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Journal of Chromatography A, 1321 (2013) 80– 87

Contents lists available at ScienceDirect

Journal of Chromatography A

j our nal homep age: www.elsev ier .com/ locate /chroma

ighly crosslinked polymeric monoliths with various C6 functionalroups for reversed-phase capillary liquid chromatography ofmall molecules

un Liua, H. Dennis Tolleyb, John S. Lawsonb, Milton L. Leea,∗

Department of Chemistry and Biochemistry, Brigham Young University, Provo, UT 84602, USADepartment of Statistics, Brigham Young University, Provo, UT 84602, USA

r t i c l e i n f o

rticle history:eceived 13 September 2013eceived in revised form 18 October 2013ccepted 22 October 2013vailable online 29 October 2013

a b s t r a c t

Three crosslinking monomers, i.e., 1,6-hexanediol dimethacrylate (HDDMA), cyclohexanedioldimethacrylate (CHDDMA) and 1,4-phenylene diacrylate (PHDA), were used to synthesize highlycross-linked monolithic capillary columns for reversed-phase liquid chromatography (RPLC) of smallmolecules. Selection of porogen type and concentration was investigated in detail. Isocratic elutionof alkylbenzenes at a flow rate of 300 nL/min was performed using HDDMA and CHDDMA monolithic

eywords:iquid chromatographyolymeric monolithsapillary columnsmall molecules

columns. Gradient elution of alkylbenzenes using all three monolithic columns showed good separations.Optimized monoliths synthesized from all three crosslinking monomers possessed high permeabilities.Poly(HDDMA) monoliths demonstrated column efficiencies up to 86,000 plates/m. Column preparationof poly(HDDMA) monolithic columns was highly reproducible; the relative standard deviation (RSD)values (n = 3) for run-to-run and column-to-column were less than 0.26% and 0.70%, respectively, basedon retention times of alkylbenzenes.

. Introduction

Polymeric monolithic stationary phases characterized by con-inuous porous beds [1] were introduced in the late 1980s andarly 1990s by Hjertén [1,2], and Svec and Fréchet [3–6]. They areotentially good alternatives to packed columns for high perfor-ance liquid chromatography (LC). Compared to packed columns,onolithic columns have several attractive advantages, which have

een described by many excellent reviews [7–13]. Monoliths areasy and fast to fabricate, and do not require frits, have low backressure and can operate at high flow rates. Rapid separationsan be realized due to their high permeability. In addition, theirkeletal structures [8,10] can be varied during the preparation pro-ess [8], because the through-pores are not strictly dependent onpherical particle packing geometries, as is the case for packedolumns.

The most common monomers used to synthesize organicolymeric monoliths include styrene [14], acrylates [15], andcrylamides [16]. Polymeric monoliths are particularly suitable

tationary phases for separations of peptides [17], proteins [18],ucleic acids [19], and synthetic polymers [20]. However, their lowesoporosities and surface areas lead to poor chromatographic

∗ Corresponding author. Tel.: +1 801 422 2135.E-mail address: Milton lee@byu.edu (M.L. Lee).

021-9673/$ – see front matter © 2013 Elsevier B.V. All rights reserved.ttp://dx.doi.org/10.1016/j.chroma.2013.10.071

© 2013 Elsevier B.V. All rights reserved.

resolution for small molecules. In addition, polymeric monolithscan swell or shrink when organic solvents are added to the mobilephase. Therefore, most attention should be focused on improvingchromatographic efficiency and mechanical stability of monolithiccolumns. Recently, researchers have reported improvements inmonoliths for small molecule separations by modifying the prepa-ration conditions [14,21–24].

A typical polymerization system for monolith prepara-tion includes initiator, monomers (functional and crosslinkingmonomers), and porogen(s). Varying the porogen type and compo-sition, polymerization temperature and time, functional monomerto crosslinker ratio, and monomer to porogen ratio are the mostcommon methods used to vary the final monolith structure. Ithas been reported that high crosslinker concentration can providehigh mechanical stability and high surface area [25,26]. Actu-ally, crosslinkers not only provide monolith rigidity, but they canalso influence their surface properties by serving as functionalmonomers. Most acrylic monolith studies report the use of eth-ylene glycol diacrylate (EGDA) or ethylene glycol dimethacrylate(EGDMA) as crosslinkers [24,27–31]. Only a couple of these studiesfocused on the effect of the crosslinker [15,32]. Recent work hassuggested that use of a single-monomer/crosslinker in the synthe-

sis provides simpler optimization of the polymerization system,improved column-to-column reproducibility, better mechanicalstability and higher surface area due to the highly crosslinked net-work [22,33–35].

