.5–2 mLrange are available, the minimally needed distance between theinjector sample chamber/loop and the beginning of the samplingchannel in the chip is too large and can result in dispersion of thesample plug. In order to minimize the dispersion of the samplebefore it reaches the beginning of the channel in the chip, a volume ofsample was manipulated into the pump tubing. Subsequently, afteraInj.EOFDet.60BRel. int. (%)SDSYnet0510151015Time (s)60bInj.EOFBDet.Rel. int. (%)CTABYnet05Time (s)60cInj.BDet.Rel. int. (%)methylcelluloseYnet051015Time (s)Fig. 5. Illustration of mobilities of EOF and analytes (yellow and blue dyes) and the obtained electropherograms when channels are rinsed with SDS (a), CTAB (b) and methylcellulose(c) before electrophoresis.

184A. Gáspár et al. / Microchemical Journal 99 (2011) 180–185connecting the tubing into the inlet port of the chip (the end of thetubing already contacts the beginning of the channel causing zerodispersion before the split injection), the sample is pumped into thechannel. This simple, dispersion free manipulation of sample providesapproximately 5–10 RSD% reproducibility, resulting in similarreproducibility for the subsequent split injection.3.2. Zone electrophoresis in PDMS chip after surface modificationAfter the sample was split into the crossing area of the chip and asmall volume of the original sample was manipulated into theseparation channel (Fig. 3a–d), the peristaltic pump was stopped andthe electrophoretic separation initiated by applying 400 V to the endsof the separation channel (Fig. 3e–f). Since the test components(yellow and blue food dyes) are anionic, both dyes migrated towardthe positively charged electrode and the components separated after1 mm separation length. However, because the components countermigrate with the electroosmotic flow (EOF), the net mobilities of thecomponents are strongly influenced by the EOF formed due to thesurface charge of the PDMS channel. Due to fluctuations in the EOF,poor precision of the CZE was obtained. To improve the reproducibility of the measurements, a reproducible EOF (that is, a constantsurface charge on the channel surface) should be attained.PDMS is a favorite material to construct microfluidic chips due toits low cost and ease of fabrication. Although air plasma oxidation ofPDMS is a fast and convenient way to modify the hydrophobic surface,the induced hydrophilicity is shortlived [17,21]. It is well known thataRel. int. (%)60051015PDMS adsorbs various materials resulting in changes to its surfacecharge creating variances in EOF. Hence, control of EOF in chips ismore critical than in conventional CE.The most efficient way to provide constant EOF is by formation of astable dynamic coating on the channel wall. Numerous surfactantshave been adsorbed on PDMS to control the EOF [22,23]. When ananionic detergent (SDS) is used, the negatively charged surface issimilar as that used in fused silica capillaries (under neutral or basicconditions). In this case the anionic test components counter-migratewith the cathode directed EOF and the more anionic yellow dyesmigrate slower (Fig. 5a). The use of a cationic detergent (CTAB) leadsto a positive charge on the channel surface and anionic componentsco-migrate with the EOF (Fig. 5b). As expected, the neutral polymermethylcellulose largely reduces EOF and the components migratewith their own, effective electrophoretic velocity (Fig. 5c). Fig. 5shows that methylcellulose yielded the best separation of the twoanionic components.Detergents were only used for pre-washing the channel and not inthe running buffer during the separation to avoid formation ofmicelles (micellar electrokinetic chromatography [MEKC] mechanism). The detergents can be washed out from the chip with organicsolvents (e.g., methanol). In