Analytica Chimica Acta 883 (2015) 90–98

A novel polymeric monolith prepared with multi-acrylate crosslinkerfor retention-independent efficient separation of small molecules incapillary liquid chromatography

Haiyang Zhang a,b, Junjie Ou b,*, Yinmao Wei a,**, Hongwei Wang b,c, Zhongshan Liu b,c,Lianfang Chen b,c, Hanfa Zou b,***aKey Laboratory of Synthetic and Natural Function Molecule Chemistry of Ministry of Education, College of Chemistry and Materials Science, Northwest University,Xi’an 710069, ChinabKey Laboratory of Separation Science for Analytical Chemistry, Dalian Institute of Chemical Physics, Chinese Academy of Sciences (CAS), Dalian 116023, ChinacUniversity of Chinese Academy of Sciences, Beijing 100049, China

H I G H L I G H T S G R A P H I C A L A B S T R A C T

� A crosslinker with multiple acrylategroups was first used for preparingpolymeric monoliths.

� The poly(LMA-co-DPEPA) monolithsexhibited the column efficiencies of111,000–165,000 N m�1 for alkylben-zenes in cLC.

� Highly crosslinked monoliths alsoexhibited retention-independentefficient chromatographic perfor-mance.

A R T I C L E I N F O

Article history:Received 6 February 2015Received in revised form 31 March 2015Accepted 1 April 2015Available online 3 April 2015

Keywords:Polymer monolithsCapillary liquid chromatography

A B S T R A C T

Low column efficiency for small molecules in reversed-phase chromatography is a major problemcommonly encountered in polymer-based monoliths. Herein, a novel highly crosslinked porous polymericmonolith was in situ prepared by using a multi-acrylate monomer, dipentaerythritol penta-/hexa-acrylate(DPEPA), as crosslinker, which copolymerized with lauryl methacrylate (LMA) as functional monomer in aUV-transparent fused-silica capillary via photo-initiated free-radical polymerization within 5 min. Themechanical stability and permeability of the resulting poly(LMA-co-DPEPA) monolith were characterizedin detail. One series of highly crosslinked poly(LMA-co-DPEPA) columns were prepared with relativelyhigher content of crosslinker (63.3%) in the precursor. Although they exhibited lower permeability, highcolumn efficiency for alkylbenzenes was acquired in cLC, and the minimum plate height (column B) was in

* Corresponding author. Tel.: +86 411 84379576; fax: +86 411 84379620.** Corresponding author. Tel.: +86 29 81535026; fax: +86 29 81535026.

Contents lists available at ScienceDirect

Analytica Chimica Acta

journal homepa ge: www.elsev ier .com/locate /aca

*** Corresponding author. Tel.: +86 411 84379610; fax: +86 411 84379620.E-mail addresses: junjieou@dicp.ac.cn (J. Ou), ymwei@nwu.edu.cn (Y. Wei),

hanfazou@dicp.ac.cn (H. Zou).

http://dx.doi.org/10.1016/j.aca.2015.04.0010003-2670/ã 2015 Elsevier B.V. All rights reserved.

H. Zhang et al. / Analytica Chimica Acta 883 (2015) 90–98 91

Dipentaerythritol penta-/hexa-acrylatePhoto-initiated polymerization

the range of 6.04–9.00 mm, corresponding to 111,000–165,000 N m�1. Meanwhile, another series ofpoly(LMA-co-DPEPA) columns prepared with relatively lower content of crosslinker (52.7%) in theprecursor exhibited higher permeability, but the minimum plate height (column E) was relatively low inthe range of 10.75–20.04 mm for alkylbenzenes, corresponding to 50,000–93,000 N m�1. Compared withcommon poly(LMA-co-EDMA) columns previously reported, the highly crosslinked poly(LMA-co-DPEPA)columns using a multi-acrylate monomer as crosslinker possessed remarkably high column efficiencyfor small molecules in cLC. By plotting of plate height (H) of alkylbenzenes versus the linear velocity (u) ofmobile phase, the results revealed a retention-independent efficient performance of small molecules inthe isocratic elution, indicating that the use of multi-functional crosslinker possibly prevents thegeneration of gel-like micropores in the poly(LMA-co-DPEPA) monolith, reducing the mass transferresistance (C-term).

ã 2015 Elsevier B.V. All rights reserved.

1. Introduction

In recent years, monolithic columns have developed rapidly inpreparation, characterization and application in separation ofsmall molecules and macromolecules due to their excellentchromatographic properties [1,2], such as facile preparation, fastseparation at high flow rate, varieties of functionality and goodpermeability [3,4]. Generally speaking, based on the naturechemistry of the monolithic matrix, monolithic columns can bedivided into three types, including silica-based monolith, organic–inorganic hybrid monolith and polymer-based monolith. Silicamonolith provides good mechanical stability, permeability andhigh column efficiency for fast separation of small molecules [5,6].However, functionalization of the surface of silica monolith islabor-intensive and time-consuming, limiting its development[7,8]. Although the organic–inorganic hybrid monolith presentssome outstanding features such as facility of preparation, lessshrinkage, good mechanical stability and high surface area, thelacking types of commercial organic-trialkoxysilanes also limitsthe development of organic–inorganic hybrid monolith [9–11].

Compared with silica-based monolith and organic–inorganichybrid monolith, polymer-based monolith, including polymetha-crylate, polyacrylamide and polystyrene monoliths [12–14], showssignificant advantages, such as good chemical stability, simplepreparation and variety of functionality [15,16]. On account of thediversity of functional monomers, polymer-based monolith hasbeen widely applied to several chromatographic modes includingreversed-phase liquid chromatography (RPLC) [17], hydrophilicinteraction chromatography (HILIC) [18] and ion-exchangechromatography (IEC) [19] etc. However, it presents low columnefficiency as well as retention-dependent column performance for

Scheme 1. Schematic preparation of polymeric m

small molecules especially in the isocratic elution because of itsless mesopores and lower specific surface areas [20]. The presenceof micropores will be harmful to the separation efficiency sincethey allow small molecules to permeate in the gel structure andenhance chromatographic dispersion due to the stagnant masstransfer resistance [21,22]. Additionally, polymer-based monolithwill shrink or swell when the mobile phase contains highcontent of organic solvents. For these reasons, improving thechromatographic efficiency and mechanical stability of polymer-based monolith is still a research focus [20,23–25].

