Ionic liquids as solvents for in situ dispersive liquid–liquid microextraction of DNA

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<ul><li><p>Journal of Chromatography A, 1272 (2013) 8 14</p><p>Contents lists available at SciVerse ScienceDirect</p><p>Journal of Chromatography A</p><p>j our na l ho me p ag e: www.elsev ier .com</p><p>Ionic liquids as solvents for in situ dispersive liquDNA</p><p>Tianhao reda Department o 06, USb School of Gree o, OH </p><p>a r t i c l</p><p>Article history:Received 18 SeReceived in re19 November Accepted 20 NAvailable onlin</p><p>Keywords:Ionic liquidDispersive liquDNA extractioNucleic acid</p><p> the in sits andfcie</p><p>16POHcies actioot intg thebina</p><p> is co</p><p>1. Introduction</p><p>Deoxyribonucleic acid (DNA) is an important biomolecule con-taining thevirtually evical and lifeproling [2biological svariety of mcan be charesults [4]. small sampstream expvital, particpolymerase</p><p>A varietof DNA fromnol/chlorofoDNAproteproteins cansolvent (ph</p><p> Corresponand Engineeri43606, USA. Te</p><p>E-mail add</p><p>DNA remains in aqueous solution. Eventually, DNA is precipitatedby adding ethanol to the aqueous solution. This method has beensuccessfully adopted to isolate DNA from a wide variety of samples</p><p>0021-9673/$ http://dx.doi.o genetic information necessary for the viability ofery organism. It is widely investigated within biolog-</p><p> sciences elds, including genetic engineering [1], DNA], and DNA nanotechnology [3]. In chemically complexamples that contain proteins, polysaccharides and aetabolites, extracting nucleic acids from this matrix</p><p>llenging and can signicantly inuence experimentalAdditionally, many experiments are performed on veryles of DNA. When extracting DNA for challenging down-eriments, purication and preconcentration of DNA isularly for trace genetic analysis and amplication using</p><p> chain reaction.y of methods have been developed for the extraction</p><p> different biological matrices. Traditionally, the phe-rm method was applied for the isolation of DNA from</p><p>in complexes [5]. This method is based on the fact that generally be denatured and dissolved into an organicenolchloroformisopropanol (25:24:1, v/v/v)), while</p><p>ding author at: Department of Chemistry, School of Green Chemistryng, The University of Toledo, 2801 W. Bancroft Street, Toledo, OHl.: +1 419 530 1508; fax: +1 419 530 4033.ress: (J.L. Anderson).</p><p>including whole blood, platelets, lymph nodes, and bone marrow[6]. Different surfactants, such as cetyltrimethylammonium bro-mide (CTAB) or sodium dodecyl sulfate (SDS), have been employedin this extraction method [6]. However, this approach has severaldisadvantages. Firstly, organic solvents are used during the extrac-tion procedures and are often not environmentally benign. Also,the entire extraction process is time consuming (generally requir-ing 34 h), tedious, and requires multiple steps [6]. Several washingand centrifugation steps are often needed for the extraction processthereby increasing the risk of sample contamination or damage [7].Control over temperature as well as special buffer solutions is oftenrequired during the extraction process [5,6].</p><p>Currently, commercial DNA extraction kits are available whichminimize the use of organic solvent, decrease the risk of samplecontamination, and accelerate the extraction process. However,the price of these kits is high and the number of extractions thatcan be performed is limited. Additionally, the recovery, sensitivity,and purity of DNA extracted using different commercial kits can behighly variable. Some extraction kits require specialized equipment[8,9].</p><p>Recently, ionic liquids (ILs) have emerged as novel solventsystems employed in DNA separations [10], ion conductive DNAlms [11], and DNA biosensors [12]. ILs are a class of non-molecular solvents with low melting points (</p></li><li><p>T. Li et al. / J. Chromatogr. A 1272 (2013) 8 14 9</p><p>CN+MeOH</p><p>45 oC, 5 h, N21</p><p>R-Br</p><p>IPA, 60 oC, 8 h</p><p>N NR</p><p>Br-</p><p>2, 3</p><p>HO</p><p>NCHN N N N</p><p>NC</p><p>+</p><p>Fig. 1. Synthe -(1,2-ILs.