ionic liquids as solvents for in situ dispersive liquid–liquid microextraction of dna
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Journal of Chromatography A, 1272 (2013) 8 14
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Journal of Chromatography A
j our na l ho me p ag e: www.elsev ier .com
Ionic liquids as solvents for in situ dispersive liquDNA
Tianhao reda Department o 06, USb School of Gree o, OH
a r t i c l
Article history:Received 18 SeReceived in re19 November Accepted 20 NAvailable onlin
Keywords:Ionic liquidDispersive liquDNA extractioNucleic acid
the in sits andfcie
16POHcies actioot intg thebina
Deoxyribonucleic acid (DNA) is an important biomolecule con-taining thevirtually evical and lifeproling [2biological svariety of mcan be charesults . small sampstream expvital, particpolymerase
A varietof DNA fromnol/chlorofoDNAproteproteins cansolvent (ph
Corresponand Engineeri43606, USA. Te
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
0021-9673/$ http://dx.doi.o genetic information necessary for the viability ofery organism. It is widely investigated within biolog-
sciences elds, including genetic engineering , DNA], and DNA nanotechnology . In chemically complexamples that contain proteins, polysaccharides and aetabolites, extracting nucleic acids from this matrix
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
chain reaction.y of methods have been developed for the extraction
different biological matrices. Traditionally, the phe-rm method was applied for the isolation of DNA from
in complexes . This method is based on the fact that generally be denatured and dissolved into an organicenolchloroformisopropanol (25:24:1, v/v/v)), while
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: Jared.Anderson@UToledo.edu (J.L. Anderson).
including whole blood, platelets, lymph nodes, and bone marrow. Different surfactants, such as cetyltrimethylammonium bro-mide (CTAB) or sodium dodecyl sulfate (SDS), have been employedin this extraction method . 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 . Several washingand centrifugation steps are often needed for the extraction processthereby increasing the risk of sample contamination or damage .Control over temperature as well as special buffer solutions is oftenrequired during the extraction process [5,6].
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].
Recently, ionic liquids (ILs) have emerged as novel solventsystems employed in DNA separations , ion conductive DNAlms , and DNA biosensors . ILs are a class of non-molecular solvents with low melting points (
T. Li et al. / J. Chromatogr. A 1272 (2013) 8 14 9
45 oC, 5 h, N21
IPA, 60 oC, 8 h
NCHN N N N
Fig. 1. Synthe -(1,2-ILs.
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
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, tempemicroextracuid dispersimethods utIL phase, resthe increassitu DLLMEtion and oftIn additionall extractiosolvent.
dazoodepance suis, btai
= 1.3Aldrgen (repaM T
lutioNA gn in ng NR = C10H21, C16H33
6, 770 oC, 7 days
sis of 1-(1,2-dihydroxypropyl)-3-decylimidazolium bromide (C10POHIM-Br) and 1
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-
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
employed the 1-butyl-3-methylimidazolium hexau-te (BMIM-PF6) IL for the direct extraction of DNA fromlution using liquidliquid extraction (LLE) . It was
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
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 ,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-
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
dispersive liquidliquid microextraction (IL-DLLME)
In methyazoliumbromidazolium3-hexaN,N-diBr] weThis cotion ofappenfrom awas st
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) , and ultrasound-assisted ionic liq-ve liquid-phase microextraction (UILDLME) . 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
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
(18.2 M cm(Bedford, M
Six diffestudy for inand (C10)2Nies [14,18,1and C16POHlonitrile (0NaOH (aq)
15 % (w/w)
dihydroxypropyl)-3-hexadecylimidazolium bromide (C16POHIM-Br)
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.
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.
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
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).
sis of ionic liquids