K. Liu et al. / J. Chromatogr.

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3.1. Selection of porogens

ig. 1. Chemical structures of dimethacrylate/diacrylate monomers with different6 functional groups.

In this study, we introduce a group of highly cross-linkedolymeric monolithic stationary phases prepared from singleethacrylate/acrylate based monomers. The structures of theseonomers are shown in Fig. 1. The separation performances of

hese monoliths were studied using a standard mixture of low-olecular-weight alkylbenzenes.

. Experimental

.1. Chemicals and reagents

2,2-Dimethoxy-2-phenylacetophenone (DMPA, 99%) and 3-trimethoxysilyl)propyl methacrylate (TPM, 98%) were pur-hased from Sigma-Aldrich (St Louis, MO, USA); 1,6-hexanediolimethacrylate (HDDMA) (see Fig. 1) was a gift from SartomerExton, PA, USA); 1,4-cyclohexanediol dimethacrylate (CHDDMA)nd 1,4-phenylene diacrylate (PHDA) (see Fig. 1) were pur-hased from Polysciences (Warrington, PA, USA). Water, methanol,ecanol, dodecanol, propylbenzene, butylbenzene, amylbenzenend uracil were also obtained from Sigma-Aldrich; acetonitrileACN), isobutanol, N,N-dimethylformamide (DMF) and ethylben-ene were purchased from Fisher Scientific (Pittsburgh, PA, USA);oluene was purchased from Mallinckrodt (Phillipsburg, NJ, USA);etrahydrofuran (THF) was purchased from Curtin Matheson Sci-

ntific (Houston, TX, USA). All porogenic solvents and chemicalsor monolith and mobile phase buffer preparations were HPLC ornalytical reagent grade, and were used as received.

A 1321 (2013) 80– 87 81

2.2. Fused silica capillary pretreatment

UV-transparent fused silica capillary tubing (75-�m i.d., 375-�m o.d., Polymicro Technologies, Phoenix, AZ, USA) was treatedwith TPM in order to attach the polymer to the capillary wall. Thetreatment procedure was reported by Vidic et al. [36] and Coutioset al. [37]. The capillary was connected to a syringe pump forwashing with ethanol and water for 30 min, respectively. The innersurface of the capillary tubing was treated with 1 M NaOH solutionfor 1 h. Both ends were sealed with GC septa and the capillary washeated in a GC oven at 120 ◦C for 3 h. Then, the tubing was washedwith water to remove NaOH, filled with 2 M HCl solution, and placedin a GC oven at 110 ◦C for 3 h. The tubing was rinsed with water andethanol and dried at 110 ◦C with a flow of nitrogen gas overnight.Then the capillary tubing was filled with 15% TPM/toluene (wt/wt)solution and placed in a GC oven at 35 ◦C overnight. Finally, the tub-ing was washed with toluene and acetone, successively, and driedwith nitrogen gas at room temperature.

2.3. Polymeric monolith preparation

All monomer solutions were prepared in 1-dram (4 mL) glassvials by admixing initiator, monomer, and porogen solvents (seeTable 1 for reagent compositions). All solutions were vortexed andthen degassed by sonication for a few seconds to avoid excessiveevaporation of porogenic solvents. The prepolymer mixture con-taining PHDA was heated in a GC oven at 70 ◦C for 30 s to promotedissolution of PHDA in the porogens, and the resultant solution wasvortexed for 30 s to ensure that it was well-mixed. Then, the reac-tion mixture was introduced into one end of the silanized capillaryby helium gas pressure. A 5-cm long empty section was left at theexit end of the capillary to provide for a detection window at theend of the monolith bed. The capillary was sealed with rubber septaat both ends and placed directly under a PRX 1000-20 exposureunit UV lamp (390 ± 15 nm, 1000 W, TAMARACK Scientific, Corona,CA, USA). Polymerization times of 1 to 6 min were evaluated. Theresultant monolith was flushed with methanol and then water toremove any porogens and unreacted residual monomers until astable pressure reading was obtained. Similar back pressures (perunit column length) were observed when the polymerization timewas longer than 3 min for HDDMA and CHDDMA, and 3.5 min forPHDA. Therefore, 3.5 min for HDDMA and CHDDMA monoliths and4 min for PHDA monoliths were selected as polymerization times toensure complete conversion of the monomers. The monoliths werecharacterized using an FEI Helios Nanolab 600 (Hillsboro, OR, USA)scanning electron microscope after coating with a thin (∼10 nm)conducting layer of gold.