It is well known that crosslinker plays a significant role in thepreparation of polymeric monolith. The size, polarity, functionalgroups and content of crosslinker in polymerization mixture couldaffect the size of pores, permeability and column efficiency. Morenumbers of functional groups of crosslinker participated in thereaction, and higher crosslinking density was obtained. Then theseparation efficiency of monolithic column for small moleculessignificantly improved with the increase in the number offunctional groups [2,26]. Commercially available N,N0-methylene-bisacrylamide, divinylbenzene and ethylene dimethacrylate aregenerally selected as crosslinker to prepare various kind ofmonolithic columns [1,27–29]. Several attempts to optimize thecapillary monolith have led to columns affording the columnefficiencies of 35,000–50,000 N m�1 for benzene [24,25,27–32].Recently, Lee and co-workers [20] prepared a highly crosslinkedpolymeric monolith with 1,6-hexanediol dimethacrylate (HDDMA)as crosslinker, and the resulting monolithic column demonstratedthe column efficiency up to 86,000 N m�1.

In this study, we first selected dipentaerythritol penta-/hexa-acrylate (DPEPA) as crosslinker, which has five or six acrylategroups to participate in the polymerization for the formation of

onoliths via photo-initiated polymerization.

92 H. Zhang et al. / Analytica Chimica Acta 883 (2015) 90–98

highly crosslinked polymeric monolith (Scheme 1). This novelpolymeric monolith was fast fabricated by copolymerization ofDPEPA and lauryl methacrylate (LMA) as functional monomer viaphoto-initiated polymerization reaction. The optimal highlycrosslinked polymeric monoliths allowed for the separationof alkylbenzenes with a retention-independent efficientperformance. The resulting poly(LMA-co-DPEPA) monolith wascharacterized by Fourier-transformed infrared spectrum (FT-IR),scanning electron microscopy (SEM) and mercury intrusionporosimetry (MIP), etc. The chromatographic performance wasalso evaluated by separation of phenols, basic compounds andintact proteins by capillary liquid chromatography (cLC).

2. Experimental

2.1. Chemicals and materials

DPEPA (contains �650 ppm MEHQ as inhibitor), LMA (contains500 ppm MEHQ as inhibitor, technical grade), 3-(trimethoxysilyl)propyl methacrylate (g-MAPS �98%), formic acid (FA, for massspectrometry, �98%) and polystyrene standards (Mw = 800, 4000,13,200, 50,000, 90,000, 280,000 and 900,000) were purchasedfrom Sigma (St. Louis, MO, USA), and used directly without furtherpurification. 2,2-Dimethoxy-2-phenylacetopheone (DMPA, 99%)was gotten from Acros Organics (New Jersey, USA), and dissolved inhexyl alcohol (10%, w/w) prior to use. Lysozyme (chicken eggwhite), bovine serum albumin (BSA), myoglobin (horse heart) andRNase A were obtained from Sigma–Aldrich (St. Louis, Mo, USA).Cytochrome c (bovine heart) was from Aladdin (Shanghai, China).Tetrahydrofuran (THF), methanol and acetonitrile (ACN) wereHPLC-grade and acquired from Yuwang Group (Shandong, China).Thiourea, benzene, toluene, ethylbenzene, propylbenzene, butyl-benzene, phloroglucinol, pyrocatechol, para-cresol, 2,6-dimethylphenol, 2,4-dichlorophenol, caffeine, carbamazepine, 2,4-dinitroa-niline, 4-aminobiphenyl and 2,6-dichloro-4-nitroaniline were ofanalytical grade, and obtained from Tianjin Kermel Chemical Plant(Tianjin, China). Deionized water was prepared with a Milli-Qsystem (Milli-pore, MA, USA). The UV-transparent fused-silicacapillary with 75 mm i.d. and 365 mm o.d. was the product ofReafine Chromatography Ltd. (Hebei, China).

2.2. Preparation of polymeric monolith via photo-initiatedpolymerization

Prior to the preparation of a polymeric monolithic column, theinner wall of fused-silica capillary was pretreated and modifiedwith a layer of methacrylate groups. Briefly, the capillary was firstrinsed with 0.1 mol L�1 NaOH for 1 h, then washed with water tillpH 7, followed by 0.1 mol L�1 HCl for 5 h, and finally rinsed withwater and methanol. After that the capillary was dried by nitrogenstream at room temperature. Then the capillary was filled with asolution of g-MAPS and methanol (50/50, v/v) with a syringe andkept in the water bath at 50 �C overnight with both ends sealed

Table 1Detailed composition of the polymerization mixture and the permeability of obtained

Column DPEPA(mg)

LMA(mg)

Crosslinker content (w/w, %

A 35 12.4 73.8

B 30 17.4 63.3

C 30 17.4 63.3

D 30 17.4 63.3

E 25 22.4 52.7

F 25 22.4 52.7

G 25 22.4 52.7

H 20 27.4 42.2

Polymerization mixture contains 1.5 mL 10% DMPA (w/w, DMPA/hexyl alcohol).

with silicon rubber. Finally, the capillary was rinsed with methanolto flush out the residual reagents and dried with a stream ofnitrogen gas.