</p><p>from the coinorganic adinium, pyrhalides, tetrbis[(triuornation of cphysicochesure at roomand thermaof moleculeicity than sco-workersorophosphaaqueous sostated thatIL and the the extractition with ILonly one ILIL volumesextraction aat low conccontaining examined, twhen 700 </p><p>In an attas well as traction mstudy descr(DLLME) mIL-based in employs a promotes isis reagent(LiNTf2), is sis reactionsolution ofing metathapproach dionic liquid[15], tempemicroextracuid dispersimethods utIL phase, resthe increassitu DLLMEtion and oftIn additionall extractiosolvent.</p><p>thlimid</p><p> broe </p><p> bdecydecyre apnsti</p><p> nucded t</p><p> comudied</p><p>erim</p><p>agen</p><p>dazoodepance suis, btai</p><p> chhlordroxuppluorest Lhite, </p><p> = 1.3Aldrgen (repaM T</p><p>lutioNA gn in ng NR = C10H21, C16H33</p><p>IPAN N</p><p>R</p><p>Br-</p><p>HOOH</p><p>6, 770 oC, 7 days</p><p>sis of 1-(1,2-dihydroxypropyl)-3-decylimidazolium bromide (C10POHIM-Br) and 1</p><p>mbination of various organic cations and organic ornions. Common IL cations include imidazolium, pyri-rolidinium, and phosphonium whereas anions includeauoroborate (BF4), hexauorophosphate (PF6), andomethyl)sulfonyl]imide (NTf2). The different combi-ations and anions produces ILs which possess uniquemical properties including nearly negligible vapor pres-</p><p> temperature, wide ranges of viscosity, high chemicall stabilities, and the ability to solvate a wide varietys. In addition, some classes of ILs exhibit lower tox-ome organic solvents. A previous study by Wang and</p><p> employed the 1-butyl-3-methylimidazolium hexau-te (BMIM-PF6) IL for the direct extraction of DNA fromlution using liquidliquid extraction (LLE) [13]. It was</p><p> electrostatic interactions between the cation of thephosphate groups within DNA played a major role inon. While the study showed the utility of DNA extrac-s, more can be done. For example, the authors studied</p><p> as the extraction solvent. In addition, relatively large in the range of 500700 L were employed for eachnd the extraction was only suitable for DNA samplesentrations (lower than 0.01 mg mL1). When samplesa higher concentration of DNA (0.1 mg mL1) werehe extraction efciency of DNA decreased to below 70%L of the IL was used.empt to examine a larger scope of IL extraction solventsinvestigate the feasibility of employing a microex-ethod that consumes a smaller amount of IL, thisibes an in situ dispersive liquidliquid microextractionethod for the extraction and preconcentration of DNA.situ DLLME, rst developed by our group in 2009 [14],hydrophilic IL dissolved in an aqueous solution thatnteractions between analytes and the IL. A metathe-, such as lithium bis[(triuoromethyl)sulfonyl)]imideadded to the solution to perform an in situ metathe-</p><p> producing a water-immiscible IL. Typically, a turbid ne IL microdroplets is formed during the ensu-esis reaction that facilitates preconcentration. Thisiffers from other IL-based DLLME methods including</p><p> dispersive liquidliquid microextraction (IL-DLLME)</p><p>In methyazoliumbromidazolium3-hexaN,N-diBr] weThis cotion ofappenfrom awas st</p><p>2. Exp</p><p>2.1. Re</p><p>Imi1-brom1,2-proreferen(St. Lowas oSodiumcium ctris(hywere sbis[(triSynQuegg wweightSigmaInvitrowere pof 20 mThe soSafe Dsolutio8.0 usirature-controlled ionic liquid dispersive liquid phasetion (TILDLME) [16], and ultrasound-assisted ionic liq-ve liquid-phase microextraction (UILDLME) [17]. Theseilize organic solvent, heat or ultrasound to disperse thepectively. In comparison with the IL-based LLE method,ed surface area of the IL extraction solvent in the in</p><p> method often results in higher analyte preconcentra-en eliminates the need of organic dispersive solvents., the in situ DLLME method often decreases the over-n time and requires smaller volumes of the