整体柱

Journal of Chromatography A,1394(2015)103–110Contents lists available at ScienceDirectJournal of ChromatographyAj o u r n a l h o m e p a g e :w w w.e l s e v i e r.c o m /l o c a t e /c h r o maFast preparation of a highly efficient organic monolith via photo-initiated thiol-ene click polymerization for capillary liquid chromatographyLianfang Chen a ,b ,Junjie Ou a ,∗,Zhongshan Liu a ,b ,Hui Lin a ,b ,Hongwei Wang a ,Jing Dong a ,Hanfa Zou a ,∗∗a Key Laboratory of Separation Science for Analytical Chemistry,Dalian Institute of Chemical Physics,Chinese Academy of Sciences,Dalian 116023,China bGraduate School of Chinese Academy of Sciences,Beijing 100049,Chinaa r t i c l ei n f oArticle history:Received 20January 2015Received in revised form 17March 2015Accepted 20March 2015Available online 27March 2015Keywords:Organic monolithThiol-ene click reactionCapillary liquid chromatography Photo-initiated polymerization 1,2,4-Trivinylcyclohexanea b s t r a c tA novel organic monolith was firstly prepared in a UV-transparent fused-silica capillary by a single-step approach via photo-initiated thiol-ene click polymerization reaction of 1,2,4-trivinylcyclohexane (TVCH)and pentaerythriol tetra(3-mercaptopropionate)(4SH)within 10min.The effects of both composition of prepolymerization solution and polymerization time on the morphology and permeability of monolithic column were investigated in detail.Then,the optimal condition was acquired to fabricate a homogeneous and permeable organic monolith.The chemical groups of the monolithic column were confirmed by Fourier transform infrared spectroscopy (FT-IR).The SEM graphs showed the organic monolith possessed a uniform porous structure,which promotes the highest column efficiency of ∼133,000plates per meter for alkylbenzenes at the linear velocity of 0.65mm/s in reversed-phase liquid chromatography.Finally,the organic monolithic column was further applied for separation of basic compounds,pesticides and EPA610,indicating satisfactory separation ability.©2015Elsevier B.V.All rights reserved.1.IntroductionWith the development of the continuous bed of Hjertén and the rigid polymer rod of Svec and Fréchet,monolithic stationary phases have been regarded as a major breakthrough in chromatographic technology that emerged in the late 1980s and early 1990s [1–4].Due to the highly porous structure of monoliths,long columns can be employed to obtain high separation efficiency.Addition-ally,fast separation and high-throughput application are possible because of their high flow rate compatibility.The in situ prepara-tion of a monolithic bed in capillary columns or microfluidic devices is another advantage compared to either conventional particle-packed columns or open-tubular columns.Currently,monolithic columns with unusual and attractive properties have been widely applied in capillary liquid chromatography (cLC)and capillary elec-trochromatography (CEC)[5–10].Dependent on the nature of the precursors used,the monolithic columns can be categorized into two major types:the inorganic∗Corresponding author.Tel.:+8641184379576;fax:+8641184379620.∗∗Corresponding author.Tel.:+8641184379610.E-mail addresses:junjieou@ (J.Ou),hanfazou@ (H.Zou).silica-based and the organic monolithic columns [11,12].Inorganic monoliths are generally prepared by sol–gel technology,and their morphology can be tailored by controlling the starting pared to inorganic silica-based monoliths,organic monoliths are more attractive due to the wide varieties of available monomers and the facile preparation via thermally initiated free radical poly-merization.Different types of polymer-based monolithic stationary phases,including polymethacrylates [13],polyacrylamides [14]and polystyrenes [15],have been developed by several research groups.Generally,organic monoliths prepared from hydrophobic,hydrophilic,or charged monomers can be employed in different chromatographic modes such as reversed-phase liquid chromatog-raphy (RPLC)[16],hydrophilic interaction chromatography (HILIC)[17]and ion-exchange chromatography (IEC)[18].However,the observed plate heights differ greatly.In general,organic monoliths contain little mesopores and possess relatively low surface area.The presence of significant amounts of macropores turns out to be favorable in the very fast separations of large molecules includ-ing proteins,nucleic acids,and synthetic polymers [7,19,20]but