2.4. Capillary liquid chromatography

An Eksigent Nano 2D LC system (Dublin, CA, USA) was used forthe chromatographic experiments. The injection volume was 30 nL.The two mobile phase components for elution of alkylbenzenes inRPLC were water (mobile phase A) and acetonitrile (mobile phaseB). On-column detection was performed using a Crystal 100 vari-able wavelength UV–Vis absorbance detector (Thermo SeparationProducts, MA, USA). Chrom Perfect software (Mountain View, CA,USA) was used for data collection and treatment. UV absorbancewas monitored at 214 nm.

3. Results and discussion

The selection of porogenic solvent(s) is an important step inthe preparation of monoliths. One of the monomers, CHDDMA,

82 K. Liu et al. / J. Chromatogr. A 1321 (2013) 80– 87

Table 1Compositions of reagent solutions.

Monolith Compositiona (g/wt%)b

Monomer Methanol Dodecanol DMF

HDDMA 0.36/33.03 0.49/44.95 0.24/22.02 –CHDDMA 0.36/40.00 0.33/36.67 0.21/23.33 –PHDA 0.36/22.93 – 0.50/31.85 0.71/45.22

wvmpwwmirmbmgtlasptcmiist4

dumdsptPteidu

TEs

fl

a All monoliths contained 1 wt% DMPA to monomer.b wt% related to total polymerization mixture.

as chosen for detailed study of porogen selection. Several sol-ents with different polarities were used in the synthesis of theonoliths. It was found that a transparent gel was obtained after

olymerization using decanol or dodecanol, indicating that theseere potentially “good” solvents for CHDDMA. The monomerould not polymerize using toluene or ACN. CHDDMA formed aonolith when dissolved in methanol or isobutanol after UV light

nitiation. However, the structures of these monoliths were notigid enough to be used as stationary phases. Rigid macroporousonoliths were obtained when methanol or isobutanol were com-

ined with decanol or dodecanol. Although CHDDMA could formonoliths with decanol, the monoliths gave very poor chromato-

raphic efficiency. When isobutanol was combined with dodecanol,he final monolith gave much higher back pressure than mono-iths prepared from the mixture of methanol and dodecanol. Inddition, it was much easier to fill the capillary using monomerolutions that contained methanol, and to flush the columns afterolymerization, as the viscosity of methanol is much lower thanhat of isobutanol. Therefore, a combination of methanol and dode-anol appeared to be the best porogen system for the CHDDMAonolith. The data in Table 2 show how the column back pressure

ncreases with an increase in the amount of dodecanol. In form-ng rigid monoliths from CHDDMA, dodecanol behaves as a “good”olvent and methanol as a “poor” solvent. The ratio of monomero total porogens was investigated, and the final ratio was set at0.0:60.0 (wt/wt).

At room temperature, PHDA is a solid and is very difficult toissolve in mixtures of methanol and dodecanol. Although its sol-bility improves with increasing temperature, the boiling point ofethanol is only 67.4 ◦C, which means that methanol evaporates

uring heating, which changes the composition of the monomerolutions. Therefore, methanol could not be used for synthesis ofoly(PHDA) monoliths. Isobutanol (boiling point 107.89 ◦C) was,herefore, tested as an alternative to methanol. Unfortunately,HDA did not totally dissolve until the temperature was increasedo 100 ◦C, which indicated that isobutanol was not a good choiceither. By using a mixture of dodecanol and DMF (individual boil-ng points of 259 and 153 ◦C, respectively), PHDA could be totally

issolved in 15 s at 70 ◦C. Consequently, dodecanol and DMF weresed as porogens for preparation of poly(PHDA) monoliths.

able 2ffect of dodecanol percentage of the total porogen solution on column back pres-ure for poly(CHDDMA) monoliths.a

% Dodecanol (wt%) Column back pressure (MPa)b

34.99 4.27 ± 0.0237.05 5.07 ± 0.0239.07 5.33 ± 0.0241.01 7.49 ± 0.0243.39 9.31 ± 0.03

a Conditions: 10 cm × 75 �m i.d. monolithic columns, acetonitrile, 300 nL/minow rate.b Average of three trials ± standard deviation.