extraction</p><p>(18.2 M cm(Bedford, M</p><p>2.2. Synthe</p><p>Six diffestudy for inand (C10)2Nies [14,18,1and C16POHlonitrile (0NaOH (aq)</p><p>15 % (w/w)</p><p>N NR</p><p>BrOH</p><p>4, 5</p><p>+</p><p>dihydroxypropyl)-3-hexadecylimidazolium bromide (C16POHIM-Br)</p><p>is study, six ILs, namely, 1-butyl-3-azolium chloride (BMIM-Cl), 1-decyl-3-methylimid-mide (C10MIM-Br), 1-hexadecyl-3-methylimidazolium(C16MIM-Br), 1-(1,2-dihydroxypropyl)-3-decylimid-romide (C10POHIM-Br), 1-(1,2-dihydroxypropyl)-limidazolium bromide (C16POHIM-Br) andl-N-methyl-d-glucaminium bromide [(C10)2NMDG-plied as extraction solvents in the extraction of DNA.</p><p>tutes the rst study to employ DLLME in the extrac-leic acids using ILs comprised of various substituentso the cation. Using this approach, the extraction of DNAplex sample matrix containing metal ions and proteins.</p><p>ental</p><p>ts</p><p>le, 1-methylimidazole, 1-chlorobutane,cane, 1-bromohexadecane, acrylonitrile, 3-bromo-ediol, benzene and phosphoric acid solution (NMRtandard, 85% in D2O) were obtained by SigmaAldrichMO, USA). Deuterated dimethylsulfoxide (d6-DMSO)ned by Cambridge Isotope (Andover, MA, USA).loride, sodium hydroxide, potassium chloride, cal-ide dihydrate, magnesium chloride hexahydrate, andymethyl)aminomethane hydrochloride (TrisHCl)ied by Fisher Scientic (Fair Lawn, NJ, USA). Lithiumomethyl)sulfonyl)]imide (LiNTf2) was purchased fromabs, Inc. (Alachua, FL, USA). Albumin, from chickenand DNA sodium salt from salmon testes (molecular</p><p> 106, approximately 2000 bp) were supplied byich. SYBR Safe DNA gel stain was purchased fromCarlsbad, CA, USA). Stock solutions of albumin and DNAred individually by dissolving 500 g of each in 1 mLrisHCl buffer and the pH adjusted to 8.0 using NaOH.ns were stored at 38 C. A working solution of SYBRel stain was prepared by dissolving 1.0 L of stock10 mL of 20 mM TrisHCl buffer and the pH adjusted toaOH. All solutions were prepared with deionized water) obtained from a Milli-Q water purication systemA, USA).</p><p>sis of ionic liquids</p><p>rent ILs were examined as extraction solvents in this situ DLLME of DNA. BMIM-Cl, C10MIM-Br, C16MIM-Br,MDG-Br were synthesized according to previous stud-9]. The synthesis of two novel ILs, namely C10POHIM-BrIM-Br, is shown in Fig. 1. Imidazole (0.10 mol) and acry-</p><p>.13 mol) were mixed and stirred in methanol (10 mL)</p></li><li><p>10 T. Li et al. / J. Chromatogr. A 1272 (2013) 8 14</p><p>at 45 C for 5 h under nitrogen to obtain 1-cyanoethylimidazole 1.Methanol and residual acrylonitrile were removed under reducedpressure for 3 h at 65 C. 1-Bromodecane or 1-bromohexadecane(0.11 mol) was mixed with compound 1 and isopropanol (20 mL).This reaction mixture was reuxed at 60 C for 8 h to obtain1-cyanoethyl-3-decylimidazolium bromide 2 or 1-cyanoethyl-3-hexadecylimidazolium bromide 3, respectively. After reux, theresidue was dissolved in chloroform and a 15% (w/w) NaOH aque-ous solution was added. After stirring for 5 h, the aqueous layerwas removed. The chloroform layer was washed using ve aliquots(10 mL) of deionized water until a neutral pH was achieved. Theproduct was dried under reduced pressure for 24 h at 70 C result-ing in 1-decylimidazole 4 or 1-hexadecylimidazole 5. Compound4 or 5 (0.038 mol) was then dissolved in isopropanol (35 mL) anda 10 mL isopropanol solution containing 3-bromo-1,2-propanediol(0.038 mol) was added slowly to the reaction mixture over a spanof 1.5 h at 70 C. This solution was then reuxed for 7 days at 70 C.Isopropanol was then removed under reduced pressure at 60 Cfor 3 h. The crude product was dissolved in water (150 mL) andwashed seven times with 100 mL of ethyl acetate. After purica-tion, water was removed under reduced pressure for 24 h at 70 Cand the product subsequently dried in a vacuum oven for 3 daysto afford C10POHIM-Br 6 or C16POHIM-Br 7 in high purity. Thesetwo ILs were characterized by 1H NMR and ESI-MS, as shown inFig. S1 of the supplemental information. The structures of all six ILsexamined in this study are shown in Fig. 2.