The prepolymerization solution consisting of functionalmonomer, crosslinker, initiator and porogenic solvents was mixedin a vial to form a homogeneous solution (the detail composition ofdifferent prepolymerization solution is listed in Table 1). Then theprepolymerization solution was introduced into the pretreatedcapillary with a syringe. After both ends were sealed with siliconrubber, the capillary was put in the UV crosslinkers (XL-1500A,l = 365 nm, Spectronics Corporation, New York, USA) at 365 nm for300 s. The capillary was then flushed with methanol to remove theresidual reagents. Finally, both ends of the capillary was kept in thewater for usage.

The rest of prepolymerization solution in the vial was also curedin the UV crosslinkers at 365 nm for 300 s to form bulk monolithicmaterial, which would be rinsed with ethanol three times, cut intosmall pieces, grinded using mortar and pestle, and then dried in avacuum at 50 �C for 2 days.

2.3. Structural characterization of the monolith

The microscopic morphology of polymeric monoliths wasobtained by SEM (JEOL JSM-5600, Tokyo, Japan). FT-IR characteriza-tion was carried out on Thermo Nicolet 380 spectrometer using KBrpellets (Nicolet, Wisconsin, USA). The specific surface area wascalculated from nitrogen adsorption/desorption measurements ofdry bulk monoliths using a Quadrasorb SI surface area analyzer(Quantachrome, Boynton Beach, USA). Pore size distribution wasmeasured by MIPon a PoreMasterGT-60(OuantachromeInstrumentCorporation, USA).

2.4. Chromatographic characterization of the monolith

The HPLC experiment was performed on an LC system coupledwith an Agilent 1100 micropump, a 7725i injector with a 20 mLsample loop and a UV detector (K-2501, Knauer, Berlin, Germany), inwhich the detection window was made by removing the polyimidecoating of fused-silica capillary tube with 50 mm i.d. in a position5.0 cm from the separation monolithic column outlet, and thedetection wavelength was set at 214 nm. A T-union connector wasused as a splitter, with one end connected to the capillary monolithiccolumn and the other end to a blank capillary (50mm i.d. and 150 cmin length). The flow rates of pump were set at 50–230mL min�1, theactual flow rates in the monolithic column were 80–380 nL min�1,resulting in split ratio about 1/600. All chromatographic data werecollected and analyzed using the software program HW-2000 fromQianpu Software (Shanghai, China).

The permeability was calculated according to Darcy’s law [33]by the equation: B0 = FhL/(pr2DP), where F (m3 s�1) is the flow rateof mobile phase, h is the viscosity of mobile phase (0.38 � 10�3 Pa sfor ACN), L and r (m) are effective length and inner diameter of thecolumn, DP (Pa) is the pressure drop of column. The data of DP and

polymeric monolithic columns.

) Hexyl alcohol(mL)

Ethylene glycol(mL)

Permeability(10�14m2)

140 20 –

140 20 0.83130 30 0.90120 40 0.92140 20 1.30130 30 1.45120 40 1.63140 20 2.13

H. Zhang et al. / Analytica Chimica Acta 883 (2015) 90–98 93

F were obtained on an ACQUITY UltroPerformance LC (Waters,USA). The flow rates of mobile phase were set at 0.1–1.5 mL min�1.

3. Results and discussion

3.1. Preparation of polymeric monoliths

The preparation of polymeric monoliths is illustrated inScheme 1. As expected, the morphology and permeability ofthe polymeric monolithic columns were obviously controlled bythe composition of prepolymerization solution, which wasinvestigated in detail. We selected DPEPA as crosslinker andLMA as functional monomer to prepare the polymeric monolithiccolumns via photo-initiated free-radical polymerization in aUV-transparent capillary. Meanwhile, the mixture of hexyl alcoholand ethylene glycol was selected as porogenic solvents, and theratio of porogenic solvents was optimized to achieve the polymericmonolithic columns with well-defined porous structure. Thepercentage of total monomers in polymerization mixture waskept at 25.8% (wt%). The polymerization mixture was directlyilluminated with UV radiation (l = 365 nm, 120 mJ cm�2) in thepresence of photo-initiator (1.5 mL, 10% DMPA, wt%). It could beobserved that white solid appeared in the vials after illuminatingfor 50 s, indicating rapid polymerization with photo initiationmode.

As shown in Table 1, a series of polymeric monolithic columnswere synthesized with different contents of crosslinker andfunctional monomer. It was found that the permeability wassignificantly increased from 0.83 to 2.13 � 10�14m2 when thecontent of DPEPA (in the total monomers, w/w%) decreased from63.3% to 42.2%, which were prepared with the same porogenicsolvents (columns A, B, E and H, Table 1). It was worth noting thathigher content of crosslinker in the precursor inclined to formmicroporous structure, resulting in lower permeability. The SEMmicrographs for columns A, B and E (Fig. 1) also proved that largermacropores emerged with a decrease of DPEPA content. What isworse, high content of DPEPA (column A) would lead to monolithmatrix detaching from the inner wall of fused-silica capillary(Fig. 1a and d).

The composition of porogenic solvents (hexyl alcohol andethylene glycol) is another important factor affecting the

Fig. 1. SEM micrographs of polymeric monolithic columns w

formation of poly(LMA-co-DPEPA) monolithic columns. As forthe columns B, C and D shown in Table 1, which were prepared withrelatively higher content of crosslinker (63.3%), the permeabilityslightly increased from 0.83 to 0.92 �10�14m2 when the propor-tion of ethylene glycol was increased from 12.5% to 25.0% (v/v).Meanwhile, as for the columns E–G, which were prepared withrelatively lower content of crosslinker (52.7%), the permeabilityremarkably increased from 1.30 to 1.63 � 10�14m2 when theproportion of ethylene glycol was increased from 12.5% to 25.0%(v/v). As a result, the ethylene glycol served as macroporogenicsolvent (poor solvent), while hexyl alcohol as microporogenicsolvent (good solvent). Additionally, it proved that the proportionof porogenic solvents had a little influence on the permeability ofhighly crosslinked polymeric monoliths fabricated with highcontent of crosslinker.