less efficient in the isocratic separation of small molecules [21].To overcome this limitation,several strategies have been developed including the optimization of composition of polymerization mix-ture,the hypercrosslinking technique and the addition of carbon/10.1016/j.chroma.2015.03.0540021-9673/©2015Elsevier B.V.All rights reserved.104L.Chen et al./J.Chromatogr.A1394(2015)103–110nanostructures[21–24].These efforts offer column efficiency up to tens of thousands of plates per meter.However,it is still desirable to explore facile approaches for improvement of separation efficiency of organic monoliths.Click chemistry,focusing on highly efficient reactions that reach quantitative conversion under mild conditions and require only facile separation,has been widely applied in the chemical,bio-logical,physical and engineeringfields[25–29].Especially the thiol-ene click chemistry without metal catalysis features high efficiency,simplicity and insensitivity to oxygen or water[30]. These advantages have made thiol-ene click reactions gradually apply in the modification and functionalization of separation media for chromatography.For example,Lindner’s group[31]pioneered to fabricate particulate silica-based chiral stationary phases via thiol-ene chemistry.Then,similar approach was also adopted for the preparation of stationary phases in hydrophilic interaction chromatography(HILIC)[32].Later,Alves and Nischang[33]have synthesized hybrid porous materials via thiol-ene click reaction of polyhedral oligomeric vinylsilsesquioxane(vinylPOSS)and thiol monomers.Recently,our group has developed a facile approach for preparation of macroporous hybrid monoliths via photoin-duced thiol-ene polymerization reaction[34].The resulting hybrid monoliths demonstrated high efficiency for separation of small molecules.This inspired us to prepare highly efficient organic monolith via thiol-ene polymerization reaction.Herein,a novel porous organic monolith was successfully pre-pared by using a multi-ene,1,2,4-trivinylcyclohexane(TVCH),and a multi-thiol,pentaerythriol tetra(3-mercaptopropionate)(4SH),as the precursors and directly formed in a UV transparent fused-silica capillary via photo-initiated thiol-ene click polymerization(as shown in Fig.1).The effects of fabrication conditions on morphol-ogy and permeability of monolith were systematically investigated. The resulting organic monolith was characterized by SEM and IR, and also applied for the separation of small molecules in cLC.2.Experimental2.1.Chemicals and materialsTVCH and4SH(95%),trimethylolpropane tris(3-mercaptopropionate)(3SH),diethylene glycol diethyl ether (DEGDE,98%)and polyethylene glycol200(PEG200)were obtained from Sigma(St.Louis,MO,USA).2,2-Dimethoxy-2-phenylacetophone(DMPA,99%)was purchased from Acros Organics(New Jersey,USA).1,6-Hexanedithiol(2SH,98%)and vinyltrimethoxysilane(VTMS,97.5%)were purchased from J&K Scientific Ltd.(Beijing,China).Thiourea,benzene,toluene,ethyl-benzene,propylbenzene,butylbenzene,caffeine,carbamazepine, 2,4-dinitroaniline,4-aminobiphenyl,2,6-dichloro-4-nitroanine and other standard analytes were of analytical grade,and gotten from Tianjin Kermel Chemical Plant(Tianjin,China).Imidaclo-prid,carbaryl,thiram,and parathion-methyl were from J&K Scientific Ltd.(Beijing,China).Polystyrene standards(MW=800, 4000,13,200,50,000,90,000,280,000,900,000)were obtained from Sigma(St Louis,MO,USA).The fused-silica capillary(UV transparent coating)with75␮m i.d.was purchased from Reafine Chromatography Ltd.(Hebei,China).HPLC-grade acetonitrile(ACN) was obtained from Yuwang Group(Shandong,China).Water used in all experiments was purified by a Milli-Q system(Millipore Inc., Milford,MA).Other chemical reagents were all of analytical grade.2.2.Preparation of organic monoliths via photo-initiatedthiol-ene click polymerizationThe fused-silica capillary wasfirst pretreated and rinsed by0.1mol L−1NaOH for1h,washed with water,followed by 0.1mol L−1HCl for3h,and rinsed with water and methanol suc-cessively.Then,the capillary wasfilled with VTMS solution in methanol(50/50,v/v),and kept in water bath at50◦C for12h with both ends sealed by rubbers.Finally,the capillary was again rinsed with methanol toflush out the residuals and dried under nitrogen flow.An alkene monomer(TVCH)and a thiol-containing monomer (4SH)were used to prepare organic monolith as shown in Fig.1. The pre-polymerization mixtures,consisting