3.2. Monolith morphologies

Fig. 2 shows SEM images of monoliths synthesized fromHDDMA, CHDDMA, and PHDA. From the SEM images, it is clearthat all three monoliths had skeletal structures composed of smallglobules. Poly(PHDA) had smaller throughpores than the other twomonoliths, which resulted in higher back pressure (17.24 MPa at300 nL/min flow rate). Poly(HDDMA) contained some large pores(Fig. 2A), which led to low back pressure (2.24 MPa at 300 nL/minflow rate).

3.3. Separation of alkylbenzenes

We obtained rigid structural monoliths using all three of themonomers, and all of the monolithic columns could be used to sep-arate alkylbenzenes. Fig. 3 shows gradient elution chromatogramsof uracil, toluene, ethylbenzene, propylbenzene, butylbenzene, andamylbenzene with the monoliths formed from the reagents listedin Table 1. The flow rate was 300 nL/min and the gradient was40–100% B in 10 min. As can be seen in Fig. 3, all peaks had goodsymmetries and narrow peak widths, ranging between 3.4 and 5.6 sat half height.

Fig. 4 illustrates the elution of alkylbenzenes using an HDDMAmonolithic column with different gradients and flow rates. The sixcompounds were eluted within 4 min using a 5 min gradient from40% to 100% B and a flow rate of 600 nL/min (Fig. 4A). As expected,a shallower gradient led to longer elution time, but providedbetter resolution. For example, resolution values for ethylben-zene and propylbenzene were 3.75 and 4.03 in Fig. 4A and B,respectively.

3.4. Chromatographic efficiency measurements

Fig. 5 shows isocratic elution of alkylbenzenes using HDDMA,CHDDMA, and PHDA monoliths at 300 nL/min (i.e., 1.13 mm/s),which was the optimized flow rate for CHDDMA mono-lithic columns. HDDMA and CHDDMA monoliths showed goodseparation performance for alkylbenzenes under isocratic con-ditions. However, the alkylbenzenes could not be baselineseparated using the PHDA monolith. The plate numbers for allof the monolithic columns were between 20,000 and 47,000plates/m measured using uracil as a nonretained compoundand between 40,000 to 65,000 plates/m for HDDMA and CHD-DMA columns using alkylbenzenes as retained compounds at300 nL/min.

The maximum theoretical plate numbers were 86,000(k = 0.364), 62,000 (k = 0.334), and 54,000 (k = 0.216) plates/mfor toluene as a retained compound using HDDMA, CHDDMA, andPHDA, respectively. A van Deemter curve for the HDDMA columnis shown in Fig. 6. The performance of the HDDMA monolith was

comparable to the performance of polymeric monoliths reportedpreviously [22,30].

All of the monomers used to prepare monoliths in this workhave C6 groups, albeit with different structures. The monolith with

K. Liu et al. / J. Chromatogr. A 1321 (2013) 80– 87 83

Fig. 2. SEM images of monoliths. (A) and (B) poly(HDDMA), (C) and (D) poly(CHDDMA), (E) and (F) poly(PHDA) (see structures in Fig. 1).

atflisgpiP

linear C6 structure demonstrated the best efficiency, followed byhose with cyclohexyl and then phenyl groups. It appears that theexibility of the linear C6 groups allows better analyte diffusion

nto the stationary phase (i.e., better mass transfer). In contrast, thetructures of the monoliths became more rigid when the functional

roups in the monoliths changed from linear C6 to cyclohexyl andhenyl groups, which made it more difficult for analyte diffusion

nto the polymer and interaction with the functional groups. TheHDA monolith had quite different properties compared to the

other monoliths. It was formed from a diacrylate monomer, whichdoes not contain the two methyl groups that are characteristicof the dimethacrylate monomers (i.e., HDDMA and CHDDMA).In addition, due to the conjugated effect of the phenyl moiety,the phenoxy group is a weaker electron donor than the alkoxy

group. These properties cause the PHDA molecule to have theleast hydrophobicity among the three monomers, which leads to amonolithic column that has the lowest selectivity for alkylbenzenes(Fig. 5C).