Considering both separation efficiency and permeability,columns B and E (Table 1) were employed for further characteriza-tion and evaluation by cLC in the following experiments. Therepeatability and reproducibility of poly(LMA-co-DPEPA) columns(columns B and E) were also characterized by measuring the relativestandard deviations (RSDs) of the retention factor (k) of benzene(thiourea as the void time maker) under the mobile phase of 60%ACN. Two columns exhibited good repeatability by cLC separation.The RSDs of run-to-run repeatability, column-to-column andbatch-to-batch reproducibility were all less than 0.2%, 1.3%, 2.1%(column B, n = 5) and 1.1%, 3.2%, 3.9% (column E, n = 5), respectively.The results demonstrated that the repeatability and reproducibilityof polymeric monolithic columns were acceptable.

3.2. Characterization of polymeric monoliths

The corresponding SEM micrographs of the poly(LMA-co-DPEPA) monolithic columns are shown in Fig. 1. The SEM imagesdemonstrated that column B (Fig. 1b and e) and column E (Fig. 1cand f) possessed uniform porosity structure and well linked to theinner wall of capillary. However, the monolithic matrix of column Adetached from the inner wall of fused-silica capillary as shown inFig. 1a and d. It was concluded that too high content of crosslinkerwould cause the polymer to shrink. Compared with column B,larger size of through-pores was found in column E, which allowedthe fluid to rapidly pass the capillary and provided a better

ith (a, d) column A, (b, e) column B and (c, f) column E.

Fig. 2. Pore size distribution of (a) column B and (b) column E; calibration curve of column B (c) and column E (d) by size exclusion chromatography. Experimental conditions:column dimension, 20.0 cm � 75 mm i.d.; off column dimension, 5.0 cm � 75 mm i.d.; flow rates, 66.7 nL min�1 (column B) and 140.1 nL min�1 (column E); mobile phase, THF;injection volume, 2.5 mL in spilt mode; detection wavelength, 214 nm; temperature, 25 �C; solutes, benzene, polystyrenes (Mw = 800, 4000, 13,200, 50,000, 90,000, 280,000and 900,000).

Fig. 3. FT-IR spectra of (a) DPEPA (crosslinker), (b) LMA (functional monomer) and(c) polymeric monolith.

Fig. 4. Back pressure against flow rate for polymeric monolithic columns (column Band E). Experimental conditions: effective length of 18.0 cm � 75 mm i.d.; mobilephase, ACN.

94 H. Zhang et al. / Analytica Chimica Acta 883 (2015) 90–98

permeability. Pore size measurement also indicated that largemacropores (>1.0 mm in diameter) existed in columns B and E, asshown in Fig. 2a and b. Their specific surface areas werecalculated as 27.4 (column B) and 19.8 (column E) m2g�1 basedon nitrogen adsorption/desorption isotherm. The size exclusionchromatography using THF as mobile phase also provided theporosity of the polymer monolithic columns as shown in Fig. 2cand d. The porosity of column B was measured at 45.2%. In contrastwith column B, the porosity of column E was higher, and couldreach 57.7%.

The IR spectrum of the polymeric monolith is shown in Fig. 3.The apparent peaks in the region of 1700–1750 cm�1 are stretchingvibration of the C¼O bond. The existence of a,b-unsaturatedcarbonyl bond (C¼O) in the structure of DPEPA and LMA is provedby a characteristic peak position at 1728 cm�1 and 1722 cm�1

(Fig. 3a and b), respectively. However, the stretching vibration ofthe C¼O band of poly(LMA-co-DPEPA) monolith appeared at1736 cm�1 (Fig. 3c). Meanwhile, the characteristic peak of thestretching vibration of C¼C bond of poly(LMA-co-DPEPA) monolithchanged slightly from 1635 cm�1 (DPEPA) and 1639 cm�1 (LMA) to1636 cm�1, and its intensity was remarkably decreased. Theseresults demonstrated that polymerization reaction successfullyoccurred, but a few unreacted C¼C bonds still existed.

As shown in Fig. 4, the mechanical stability of polymericmonolithic columns (columns B and E) was studied via the back-pressure measurement by connecting an 18-cm-long polymericmonolithic column to a NanoLC pump with ACN as the mobilephase. The back-pressure of two columns up to 40 MPa(R1 = 0.996 and R2 = 0.999) increased linearly with the increase offlow rate from 0.1 to 1.8 and 0.2 to 3.0 mL min�1, respectively.The results suggested that the resulting poly(LMA-co-DPEPA)monolithic columns owned satisfactory mechanical stability.

The chromatographic evaluation of two poly(LMA-co-DPEPA)monolithic columns (B and E) was performed by cLC separation ofalkylbenzenes as probes. As shown in Fig. 5a and b, fivealkylbenzenes were baseline-separated with good peak shapesusing ACN/H2O (60/40, v/v) as the mobile phase at 170 mL min�1

(before split) (thiourea as the void time marker). The elution ofalkylbenzenes was in the order of thiourea < benzene < toluene <

ethylbenzene < propylbenzene < butylbenzene according to their

Fig. 5. (a, b) Separation of alkylbenzenes on polymeric monolithic columns by cLC and (c, d) dependence of the plate height (H) of analytes on the linear velocity (u) of mobilephase on polymeric monolithic columns. Analytes: (1) thiourea, (2) benzene, (3) toluene, (4) ethylbenzene, (5) propylbenzene and (6) butylbenzene. Experimental conditions:columns, (a, c) column B and (b, d) column E; effective length of 18.0 cm � 75 mm i.d.; mobile phase, ACN/H2O (60/40, v/v); flow rates for (a, b), 170 mL min�1 (before split);detection wavelength, 214 nm.