of monomers,poro-gens and photo-initiator(DMPA)are listed in Table1.In an example preparation,two monomers(TVCH/4SH,7.9/17.4,wt%) werefirst dissolved in binary porogenic solvents(DEGDE/PEG200, 45.2/29.4,wt%),followed by addition of0.2mg(0.37%,with respect to the monomer mass)DMPA for initiation.Then the single phase homogeneous polymerization mixture was introduced into the above-mentioned pretreated capillary with a syringe.With both sides sealed with rubbers,the capillary was irradiated with a UV-lamp( =365nm,120mJ/cm2)for6min.After polymerization,the obtained organic monolith was thenflushed with methanol to remove residuals.The remaining polymerization mixture was poly-merized to form bulk monolithic materials in a centrifuge tube under the same conditions.After reaction,the molded bulk polymer was cut into pieces,washed with ethanol three times,and dried ina vacuum oven at50◦C overnight for further characterization.2.3.Instruments and methodsThe thiol-ene polymerization was irradiated in UV crosslink-ers(XL-1500A, =365nm,Spectronics Corporation,New York, USA).Fourier-transformed infrared spectroscopy(FT-IR)charac-terization was carried out on Thermo Nicolet380spectrometer with KBr pellets(Nicolet,Wisconsin,USA).KBr pellets,containing around1mg monolithic polymer and100mg KBr,were prepared by powder compressing machine.The microscopic morphology of monolith was examined by scanning electron microscopy(SEM, JEOL JSM-5600,and Tokyo,Japan).Metal spraying of capillary monoliths was necessary for SEM.Water contact angle was mea-sured on a contact angle measurement machine DSA100with5␮L water drops(Kruss,Germany).The permeability(B0),reflecting through-pore size and external porosity,can be calculated according to Darcy’s law[35]by the following equation:B0=FÁL/(␲r2 P),where F(m3s−1)is theflow rate of mobile phase,Á(Pa s)is the dynamic viscosity of the mobile phase(0.38Pa s for ACN[36]),L(m)is the effective length of the column,r(m)is the inner radius of the column and P(Pa)is the pressure drop across the column.ACN was pumped through the prepared monolithic columns at aflow rate of0.5–12␮L/min.Back-pressure was recorded when the pressure stabilized.The data of P and F were obtained on nanoACQUITY Ultro Performance LC (Waters,Japan).The oligomersflushed out of the monolith were collected and analyzed by an AB Sciex5800MALDI-TOF/TOF mass spectrometer (AB Sciex,CA)equipped with a pulsed Nd/YAG laser at355nm in linear positive ion mode.The mixture of oligomers in methanol (0.5␮L)and the matrix(0.5␮L of25mg/mL2,5-dihydroxybenzonic acid(DHB)in ACN/H2O/H3PO4(70/29/1,v/v/v)were spotted on the MALDI plate for MS analysis.All cLC experiments were performed on a HPLC system consist-ing of a7725i injector with a20␮L sample loop,two LC-10ADVP pumps(Shimadzu,Kyoto,Japan)and a K-2501UV detector(Knauer, Berlin,Germany)operated at214nm.For obtaining aflow rate of nanoliters per minute(nL/min),a T-union connector was used to serve as a splitter with one end connected to the capillary mono-lithic column and the other end to a blank capillary(95cm×50␮m i.d.).The split ratio was controlled at about1/400for the50–200␮mL.Chen et al./J.Chromatogr.A1394(2015)103–110105Fig.1.Preparation of organic monolith via thiol-ene click polymerization.i.d.capillary columns.The outlet of the monolithic column was con-nected to an empty fused-silica capillary(50␮m i.d.and365␮m o.d.)with a Teflon tube,where a detection window was made by removing a2mm length of the polyimide coating in a posi-tion of5.0cm from the separation monolithic column outlet.All chromatographic data were obtained by using the software pro-gram HW-2000from Qianpu Software(Shanghai,China)at room temperature.3.Results and discussion3.1.Preparation of organic monoliths via photo-initiatedthiol-ene click polymerizationThe organic monolith was prepared by a single-step approach via thiol-ene click polymerization of TVCH and4SH.As both com-position of pre-polymerization mixture and UV light irradiation time have significant impact on the porous structure and chromato-graphic properties of monolithic columns,a combination of these parameters including porogenic system,initiator content,polymer-ization time and monomer content was investigated in detail to obtain satisfactory monolithic materials.Atfirst,the molar ratio of thiol group to alkene group was set at1:1in the polymerization mixture.It is well