order to provide a stable, dynamiccoating, the channel was washed with detergent for 1 minute and wasrepeated every 3 hours.In chip electrophoresis, the shortest distance to achieve completeseparation of the test mixture was approximately 10 mm (separationtime: 15 s) (Fig. 6a). When the detection position was shifted to45 mm from the injection crossing, better resolution was obtained,but the analysis time increased to 70 s (Fig. 6b). In a conventional CEinstrument, the minimal separation length is 85 mm (sampleinjection is carried out at the detection end (“short-end” injection,Agilent instruments) and a 130 s analysis time provides good resolution (Fig. 6c).The electrophoretic profiles obtained in chip and capillaries weresimilar due to similar separation mechanisms and running buffer.Differences could be expected from the various contributions of EOFand varying degrees of adsorption onto PDMS and fused silica.4. ConclusionsTime (s)bRel. int. (%)60020406080Time (s)cmAU15050050100150Time (s)Fig. 6. Electropherograms obtained in PDMS chip along 10 mm (a) and 45 mm(b) separation length and in fused silica capillary of 85 mm length (sample injection atthe detection end (“short-end” injection, 3DCE instrument, Agilent) (c).We have developed a simple injection method based on Hagen–Poiseuille's law which is similar to split-injection commonly appliedin gas chromatography. The injection procedure makes it possible toinject a plug of sample of as little as 100–300 μm in length ( 200 pLvolume). Here, the injected amount can be as little as 1% of the totalvolume of the separation channel (as in conventional CE). Withproper choice of diameters and lengths of channels of a crossing largerange of sample volumes can be created for electrophoreticseparations. The amount of solution injected into the separationchannel does not depend on the pumping rate but is defined by thedimensions of the crossing channels and volume of the originalsample introduced into the inlet port. In this injection method onlypressure is applied; therefore, the known quantitation errors (biases)of electrokinetic injection do not occur.Studying the effects of different surface modifying agents on thezone electrophoretic separation was obtained and optimal separationcan be achieved by forming a dynamic coating of methylcellulose onthe PDMS channel. Similar electropherograms were obtained in chipand fused silica capillary but in chips the separation length can beeasily changed even within 5 cm (not possible with commercial CEinstruments).In this work, a simple, fast detection mode is proposed for flowmanipulation and separation studies in microfluidic chips throughmonitoring visible dyes and computational evaluation of the imagescaptured by microscopic means.

A. Gáspár et al. / Microchemical Journal 99 (2011) 180–185AcknowledgmentsThe authors gratefully acknowledge financial support for thisresearch by grants from the National Scientific Research Fund,Hungary (K75286), TÁMOP 4.2.1./B-09/1/KONV-2010-0007 projectand the National Science Foundation (OISE-0754138). The authorsalso would like to express their thanks to Dr. Melinda Andrasi(University of Debrecen, Hungary) and Maria Ortega (California StateUniversity, Los Angeles) for their assistance.Appendix A. Supplementary materialSupplementary data to this article can be found online atdoi:10.1016/j.microc.2011.05.001.References[1] E.S. Roddy, H. Xu, A.G. Ewing, Sample introduction techniques for microfabricatedseparation devices, Electrophoresis 25 (2004) 229–242.[2] D. Wu, J. Qin, B. Lin, Electrophoretic separations on microfluidic chips, J. Chromatogr.A 1184 (2008) 542–559.[3] S.C. Jacobson, R. Hergenroder, L.B. Koutny, R.J. Warmack, J.M. Ramsey, Effects ofinjection schemes and column geometry on the performance of microchipelectrophoresis devices, Anal. Chem. 66 (1994) 1107–1113.