Table 2Fitted values of A,B and C terms in van Deemter equation: H = A + B/u + Cu.

Analytes Column B Column E

A(mm)

B(mm2s�1)

C(ms)

A(mm)

B(mm2 s�1)

C(ms)

Benzene 2.40 1780 6.76 �13.92 12500 19.99Toluene 2.85 1420 5.67 �7.50 11000 14.95Ethylbenzene 2.13 1490 5.60 �4.76 8990 11.98Propylbenzene 2.93 1240 3.46 �0.31 5810 7.89Butylbenzene 0.39 1720 2.52 0.44 4450 6.16

H. Zhang et al. / Analytica Chimica Acta 883 (2015) 90–98 95

hydrophobicity, indicating typical reversed-phase separationmechanism. The long carbon chain of LMA contributed tothe hydrophobic property of poly(LMA-co-DPEPA) monolithiccolumns. It can be clearly observed that the separation ofalkylbenzenes on column B was performed within 13 min(Fig. 5a), while the separation of alkylbenzenes on column Ewas finished within 9 min (Fig. 5b).

The column efficiencies of two poly(LMA-co-DPEPA) columnswere evaluated with ACN/H2O (60/40, v/v) as the mobile phaseby cLC. The curves of plate height changed over flow rate in therange of 50–230 mL min�1 (before split) are depicted inFig. 5c and d. The minimum plate height of highly crosslinkedcolumn B was in the range of 6.04–9.00 mm for alkylbenzenes,corresponding to 111,000–165,000 N m�1. However, column Eproduced the minimum plate height of 10.75–20.04 mm foralkylbenzenes, corresponding to 50,000–93,000 N m�1. A largeamount of LMA-based polymeric monolithic columns hadbeen prepared in the past decades, and ethylene dimethacrylate(EDMA) was commonly selected as crosslinker [3,32,34–39]. As onekind of polymer-based monolithic columns, the resulting poly(LMA-co-EDMA) produced low column efficiencies for smallmolecules in the isocratic elution, on which the highest columnefficiency could only reach 53,000 N m�1 for alkylbenzenes by cLC.However, in our case, the highly crosslinked poly(LMA-co-DPEPA)columns exhibited higher column efficiency than those ofpoly(LMA-co-EDMA) monolithic columns.

The values of A, B and C terms in van Deemter equation are listedin Table 2. A-term is the eddy diffusion term that is influenced byhomogeneities of chromatographic bed. B-term value representsthe longitudinal diffusion, which dependents on analyte diffusioncoefficients and decreases with the increasing molecular mass[40]. C-term is the mass transfer term in mobile and stationaryphases. As shown in Table 2, the B-term and C-term values ofcolumn E are much larger than those of column B, while the A-termof column E was lower than column B. Compared with aC18-modified silica monolith, on which the A-term value wasabout 3.0 mm, the A-term values of columns B and E were all lowerthan 3.0 mm, likely benefiting from the homogeneous structure.

The C-terms of columns B and E were also lower (�20.0 ms) thanthose of common methacrylate-based monoliths [41,42]. Thelower C-term of column B ranged from 2.52 to 6.76 ms indicated agood communication between stationary phase and analytes.However, the C-term of column E was higher than that of column B,which ranged from 6.16 to 19.99 ms. The remarkable difference incolumn efficiency and C-terms was possibly related to the porestructure and specific surface area. Porous structure acceleratesthe rate of mass transfer as a result of obviously reducedconvection [41], and the specific surface area is a good indicationof the presence of mesopores as measured in the dry state [43],while the images of SEM could not accurately reflect the realmicrostructure in the dry state. The mesopores affect the masstransfer resistance, and hence the chromatographic separationefficiency and C-term. The column E (contains lower content ofcrosslinker) had a higher porosity of 57.7%, but it had lower specificsurface area (19.8 m2g�1) and lacked mesopores. Therefore,column E produced higher plate height (lower theoretical platesper meter) and C-terms. Contrary to column E, column B (containshigher content of crosslinker) had a lower total porosity of 45.2%,but larger specific surface area (27.4 m2g�1) and much moremesopores, which facilitated the efficient separation of smallmolecules [22].

Traditional porous polymeric monoliths exhibit small specificsurface area reaching only a few tens of m2g�1 since theylack the mesopores. Nonetheless, Urban et al. [43] prepared

Fig. 6. Relationship between retention factor (k) of alkylbenzenes and ACN content on polymeric monolithic (a) column B and (b) column E; dependence of log k on thenumber of carbon in alkyl chain on (c) column B and (d) column E. Experimental conditions: effective length of 18.0 cm � 75 mm i.d.; flow rate, 140 mL min�1 (before split).

96 H. Zhang et al. / Analytica Chimica Acta 883 (2015) 90–98

hyper-crosslinked polymeric monoliths with large surface area(more than 600 m2g�1 in dry state), showing high column efficiencyfor small molecules. It demonstrated that a large number ofmesopores and micropores were existent. However, the specificsurface areas of poly(LMA-co-DPEPA) monolith were only tens ofm2 g�1, indicating the lack of micropores in the dry state.

As shown in Fig 5, it is also observed that the efficiencies forthe strong-retained compounds (such as butylbenzene) weresignificantly higher than those of weak-retained compounds (suchas benzene). The results demonstrated that poly(LMA-co-DPEPA)monolith revealed a retention-independent efficient performanceof small molecules in the isocratic elution. As shown in Table 2, it

Fig. 7. Separations of (a and b) phenols and (c and d) basic compounds on polymeric m(a, b) (1) phloroglucinol, (2) pyrocatechol, (3) para-cresol, (4) 2,6-dimethyl phenol and ((4) 4-aminobiphenyl and (5) 2,6-dichloro-4-nitroaniline. Experimental conditions: e150 mL min�1 for (c, d) (before split); mobile phases, ACN/H2O (35/65, v/v) for (a, b) an

was apparent that the C-terms of butylbenzene were lower thanthose of benzene on both column B and column E. This low masstransfer resistance is possibly caused by the lack of micropores.Therefore, it can be deduced that the employ of multi-functionalcrosslinker prevents polymeric monolith from generation ofgel-like micropores, which reduced the permeation of smallmolecules in the gel-like structure and the mass transfer resistancein the polymeric monolith [44].