known that the type and volume of porogenic solvents in the prepolymer-ization solution play a vitally important role in the formation of organic monoliths.The commonly used porogens such as toluene/dodecanol,cyclohexanol/decanol and so on have been initially selected to prepare the organic monoliths,but unfortu-nately,satisfactory monoliths were not obtained.According to the results recently reported by us[34],we also adopted DEGDE/THF as the binary porogens.However,transparent bulk monolith with-out macropores was obtained,suggesting that DEGDE/THF was not suitable for the formation of monolith.As a result,THF was replaced with PEG200,and the opaque white monolith was obtained in the porogenic system of DEGDE/PEG200.The effect of the ratio of DEGDE to PEG200on the morphology and permeability of the resulting monolith was investigated.It was found that increas-ing the content of PEG200from26.9to31.9%led to an increase of permeability from3.8to7.6×10−14m2(as for columns2–4in Table1),which was in accordance with observations by SEM(a–c in Fig.S1).These results demonstrated the sensitive influence of porogenic solvent composition on the structure of monolith.When further increasing PEG200in the polymerization mixture,a little slack monolith was obtained with a permeability of3.4×10−13m2 (as for column1in Table1).This phenomenon is related to the rel-ative speed of phase separation.Though PEG200shows excellent compatibility with DEGDE and thiol-containing monomer,it served as a macroporogenic solvent for both hydrophobic ene-containing monomer and the sulfoether oligomer generated via thiol-ene reac-tion.The above results are in agreement with that a poor solvent leads to an earlier phase separation and the formation of larger pores in organic monolith[37].As a result,the ratio of45.2/29.4 (DEGDE/PEG200,wt%/wt%,column3)was selected as porogenic system for the preparation of organic monolith in the following experiments.As discussed in many publications,photoinitiator is a vitally important component of thiol-ene polymerization reaction,and various types of photoinitiator induce thio-ene click reaction with different reactivity and efficiency.According to the literature pre-viously reported[38],we selected DMPA,with excellent initiationTable1Detail preparation conditions for organic monoliths and their permeability.Column TVCH a(wt%)Thiol b(wt%)DEGDE c(wt%)PEG200d(wt%)DMPA e(wt%)Reaction time(min)Permeability(×10−14m2)17.817.238.136.90.3763427.917.342.831.90.3767.637.917.445.229.40.376 4.848.017.547.626.90.376 3.858.117.752.521.70.3760.0967.917.445.229.40.196 5.377.917.445.229.40.756 3.487.917.445.229.4 1.316 3.297.917.445.229.4 1.876 2.8107.917.445.229.40.372 6.1117.917.445.229.40.374 5.6127.917.445.229.40.3710 4.3137.917.445.229.40.3715 4.21410.115.145.329.50.376 5.515 5.719.545.329.50.3760.3a Weight percentage of TVCH in the pre-polymerization mixture.b Weight percentage of thiol(4SH)in the pre-polymerization mixture.c Weight percentage of DEGDE in the pre-polymerization mixture.d Weight percentage of PEG200in the pre-polymerization mixture.e Weight percentage of DMPA with respect to the monomer mass.106L.Chen et al./J.Chromatogr.A 1394(2015)103–110Fig.2.SEM images of (a,b)organic monolithic column,and (c)water contact angle of the surface of organic monolith.efficiency,as initiator and probed the impact of DMPA content on the structure and permeability of organic monoliths.In the fabri-cation process of organic monoliths,different amounts of DMPA were added to the reaction system.The detailed permeability val-ues of the monoliths are shown in Table 1(columns 3,6–9).It can be obviously found that the permeability of monoliths decreased from 5.3to 2.8×10−14m 2when increasing DMPA content from 0.19to 1.87%.Moreover,SEM images provided an illustrative insight into the impact of the initiator content on the morphologies of these monoliths (b,d,and e in Fig.S1,corresponding to columns 3,7and 9,respectively).It could be seen that the pore size became smaller with increasing content of DMPA.In comparison with porogenic system,the initiator concentration only moderately influences the morphology and permeability of monolith.In our experiment,the influence of polymerization time on the formation of organic monoliths was also studied (columns 3,10–13in Table 1).It was found that the permeability of organic monoliths slightly decreased from 6.1to 4.2×10−14m 