[4] S.C. Jacobson, R. Hergenroder, A.W. Moore, J.M. Ramsey, Precolumn reactions withelectrophoretic analysis integrated on a microchip, Anal. Chem. 66 (1994)4127–4132.[5] J.P. Alarie, S.C. Jacobson, J.M. Ramsey, Electrophoretic injection bias in microchipvalving, Electrophoresis 22 (2001) 312–317.[6] B.E. Slentz, N.A. Penner, F. Regnier, Sampling BIAS at channel junctions in gatedflow injection on chips, Anal. Chem. 74 (2002) 4835–4840.[7] T.T. Lee, E.S. Yeung, Compensating for instrumental and sampling biasesaccompanying electrokinetic injection in capillary zone electrophoresis, Anal.Chem. 64 (1992) 1226–1231.[8] C.C. Lin, C.C. Chen, C.E. Lin, S.H. Chen, Microchip electrophoresis withhydrodynamic injection and waste-removing function for quantitative analysis,Chromatogr. A. 1051 (2004) 69–74.185[9] X. Chen, L. Fan, Z. Hu, The combination of flow injection with electrophoresisusing capillaries and chips, Electrophoresis 25 (2004) 3962–3969.[10] U. Backofen, F.M. Matysik, C.E. Lunte, A chip-based electrophoresis system withelectrochemical detection and hydrodynamic injection, Anal. Chem. 74 (2002)4054–4059.[11] W. Wang, F. Zhou, L. Zhao, J.R. Zhang, J.J. Zhu, Improved hydrostatic pressuresample injection by tilting the microchip towards the disposable miniaturized CEdevice, Electrophoresis 29 (2008) 561–566.[12] D. Solignac, M.A.M. Gijs, Pressure pulse injection: a powerful alternative toelectrokinetic sample loading in electrophoresis microchips, Anal. Chem. 75(2003) 1652–1657.[13] Z. Wu, H. Jensen, J. Gamby, X. Bai, H.H. Girault, A flexible sample introductionmethod for polymer microfluidic chips using a push/pull pressure pump, Lab Chip4 (2004) 512–515.[14] J.M. Karlinsey, J. Monahan, D.J. Marchiarullo, J.P. Ferrance, J.P. Landers, Pressureinjection on a valved microdevice for electrophoretic analysis of submicrolitersamples, Anal. Chem. 77 (2005) 3637–3643.[15] H. Gai, L. Yu, Z. Dai, Y. Ma, B. Lin, Injection by hydrostatic pressure in conjunctionwith electrokinetic force on a microfluidic chip, Electrophoresis 25 (2004)1884–1894.[16] S. Attiya, A.B. Jemere, T. Tang, G. Fitzpatrick, K. Seiler, N. Chiem, D.J. Harrison,Design of an interface to allow microfluidic electrophoresis chips to drink fromthe fire hose of the external environment, Electrophoresis 22 (2001) 318–327.[17] D.C. Duffy, J.C. McDonald, O.J.A. Schueller, G.M. Whitesides, Rapid prototyping ofmicrofluidic systems in poly(dimethylsiloxane), Anal. Chem. 70 (1998)4974–4984.[18] B.M. Paegel, L.D. Hutt, P.C. Simpson, R.A. Mathies, Turn geometry for minimizingband broadening in microfabricated capillary electrophoresis channels, Anal.Chem. 72 (2000) 3030–3037.[19] C.F. Poole, The Essence of Chromatography, Elsevier, Amsterdam, 2003.[20] K. Grob, Split and Splitless Injection for Quantitative Gas Chromatography:Concepts, Processes, Practical Guidelines, Sources of Error, Wiley-VCH VerlagGmbH, Weinheim, Germany, 2007.[21] I.J. Chen, E. Lindner, Stabilization of the hydrophilicity of radio-frequency plasmatreated polydimethylsiloxane surface, Langmuir 23 (2007) 3118–3122.[22] Y. Liu, J.C. Fanguy, J.M. Bledsoe, J.C. Henry, Dynamic coating using polyelectrolytemultilayers for chemical control of electroosmotic flow in capillary electrophoresis microchips, Anal. Chem. 72 (2000) 5939–5944.[23] G.T. Roman, K. McDaniel, C.T. Culbertson, High efficiency micellar electrokineticchromatography of hydrophobic analytes on poly(dimethylsiloxane) microchips,Analyst 131 (2006) 194–201.

(splitless mode) or a portion (split mode) of the sample into the column. In split mode, a part of the sample/carrier gas mixture in the injection chamber is exhausted through the split vent [19,20]. In the split mode the s

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