The effect of ACN content on the retention factor (k) of fivealkylbenzenes is shown in Fig. 6a and b. It could be found that theretention factors of five alkylbenzenes (thiourea as the void timemaker) on two columns B and E decreased with an increase of ACN

onolithic (a and c) column B and (b and d) column E by cLC, respectively. Analytes:5) 2,4-dichlorophenol; (c, d) (1) caffeine, (2) carbamazepine, (3) 2,4-dinitroaniline,ffective length of 18.0 cm � 75 mm i.d.; flow rates, 140.1 mL min�1 for (a, b) andd ACN/H2O (40/60, v/v) for (c, d); detection wavelength, 214 nm.

H. Zhang et al. / Analytica Chimica Acta 883 (2015) 90–98 97

content from 45% to 70%. The trend followed the principle ofreversed-phase chromatography for alkylbenzenes on the twopolymeric monolithic columns. The logarithm of the retentionfactors linearly decreased with an increase of ACN content.The hydrophobicity of polymeric monolithic columns could becharacterized by the methylene selectivity (aCH2 ) using thefollowing equation [45]:

logk ¼ nlogaCH2 þ logb;

where n is the carbon number of alkylbenzenes (��CH2��), andlog aCH2 and log b are constants for a given homologous series andchromatographic system (b is the retention factor of benzene). Asshown in Fig. 6c and d, the slopes of the lines represented thelogarithmic methylene selectivity of column B (the linearrelationship R > 0.996), giving the aCH2 values of 1.61, 1.52, 1.45,1.40, 1.35 and 1.32 at 45, 50, 55, 60, 65 and 70% ACN in mobilephases, respectively. Meanwhile, the aCH2 values of column E werealso calculated to be 1.67, 1.57, 1.51, 1.45, 1.40 and 1.36 at 45, 50, 55,60, 65 and 70% ACN in mobile phases, respectively. The values ofcolumn E were slightly higher than those of column B, illustratingthat increasing LMA content (decreasing the crosslinker content)would enhance the hydrophobicity of the resulting polymericmonolith.

3.3. Application of polymeric monolithic columns

For further researching the selectivity of the poly(LMA-co-DPEPA) monolithic columns, the phenols and basic compoundswere separated on both column B and column E by cLC. The resultsare shown in Fig. 7. As presented in Fig. 7a and b, baselineseparation of five phenols on column B was performed within20 min by using ACN/H2O (35/65, v/v) as the mobile phase, and thehighest column efficiency for 2,6-dimethyl phenol wascalculated at 86,000 N m�1. Comparing with column B, theseparation of these phenols on column E was also performedwithin 13 min, and the column efficiency for 2,6-dimethyl phenolwas only 52,000 N m�1. Similarly, 5 basic compounds werebaseline-separated on two poly(LMA-co-DPEPA) monolithiccolumns, as shown in Fig. 7c and d. The separation of basiccompounds on column B was completed within 25 min, exhibitingthe column efficiencies of 39,000–67,000 N m�1. Comparing withcolumn B, relative fast separation was obtained on column Ewithin 15 min, and the column efficiencies were decreased in therange of 19,000–52,000 N m�1. Although the column efficienciesfor the phenols and basic compounds on column E were lowerthan those on column B, the faster separation was obtained oncolumn E as expected. Column E was also selected to separate

Fig. 8. Separation of intact protein mixture on polymeric monolithic column E bycLC. Solute of standard protein mixture: (1) ribonuclease A, (2) cytochrome c,(3) lysozyme, (4) BSA and (5) myoglobin. Experimental conditions: effective lengthof 18.0 cm � 75 mm i.d.; flow rate, 140 mL min�1 (before split); mobile phase A, H2O,B, 0.1% FA in ACN, gradient 15% to 70% B in 20 min; detection wavelength, 214 nm.

intact proteins by cLC with gradient elution, as shown in Fig. 8. Fiveintact proteins were well separated within 16 min. These resultsshowed that potential application of such highly crosslinkedpolymeric monolithic columns in different chromatographicconditions for both large molecules and small molecules.

4. Conclusions

A novel highly crosslinked poly(LMA-co-DPEPA) monolithiccolumn has been successfully synthesized by using DPEPA ascrosslinker, which has multiple acrylate groups to manufacturehighly crosslinked monolithic columns. Since the use of multi-functional groups crosslinker could protect polymer monolith fromforming gel-like micropores and the absence of gel-like microporesreduced the mass transfer resistance, the poly(LMA-co-DPEPA)monolith exhibited the retention-independent efficient perfor-mance in the separation of small molecules. Therefore, it is deducedthat the multi-functional crosslinker could facilitate to improve thechromatographic separation efficiency for small molecules in cLC.Meanwhile, various kinds of functional monomers could becopolymerized with multi-functional crosslinker reagents likeDPEPA to fabricate a myriad of polymeric monoliths for differentchromatographic modes, such as reversed-phase liquid chroma-tography (RPLC), hydrophilic interaction chromatography (HILIC)and ion-exchange chromatography (IEC) etc.

Acknowledgments

The authors acknowledge funding support from the NationalNatural Science Foundation of China (No. 21475104) to Y. Wei theChina State Key Basic Research Program Grant (2013CB-911203,2012CB910601), the National Natural Science Foundation of China(21235006), the Creative Research Group Project of NSFC(21321064), and the Knowledge Innovation program of DICP toH. Zou as well as the National Natural Science Foundation of China(No. 21175133) to J. Ou.