2when increasing UV light irradiation time from 2to 15min.Since the thiol-ene click polymerization proceeded very rapidly,opaque white monolithic material would be observed in 60s,which indicated phase sep-aration had occurred.As seen in previously publications [30,39],polymerization of TVCH and a multi-thiol proceeds to a high conversion quite rapidly (in 300s).Moreover,a 1:1thiol-ene con-version can be observed over the course of the polymerization,and the final conversion approaches 90%in 6min.SEM photos show four monoliths synthesized with different polymerization time (f,b,g and h in Fig.S1,with time increasing in order).It can be clearly seen that the monolith displayed a more dense appear-ance as the polymerization time increased.This may be interpreted that more and more monomers converted to cross-linked polymers with the increase of polymerization time,and therefore formed a more cross-linked polymeric network.The ratio of multi-ene and multi-thiol precursors may affect the formation and permeability of the final material,in addition to its analytical performance.Three monoliths were synthesized with different ene/thiol molar ratios while keeping other experimental conditions same (columns 3,14,and 15in Table 1).As can be seen in Table 1,the permeability was dramatically decreased from 5.5to 0.3×10−14m 2with a decrease of ene/thiol molar ratio from 1.2/0.8to 0.8/1.2.The increase of multi-thiol precursor may provide high crosslinkage,endowing high mechanical strength on the monolith but leading to low permeability.On the basis of the above investigations,it is found that porogenic system mainly affects the porous structure of organic monoliths.In terms of the chromatographic application,the monolithic column should have appropriate pore structure for high permeability and separation efficiency.The macropores are needed to achieve good flow-through property.Finally,the monolith (column 3in Table 1)prepared with a mixture of TVCH/4SH/DEGDE/PEG200(7.9/17.4/45.2/29.4,wt%)and 0.2mgFig.3.Back pressure against flow rate for organic monolithic columns.Experimental conditions:column,20cm ×75␮m i.d.;mobile phase,H 2O (filled circles),MeOH (empty triangles),and ACN (filled squares).(0.37%,with respect to the monomer mass)DMPA under UV light ( =365nm,120mJ/cm 2)for 6min was employed for further exper-iment.We also selected other thiol-containing monomers,2SH and 3SH,as the control experiment.Unfortunately,solid bulk mono-lithic material was not obtained when 2SH was used as the precursor.Although white opaque bulk material formed when replaced 2SH with 3SH,the resulting monolith was either too dense or easily detached from the capillary wall.It would possi-bly take much more time to optimize the preparation conditions for acquirement of satisfactory 3SH-based organic column.Herein,4SH is the ideal multi-thiol monomer for fabrication organic monolith via photo-initiated thiol-ene click polymerization.It is deduced that higher functionality of thiol-containing monomers would facilitate to form the satisfactory monoliths via thiol-ene polymerization reaction.3.2.Characterization of organic monolithThe SEM graphs of the organic monolith were shown in Fig.2a and b.It was observed that a uniform monolithic matrix with large through-pores was obtained within the capillary,and the formed monolithic matrix was attached well to the inner wall of the cap-illary.This was attributed to the alkene group at the inner wall of the capillary had taken part in the polymerization during the for-mation of organic monolith.Additionally,Fig.3revealed a good linear relationship of back pressure against flow rate even at the high pressure of 40MPa,implying that the organic monolith pos-sessed relatively good mechanical stability.The permeability of the monolithic column was calculated as 4.8×10−14m 2according toL.Chen et al./J.Chromatogr.A1394(2015)103–110107Fig.4.FT-IR spectra of the TVCH,4SH and organic monolithic material. Darcy’s law,which indicated the good permeability of the prepared monolithic column.The monolithic material formed in centrifuge tube was char-acterized by FT-IR,providing a direct proof for the thiol-ene click polymerization reaction of TVCH and4SH.The peak at2560cm−1 is assigned to thiol group and peak at1743cm−1indicates C O stretches(Fig.4a).Characteristic peaks at1640,990and910cm−1 in Fig.4b are C C pared to Fig.4a and b,the disappear-ance