84 K. Liu et al. / J. Chromatogr. A 1321 (2013) 80– 87

0 2 4 6 8 10

0

5

10

15

20

UV

dete

ctio

n, m

V

Retention time, min

B

0 2 4 6 8 10

0

5

10

15

20

25

UV

dete

ctio

n, m

V

Retention time, min

A

0 2 4 6 8 10

0

2

4

6

8

10

12

14

16

UV

dete

ctio

n, m

V

Retention time, min

C

Fig. 3. (A–C) are RPLC separations of alkylbenzenes on monoliths synthesized from HDDMA, CHDDMA, and PHDA, respectively. Conditions: 16 cm × 75 �m i.d. monolithiccolumn; mobile phase component A was water, and B was acetonitrile; linear A–B gradient from 40% to 100% B in 10 min, and then isocratic elution with 100% B; 300 nL/minfl

TP

TR

ow rate; on-column UV detection at 214 nm. Peak identifications: uracil, toluene, ethylb

able 3ermeabilities of dimethacrylate/diacrylate monolithic columns for different liquids.

Liquid Relative polaritya Viscosity (mPa s)b

Water 1.00 0.89

Acetonitrile 0.46 0.37

Methanol 0.76 0.54

a Relative polarity data are from Ref. [38].b Viscosity, �, data are from online CRC Handbook of Chemistry and Physics, 89th ed.; Cc Permeability k = �Lu/�P, where � is the viscosity, L is the column length (16 cm in thid Average of six trials at different flow rates ± standard deviation.

able 4etention times of uracil and alkylbenzenes showing column-to-column reproducibility o

Retention time (min)

Uracil Toluene Ethyl

Column 1 1.920 4.189 4.730Column 2 1.914 4.131 4.666Column 3 1.923 4.159 4.692Mean 1.919 4.160 4.696Relative standard deviation (RSD) (%) 0.24 0.70 0.69

a Conditions are the same as in Fig. 3.

enzene, propylbenzene, butylbenzene and amylbenzene in order of elution.

Permeability (×10–14 m2)c,d

HDDMA CHDDMA PHDA

5.54 ± 1.07 1.22 ± 0.23 1.07 ± 0.232.15 ± 0.39 0.57 ± 0.12 0.43 ± 0.075.43 ± 1.30 0.83 ± 0.20 0.73 ± 0.15

RC: Boca Raton, FL, 2008–2009.s case), u is the solvent linear velocity, and �P is the column back-pressure.

f three independently prepared poly(HDDMA) columns.a

benzene Propylbenzene Butylbenzene Amylbenzene

5.333 5.966 6.561 5.265 5.908 6.505 5.293 5.932 6.521 5.297 5.935 6.529

0.65 0.49 0.44

K. Liu et al. / J. Chromatogr. A 1321 (2013) 80– 87 85

0

2

4

6

8

10

12

14U

V de

tect

ion,

mV

A

0 2 4 6 8 10

0

5

10

15

20

25

UV

det

ectio

n, m

V

Retention tim e, min

C

0

2

4

6

8

10

12

UV

dete

ctio

n, m

V

B

Fig. 4. Separations of alkylbenzenes on an HDDMA monolithic column. Conditions:linear A–B gradient from 40% to 100% B in (A) 5 min, 600 nL/min flow rate, (B) 10 min,600 nL/min flow rate, and (C) 10 min, 300 nL/min flow rate; other conditions andpeak identifications are the same as in Fig. 3.