References

[1] F. Svec, Organic polymer monoliths as stationary phases for capillary HPLC, J.Sep. Sci. 27 (2004) 1419–1430.

[2] R.D. Arrua, M. Talebi, T.J. Causon, E.F. Hilder, Review of recent advances in thepreparation of organic polymer monoliths for liquid chromatography of largemolecules, Anal. Chim. Acta. 738 (2012) 1–12.

[3] K. Liu, P. Aggarwal, J.S. Lawson, H.D. Tolley, M.L. Lee, Organic monoliths forhigh-performance reversed-phase liquid chromatography, J. Sep. Sci. 36 (2013)2767–2781.

[4] Z.J. Liu Ou, Z. Liu, J. Liu, H. Lin, F. Wang, H. Zou, Separation of intact proteins byusing polyhedral oligomeric silsesquioxane based hybrid monolithic capillarycolumns,J. Chromatogr. A 1317 (2013) 138–147.

[5] K. Cabrera, D. Lubda, H.M. Eggenweiler, H. Minakuchi, K. Nakanishi, A newmonolithic-type HPLC column for fast separations, HRC J. High Resolut.Chromatogr. 23 (2000) 93–99.

[6] N. Ishizuka, H. Minakuchi, K. Nakanishi, N. Soga, K. Hosoya, N. Tanaka,Chromatographic properties of miniaturized silica rod columns, HRC J. HighResolut. Chromatogr. 21 (1998) 477–479.

[7] Z. Lin, X. Tan, R. Yu, J. Lin, X. Yin, L. Zhang, H. Yang, One-pot preparation ofglutathione–silica hybrid monolith for mixed-mode capillary liquidchromatography based on thiol-ene click chemistry, J. Chromatogr. A 1355(2014) 228–237.

[8] P. Jandera, Advances in the development of organic polymer monolithiccolumns and their applications in food analysis—a review, J. Chromatogr. A1313 (2013) 37–53.

[9] J.D. Hayes, A. Malik, Sol–gel monolithic columns with reversed electroosmoticflow for capillary electrochromatography, Anal. Chem. 72 (2000) 4090–4099.

[10] Z. Liu, J. Ou, H. Lin, H. Wang, J. Dong, H. Zou, Preparation of polyhedraloligomeric silsesquioxane-based hybrid monolith by ring-openingpolymerization and post-functionalization via thiol-ene click reaction, J.Chromatogr. A 1342 (2014) 70–77.

[11] H. Wang, J. Ou, H. Lin, Z. Liu, G. Huang, J. Dong, H. Zou, Chromatographicassessment of two hybrid monoliths prepared via epoxy-amine ring-openingpolymerization and methacrylate-based free radical polymerization usingmethacrylate epoxy cyclosiloxane as functional monomer, J. Chromatogr. A1367 (2014) 131–140.

98 H. Zhang et al. / Analytica Chimica Acta 883 (2015) 90–98

[12] D. Guillarme, J. Ruta, S. Rudaz, J.L. Veuthey, New trends in fast andhigh-resolution liquid chromatography: a critical comparison of existingapproaches, Anal. Bioanal. Chem. 397 (2010) 1069–1082.

[13] J. Krenkova, F. Foret, F. Svec, Less common applications of monoliths: V.Monolithic scaffolds modified with nanostructures for chromatographicseparations and tissue engineering, J. Sep. Sci. 35 (2012) 1266–1283.

[14] J. Krenkova, F. Svec, Less common applications of monoliths: IV. Recentdevelopments in immobilized enzyme reactors for proteomics andbiotechnology, J. Sep. Sci. 32 (2009) 706–718.

[15] Z. Zhang, F. Wang, B. Xu, H. Qin, M. Ye, H. Zou, Preparation of capillary hybridmonolithic column with sulfonate strong cation exchanger for proteomeanalysis, J. Chromatogr. A 1256 (2012) 136–143.

[16] J. Ou, G.T. Gibson, R.D. Oleschuk, Fast preparation of photopolymerized poly(benzyl methacrylate-co-bisphenol A dimethacrylate) monoliths for capillaryelectrochromatography, J. Chromatogr. A 1217 (2010) 3628–3634.

[17] M.J. Ruiz-Angel, A. Berthod, Reversed phase liquid chromatography ofalkyl-imidazolium ionic liquids, J. Chromatogr. A 1113 (2006) 101–108.

[18] C.L. Lin, B. Singco, C.Y. Wu, P.Z. Liang, Y.J. Cheng, H.Y. Huang, Poly(triallylisocyanurate-co-ethylene dimethacrylate-co-alkyl methacrylate) stationaryphases in the chromatographic separation of hydrophilic solutes, J.Chromatogr. A 1272 (2013) 65–72.

[19] M. Talebi, A. Nordborg, A. Gaspar, N.A. Lacher, Q. Wang, X.Z. He, P.R. Haddad, E.F. Hilder, Charge heterogeneity profiling of monoclonal antibodies using lowionic strength ion-exchange chromatography and well-controlled pHgradients on monolithic columns, J. Chromatogr. A 1317 (2013) 148–154.

[20] K. Liu, H.D. Tolley, J.S. Lawson, M.L. Lee, Highly crosslinked polymeric monolithswith various C6 functional groups for reversed-phase capillary liquidchromatography of small molecules, J. Chromatogr. A 1321 (2013) 80–87.

[21] I. Nischang, O. Bruggemann, On the separation of small molecules by means ofnano-liquid chromatography with methacrylate-based macroporous polymermonoliths, J. Chromatogr. A 1217 (2010) 5389–5397.

[22] I. Nischang, O. Brueggemann, F. Svec, Advances in the preparation of porouspolymer monoliths in capillaries and microfluidic chips with focus onmorphological aspects, Anal. Bioanal. Chem. 397 (2010) 953–960.