of peak at2560cm−1and the decrease of peak at1640cm−1 in Fig.4c confirmed the thiol-ene click polymerization reaction of TVCH and4SH.Additionally,the oligomers collected in a cen-trifuge was analyzed by MALDI-TOF mass spectrometer(Fig.5). The oligomers with molecular weight ranging from1100to2000Da were detected.The repeating units with mass difference of162and 488Da were observed,corresponding to TVCH and4SH monomers, respectively.The formation of oligomers strongly indicated the occurrence of thiol-ene reaction between TVCH and4SH,which would further construct the porous monolith.The repeatability is an important factor for the effectiveness and practicability of the preparation method of monolith.In the study, the repeatability of organic monolith was assessed through the rel-ative standard deviation(RSD)for both retention factor and column efficiency of benzene as analyte(thiourea as the void time marker). The run-to-run(n=5),column-to-column(n=5)and batch-to-batch(n=5)repeatabilities for retention factor were0.2%,1.2% and2.8%(correspondingly the average retention factor values of benzene were0.67,0.64,0.65),respectively.The run-to-run(n=3), column-to-column(n=3)and batch-to-batch(n=3)repeatabilites for column efficiency were1.5%,3.1%and5.6%(correspondingly the average column efficiency values of benzene were73,116,73,044,Fig.5.MALDI-TOF mass spectrum of the oligomersflushed out of the organic mono-lith prepared with the porogenic system of pure DEGDE.72,986),respectively.This suggested the photo-initiated thiol-ene click polymerization provided a robust and practical method for fabrication of organic monolithic columns.What’s more,clear col-umn deterioration or significant decrease of column efficiency was not observed even after continuous use for three months,and the column efficiency could remain about89%(butylbenzene as the test compound),which demonstrated the high stability of organic monolith.3.3.Chromatographic performance of organic monolithFig.2c demonstrated that organic monolithic matrix was hydrophobic according to the water contact angle of126◦,so the monolith was investigated in RPLC mode using alkylbenzenes as probes.As shown in Fig.6a,five alkylbenzenes were baseline-separated in the order of benzene<toluene<ethylbenzene< propylbenzene<butylbenzene with the mobile phase of ACN/H2O (65/35,v/v).A lot of ester groups and carbon sulfur bonds should be existed in the organic monolithic skeleton,contributing the hydrophobic retention mechanism.The column efficiency of the resulting organic monolith was evaluated on cLC by changing the flow rate from40to180␮L/min(before split).Fig.6b depicts the dependence of the plate height on the linearflow velocity for alkylbenzenes.The organic monolith exhibited the minimum plate height of7.5–13.7␮m for alkylbenzenes,corresponding to 133,000–73,000theoretical plates per meter,which exhibits more excellent chromatographic performance than conventional organic monoliths such as polystyrenes and polymethacrylates[24,40–42]. The size exclusion chromatography in THF afforded total porosityofFig.6.(a)Separation of alkylbenzenes on the organic monolith by cLC.Analytes:(1)thiourea,(2)benzene,(3)toluene,(4)ethylbenzene,(5)propylbenzene and(6)butyl-benzene.(b)Dependence of the plate height of analytes on the linear velocity of mobile phase by the organic monolith capillary column.(c)The effect of ACN content in mobile phases on retention factors of alkylbenzenes.Analytes:benzene(filled squares),toluene(empty circles),ethylbenzene(filled triangles),propylbenzene(empty triangles)and butylbenzene(filled rhombuses).Experimental conditions:effective length of22cm×75␮m i.d.;mobile phase,ACN/H2O(65/35,v/v,for a,b);flow rates,200 and100␮L/min for(a)and(c)respectively(before split,);detection wavelength,214nm.108L.Chen et al./J.Chromatogr.A 1394(2015)103–110Fig.7.Calibration curve of organic monolith by size exclusion chromatography.Experimental conditions:column dimension,19.6cm ×75␮m i.d.;off column dimension,5.0cm ×75␮m i.d.;flow rate,75nL/min;mobile phase,THF;injection volume,2.5␮L in split mode;detection wavelength,214nm;temperature,25◦C;solute,benzene,polystyrenes (M w =800,4000,13,200,50,000,90,000,280,000,and 900,000).Table 2Fitted values of A ,B and C terms in van Deemter equation:H =A +B /u +Cu .A (␮m)B (␮m 2/s)C (s)Benzene 2.0934520.0039Toluene2.4627870.0038Ethylbenzene 1.9226770.0041Propylbenzene 