3

leppd(msl

0

5

10

15

20

UV

dete

ctio

n, m

V

A

0

5

10

15

20

25

UV

dete

ctio

n, m

V

B

0 1 2 3 4 5 6

0

5

10

15

20

25

30

UV

det

ectio

n, m

V

Reten tion time, min

C

Fig. 5. Separations of alkyl benzenes using (A) HDDMA, (B) CHDDMA, and (C)PDMA monolithic columns (Table 1) under isocratic elution conditions. Conditions:16 cm × 75 �m i.d. monolithic column; 30% A/70% B mobile phase; 300 nL/min flow

ducibility is a very important characteristic of any LC column.Run-to-run and column-to-column reproducibilities were mea-

.5. Column permeability and rigidity

Back pressure measurements and column permeability calcu-ations for monoliths exposed to different solvents were used tovaluate the rigidities of the monoliths. To obtain plots of backressure versus flow rate, acetonitrile, methanol and water wereumped through 16-cm lengths of monolithic columns at sixifferent flow rates from 50 to 500 nL/min. Linear relationshipsFig. 7) between back pressure and flow rate (R2 > 0.999 for all

onoliths) clearly indicated that the monoliths were mechanically

table. The permeabilities calculated based on Darcy’s law areisted in Table 3. All monolithic columns were found to slightly

rate; other conditions and peak identifications are the same as in Fig. 3.

shrink in polar solvents, with the highest permeability in waterand only slight swelling in acetonitrile.

3.6. Reproducibility of poly(HDDMA)

In addition to good chromatographic performance, repro-

sured for the poly(HDDMA) monolithic column. Chromatogramsillustrating column-to-column reproducibilities for three

86 K. Liu et al. / J. Chromatogr. A 1321 (2013) 80– 87

0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.610

11

12

13

14

15

16

17

18

H, μ

m

line ar velocity, mm/s

Fig. 6. Plot of plate height (H) versus linear velocity for an HDDMA monolithic col-umn using toluene as a retained compound. Conditions: 16 cm × 75 �m i.d. column;mobile phase component A was water, and B was acetonitrile; 30% A/70% B mobilep

iFr(pv(gtTDfr[

F1

20 C

0

2

4

6

8

10

12

UV

det

ectio

n, m

V

B

0

5

10

15

20

25

UV

det

ectio

n, m

V

A

hase; on-column UV detection at 214 nm.

ndependent poly(HDDMA) monolithic columns are shown inig. 8. The run-to-run and column-to-column RSD values based onetention times (n = 3) were less than 0.26% and 0.70%, respectivelysee Table 4). The theoretical plate number RSD values for theoly(HDDMA) column (3 replicate measurements at 10 differentelocities) ranged between 0.778% and 7.16%. Measurementsn = 3) of the maximum theoretical plate number (86,000 plates/m)ave an RSD value of 1.58%. More than 60 runs were conductedo test the robustness of the poly(HDDMA) monolithic column.here was no noticeable change observed in column performance.

ue to the highly crosslinked network, monoliths synthesized

rom single crosslinking monomers typically exhibited excellentobustness, as demonstrated here and in our previous work22,34].

0 10 0 20 0 30 0 40 0 50 00

5

10

15

20

25

HDD MA CHDDMA PH DA Acetonitrile water Methanol

Col

umn

back

pre

ssur

e, M

Pa

Flow rate, nL/min

ig. 7. Effect of mobile phase flow rate on column back pressure. Conditions:6 cm × 75 �m i.d. monolithic columns.

0 2 4 6 8 10

0

5

10

15

UV

det

ectio

n, m

V

Retention time, min

Fig. 8. Chromatograms showing column-to-column reproducibility for HDDMAmonolithic columns using uracil and alkylbenzenes as analytes. Conditions and peakidentifications are the same as in Fig. 3.

4. Conclusions

New monolithic RPLC stationary phases based on singlemonomers were synthesized using UV-initiated free radical poly-merization. The performance of these new monolithic columnswere demonstrated using alkylbenzenes under RPLC condi-tions. SEM images were taken which showed different globular

morphologies for monoliths made from different dimethacry-lates/diacrylates. Among the monoliths, those prepared from thelinear 1,6-hexanediol dimethacrylate monomer provided the high-est efficiency (86,000 plates/m) and lowest backpressure. Gradient

atogr.

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lution of alkylbenzenes was achieved with high resolution usingll three columns. The test analytes were completely separatedn 10 min using 300 nL/min (1.13 mm/s) flow rate. Good run-to-un and column-to-column (n = 3) reproducibilities were observed,hich are mainly attributed to the use of single monomers in thereparation of the monoliths.

eferences

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