[23] J. Urban, F. Svec, J.M. Frechet, Hypercrosslinking: new approach to porouspolymer monolithic capillary columns with large surface area for thehighly efficient separation of small molecules, J. Chromatogr. A 1217 (2010)8212–8221.

[24] S.H. Lubbad, M.R. Buchmeiser, Fast separation of low molecular weightanalytes on structurally optimized polymeric capillary monoliths, J.Chromatogr. A 1217 (2010) 3223–3230.

[25] Y. Li, H.D. Tolley, M.L. Lee, Preparation of monoliths from single crosslinkingmonomers for reversed-phase capillary chromatography of small molecules, J.Chromatogr. A 1218 (2011) 1399–1408.

[26] P. Jandera, M. Stankova, V. Skerikova, J. Urban, Cross-linker effects on theseparation efficiency on (poly)methacrylate capillary monolithic columns.Part I. Reversed-phase liquid chromatography, J. Chromatogr. A 1274 (2013)97–106.

[27] E.G. Vlakh, T.B. Tennikova, Preparation of methacrylate monoliths, J. Sep. Sci.30 (2007) 2801–2813.

[28] Y. Huo, P.J. Schoenmakers, W.T. Kok, Efficiency of methacrylate monolithiccolumns in reversed-phase liquid chromatographic separations, J. Chromatogr.A 1175 (2007) 81–88.

[29] R. Koeck, R. Bakry, R. Tessadri, G.K. Bonn, Monolithic poly(N-vinylcarbazole-co-1,4-divinylbenzene) capillary columns for the separation of biomolecules,Analyst 138 (2013) 5089–5098.

[30] D. Moravcová, P. Jandera, J. Urban, J. Planeta, Comparison of monolithicsilica and polymethacrylate capillary columns for LC, J. Sep. Sci. 27 (2004)789–800.

[31] H. Aoki, T. Kubo, T. Ikegami, N. Tanaka, K. Hosoya, D. Tokuda, N. Ishizuka,Preparation of glycerol dimethacrylate-based polymer monolith with unusualporous properties achieved via viscoelastic phase separation induced bymonodisperse ultra high molecular weight poly(styrene) as a porogen, J.Chromatogr. A 1119 (2006) 66–79.

[32] S.L. Lin, Y.R. Wu, T.Y. Lin, M.R. Fuh, Preparation and evaluation of1,6-hexanediol ethoxylate diacrylate-based alkyl methacrylate monolithiccapillary column for separating small molecules, J. Chromatogr. A 1298 (2013)35–43.

[33] S. Vanderwal, Low viscosity organic modifiers in reversed-phase HPLC,Chromatographia 20 (1985) 274–278.

[34] B. Buszewski, M. Szumski, Study of bed homogenity of methacrylate-basedmonolithic columns for micro-HPLC and CEC, Chromatographia 60 (2004)S261–S267.

[35] S.M. Matusova, K.I. Ivanova, I.A. D’yachkov, A.D. Smolenkov, A.V. Pirogov, O.A.Shpigun, Poly(alkyl methacrylate) monolithic columns for HPLC, Russ. Chem.Bull. 57 (2008) 2554–2560.

[36] Z.A. Alothman, A. Aqel, H.A. Al Abdelmoneim, A. Yacine Badjah-Hadj-Ahmed,A.A. Al-Warthan, Preparation and evaluation of long chain alkylmethacrylate monoliths for capillary chromatography, Chromatographia 74(2011) 1–8.

[37] S. Shu, H. Kobayashi, N. Kojima, A. Sabarudin, T. Umemura, Preparation andcharacterization of lauryl methacrylate-based monolithic microbore columnfor reversed-phase liquid chromatography, J. Chromatogr. A 1218 (2011)5228–5234.

[38] S. Shu, H. Kobayashi, M. Okubo, A. Sabarudin, M. Butsugan, T. Umemura,Chemical anchoring of lauryl methacrylate-based reversed phase monolithto 1/16 o.d. polyetheretherketone tubing, J. Chromatogr. A 1242 (2012)59–66.

[39] S.-L. Lin, Y.-R. Wu, T.-Y. Lin, M.-R. Fuh, Preparation and evaluation of poly(alkylmethacrylate-co-methacrylic acid-co-ethylene dimethacrylate) monolithiccolumns for separating polar small molecules by capillary liquidchromatography, Anal. Chim. Acta 871 (2015) 57–65.

[40] I. Nischang, Porous polymer monoliths: morphology, porous properties,polymer nanoscale gel structure and their impact on chromatographicperformance, J. Chromatogr. A 1287 (2013) 39–58.

[41] A.M. Siouffi, About the C term in the van Deemter’s equation of plate height inmonoliths, J. Chromatogr. A 1126 (2006) 86–94.

[42] E. Klodzinska, D. Moravcova, P. Jandera, B. Buszewski, Monolithic continuousbeds as a new generation of stationary phase for chromatographic andelectro-driven separations, J. Chromatogr. A 1109 (2006) 51–59.

[43] J. Urban, F. Svec, J.M.J. Frechet, Efficient separation of small molecules using alarge surface area hypercrosslinked monolithic polymer capillary column,Anal. Chem. 82 (2010) 1621–1623.

[44] I. Nischang, I. Teasdale, O. Bruggemann, Towards porous polymer monolithsfor the efficient, retention-independent performance in the isocraticseparation of small molecules by means of nano-liquid chromatography, J.Chromatogr. A 1217 (2010) 7514–7522.

[45] A.M. Weed, J. Dvornik, J.J. Stefancin, A.A. Gyapong, F. Svec, Z. Zajickova,Photopolymerized organo-silica hybrid monolithic columns: characterizationof their performance in capillary liquid chromatography, J. Sep. Sci. 36 (2013)270–278.