2.1422520.004Butylbenzene1.9420710.0039Correlation coefficients ranging from 0.91to 0.95.the organic monolith at 71.5%,while the volume of through-pores was calculated at 53.3%(as shown in Fig.7).The great pore vol-ume was beneficial to the separation of small molecules,therefore high column efficiency was achieved on this organic monolith in cLC.Additionally,the values of A,B and C terms in van Deemter equation were summarized in Table 2.The A-term describes dis-persion of analytes due to the irregularity of the stream paths in the porous medium [43].Compared to C18-modified silica mono-lith [44],the obtained organic monolith exhibited lower A-term likely benefiting from the homogeneous structure.The B-term,representing the longitudinal diffusion,depends on the analyte diffusion coefficient in the mobile phase [45].Therefore,its valuedecreases as the molecular mass increases.The C-term is close to that observed with C18-modified silica monoliths [44].Com-pared with the monolithic polymeric column recently prepared via thiol-yne click polymerization [46],this organic monolith exhib-ited higher column efficiency,which can be attributed to lager through-pores in the monolithic matrix (smaller C-term).Furthermore,the influence of ACN content on retention factors of alkylbenzenes was investigated,as shown in Fig.6c.With ACN content increasing,the values of retention factors (k )decreased as expected.The methylene selectivity (˛)[47,48],used to character-ize the hydrophobicity of stationary phase,was calculated by the following equation:log k =n log ˛+log ˇ,where n is carbon num-ber of homologous compounds and ˇis retention factor speculated to n =0.Plotting log k versus n ,the values of methylene selectivity were calculated,namely 1.43,1.38,1.34,1.31and 1.27under 60%,65%,70%,75%and 80%ACN in the mobile phases,respectively (see Fig.S2).These results again confirmed the reversed-phase retention mechanism of the organic monolith.3.4.Application of organic monolithTo further investigate the application of the fabricated organic monolith,the separations of basic compounds,pesticides and pol-yaromatic hydrocarbons were performed in cLC.It was shown in Fig.8a that five basic compounds were baseline-separated with the mobile phase of ACN/H 2O (40/60,v/v)in cLC,and the peaks were all symmetry without any peak tailing.As mentioned above,there were only several neutral methylene,ester functional groups and carbon sulfur bonds in the surface of organic monolithic skeleton,contributing the hydrophobic retention for the basic compounds.As a result,the separation of basic compounds on this TVCH-4SH-based organic monolith with RPLC does not suffer from peak tailing.Fig.8b depicts the separation of four pesticides with the mobile phase of ACN/H 2O (55/45,v/v)in cLC,also demonstrating good separation ability of this organic monolith for these pesticides.EPA 610,consisting of 16priority pollutant PAHs,can participate in the body’s metabolism and presents potential health hazards because of their carcinogenicity,teratogenicity,mutagenicity and difficult biodegradability.Fig.8c clearly shows the separation of EPA 610on the prepared organic monolith in cLC with gradient elution.It can be seen that EPA 610were well separated except that phenanthrene (analyte 5)and anthracene (analyte 6)could not be separated,sug-gesting that the organic monolith has a potential application in the field of environmental samplesseparation.Fig.8.Separations of (a)basic compounds,(b)pesticides and (c)EPA 610on organic monolithic column by cLC.Experimental conditions:effective lengths,16.5cm ×75␮m i.d.,19.5cm ×75␮m i.d.and 20cm ×75␮m i.d.,for a–c respectively;mobile phases for (a),ACN/H 2O (40/60,v/v),for (b),ACN/H 2O (55/45,v/v),for (c),mobile phase A,water with 0.1%FA;mobile phase B,ACN with 1%FA;gradient,60%B to 100%B in 20min;flow rates,80,160and 140␮L/min for a,b,and c respectively (before split);detection wavelength,214nm.Elution order:(a)caffeine,carbamazepine,2,4-dinitroaniline,4-aminobiphenyl and 2,6-dichloro-4-nitroanine;(b)imidacloprid,carbaryl,thiram,parathion-methyl;(c),(1)naphthalene,(2)acenaphthylene,(3)acenaphthene,(4)fluorene,(5)phenanthrene,(6)anthracene,(7)fluoranthene,(8)pyrene,(9)benzo(a)anthracene,(10)chrysene,(11)benzo(b)fluoranthene,(12)benzo(k)fluoranthene,(13)benzo(a)pyrene,(14)dibenzo(a,h)anthracene,(15)benzo(g,h,i)perylene and (16)indeno(1,2,3-cd)pyrene.。