</p><p>2.3. Instrumentation</p><p>High-performance liquid chromatographic analysis was per-formed using a LC-20A liquid chromatograph (Shimadzu, Japan)with two LC-20AT pumps, a SPD-20 UV/VIS detector, and aDGU-20A3 degasser. All separations were carried out using ananion exch2.5 m par5 mm 4.6 </p><p>N </p><p>HO</p><p>Fig. 2. Structbutyl-3-methybromide (C10MBr), (d) 1-(1,21-(1,2-dihydroN,N-didecyl-N</p><p>(Bellefonte, PA, USA). All separations were performed usingtwo mobile phases (A) 20 mM TrisHCl (pH = 8) and (B) 1.0 MNaCl/20 mM TrisHCl (pH = 8). In the analysis of DNA, the sepa-ration gradient started with 50:50 of mobile phases A and B, andthen was garation of Dwas graduaat 1.0 mL mplished at 2</p><p>Extractiopolypropyleusing a miCentrifugatFisher Scientra for the8452A Dioda quartz cDNAIL coma Varian Vquency of 8.5 mg mL</p><p>ized water.adding diffedimethylsurelative to standard.</p><p>2.4. Extract</p><p>2.4.1. In sitThe IL-b</p><p>in Fig. 3. Brioluti</p><p> disss LiNuge tratioution</p><p> of tge aeioni</p><p>Extraamplteinn. A5 L,</p><p> of ptrations watriH2O</p><p> mg m</p><p>ults </p><p>alua</p><p> amwas ding s soange column (TSKgel DEAE-NPR, 35 mm 4.6 mm i.d.,ticle size) with a guard column (TSKgel DEAE-NPR,mm i.d., 5 m particle size) from Tosoh Bioscience</p><p>NC4</p><p>Cl-</p><p>N NC10</p><p>Br-</p><p>N NC16</p><p>Br-</p><p>(a) (b) (c)</p><p>N NC10</p><p>Br-</p><p>OH</p><p>N NC16</p><p>Br-</p><p>HOOH</p><p>(d) (e)</p><p>Br-</p><p>OH</p><p>H</p><p>H</p><p>OH</p><p>OH</p><p>H</p><p>OH</p><p>H</p><p>OHN</p><p>C10</p><p>CH3C10+</p><p>(f)</p><p>ures of studied ILs used for the in situ DLLME of DNA: (a) 1-limidazolium chloride (BMIM-Cl), (b) 1-decyl-3-methylimidazoliumIM-Br), (c) 1-hexadecyl-3-methylimidazolium bromide (C16MIM-</p><p>-dihydroxypropyl)-3-decylimidazolium bromide (C10POHIM-Br), (e)xypropyl)-3-hexadecylimidazolium bromide (C16POHIM-Br), and (f)-methyl-d-glucaminium bromide [(C10)2NMDG-Br].</p><p>DNA spletelyaqueoucentrifmolar bid solportiona syrinwith dliquid.</p><p>2.4.2. A s</p><p>ing prosolutio1513trationconcenmetal iing a mCaCl22(0.015</p><p>3. Res</p><p>3.1. Ev</p><p>Thephase remainaqueouradually increased to 100% B over 10 min. For the sep-NA and albumin, the gradient began with 100% A andlly increased to 100% B in 20 min. The ow rate was setin1. For DNA and albumin, UV detection was accom-60 nm and 280 nm, respectively.ns were performed using either 0.6 or 2.0 mLne microcentrifuge tubes. All samples were shakenxer from Barnstead/Thermolyne (Dubuque, IA, USA).ion was performed in a model 228 centrifuge fromtic at a rate of 3400 rpm (1380 g). Absorbance spec-</p><p> ILs were recorded at 208 nm on a Hewlett Packarde Array Spectrophotometer (Santa Clara, CA, USA) withuvette (d = 1 cm). 31P NMR spectra of DNA and theplex were obtained at room temperature (293 K) on</p><p>XRS 400 MHz NMR spectrometer at a resonance fre-161.9 MHz. Stock solutions of 2.0 mg mL1 DNA and1 C16POHIM-Br IL were individually prepared in deion-</p><p> The samples for 31P NMR analysis were prepared byrent volumes of these stock solutions into deuteratedlfoxide (d6-DMSO). The chemical shifts were recorded85% phosphoric acid, which was used as the external</p><p>ion procedure</p><p>u DLLMEased in situ DLLME method used in this study is depictedey, 0.5 mg of C16POHIM-Br IL was added to an aqueouson in a 2.0 mL microcentrifuge tube. The IL was com-olved in the aqueous solution after gentle shaking. AnTf2 solution (1.0 g mL1) was then added to the micro-ube resulting in the formation of a turbid solution. The</p><p> of IL to LiNTf2 was 1:1. After shaking for 5 min, the tur- was centrifuged for 10 min at a rate of 3400 rpm. A</p><p>he upper aqueous solution (20 L) was withdrawn intond subjected to HPLC analysis. The syringe was rinsedzed water multiple times to remove any residual ionic</p><p>ction of DNA...</p></li></ul>


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