Focusing Review

CHROMATOGRAPHY, Vol.34 No.1 (2013)

Focusing Review

Simple LC using New Macroporous Polymers
Ken Hosoya
Graduate School of Life and Environmental Sciences, Kyoto Prefectural University
1−5 Shimogamo−hangi−cho, Sakyo−ku, Kyoto 606−8522, Japan

Abstract
New macroporous polymers have been prepared based on two different “platforms” to simplify rather complicated pretreatment processes for
LC analyses. For example, uniformly sized, molecularly imprinted polymer particles modified with sulfonic acids on outer surface easily removed humic acids in environmental water sample to improve detectability of really low level of the target molecule. Nicely controlled, 3D
skeletal co−continuous polymers (monolithic polymer) have been prepared using visco−elastic phase separation mechanism as well as theutilization of epoxy monomers and a diamine. In addition, a hydrophilic monolithic media based on ethylene oxide units simplified affinity
chromatography through avoiding non−specifically bound proteins even if those were utilized in protein lysate. All the macroporous polymers
have contributed to simple and accurate LC analyses.
Keywords : Macroporous polymer, HPLC, Selectivity, Monolithic media

selective concentration of target molecule is of course essential in

Introduction
Modern HPLC can realize really high performance separation

the case of low concentration of the target molecules, therefore

for highly complicated bio−related samples, for example, using

various types of pretreatment devices including particulate type,multi−dimensional, sophisticated HPLC system [1,2] coupled withmembrane type, as well as the other formats are now commercially

MS/MS detection. Environmental samples including various mis-available, while the detailed information will be published some-placed materials can be also selectively determined even if thosewhere else [3]. In recent years, the importance of analysis for rela-

are in ppt level concentration. Those analyses are now highly auto-

tively hydrophobic molecules, such as dioxins and/or poly−aro-mated, therefore, the data obtained seems to be reliable. This is be-matic hydrocarbons (PAH) has been continuing, however, highlycause the data observed was appropriately processed and calculatedhydrophilic molecules related to pharmaceuticals and personal carebased on the “sample” introduced into HPLC system and sepa-

products (PPCPs) become more important [4]. Because of highly

rated. In this context, we have to become aware of importance ofhydrophilic properties of those compounds, activated sludge treat-

the quality of sample injected. In MS detection, especially selective

ment is not effective therefore, it is now serious problem that possi-

ion mode, we tend to pay no attention to sample quality.

ble high concentration of those compounds was determined in river

If we look at overall process or strategy of LC analysis, sample

water just after sewage plant. A role of pretreatment device as well

preparation processes are not negligible to obtain suitable and ac-

as its process has been more important for LC analyses even if thecurate sample for LC system utilized. One of the important roles of

HPLC system has been highly automated. Because commerciallysample preparation processes is separation and/or isolation of theavailable solid phase extraction (SPE) media are not effective fortarget compound from matrix of impurities as much as possible andsome of PPCPs.

Corresponding author: Ken Hosoya
Tel: +81−75−703−5444
Fax: +81−75−703−5444
E−mail address: hosoya@kpu.ac.jp―１―

CHROMATOGRAPHY, Vol.34 No.1 (2013)

In this brief contribution, I will focus on preparation and applica-

bution, but this procedure is highly complicated. Therefore, sometion of new macroporous, polymer−based materials to simplify theof the special polymerization methods have been introduced to ob-

LC separation and/or analysis through unique pretreatment concept

tain uniformly sized, macroporous polymer particles through easiereffectuated by some useful traps. Two different platforms were em-

processes. I have employed one of the seed polymerization meth-ployed for the purpose, where particulate type and co−continuous

ods, namely, multi−step polymerization method [6,7]. Uniformly

type polymer−based materials will be introduced including experi-

sized polymer particles, packing materials realized high column ef-

mental details. There two platforms have been actively studied in

ficiency with much lower column pressure drop [8], however, es-

the world, however, the focus in this contribution will be thesential draw−back of the polymer−based packing materials has not

macroporous polymers having multi−dimensional separation

been improved by only size uniformity of polymer particles. An-

mechanism including retention and exclusion on one platform. Us-other draw−back of traditional polymer−based packing materials

ing these polymers, LC strategy and process must be simplified. In

was much lower column efficiency toward aromatic compounds

addition, it will be discussed that cursory pretreatments have a sig-

such as naphthalene and anthracene. I have had an improvement in

nificantly increased risk of inaccurate analytical results.

the draw−back using a hydrophilic cross−linking agent with co−monomer [9]. Fig. 1 reveals greatly improved column efficiency ofpolymer−based HPLC column packed with uniformly sized, poly-

Particulate type, polymer −based packing materialFirst of all, particulate type, macroporous polymer is briefly in-mer packing materials. This column afforded 13,300 plates/150

troduced. Usually particulate type polymer “beads” are prepared

mm length for naphthalene with larger k values compared with thatusing suspension polymerization method [5], namely pearl polym-of typical silica−based C18 column. Through this work, compatibil-

erization. This method is really easy to use, but one of the draw−

ity of the packed column in 100% water mobile phase has beenbacks of the method is to afford rather broad particle size distribu-

also proved because of hydrophilic properties of the polymer−

tion. Size classification is essential to improve particle size distri-based packing materials. This is really one of the contributions ofFig. 1 Column performance of polymer−based column and C18 column.
Column size: 2 mm ID×150 mm, Mobile phase: 50% (v/v) aqueous acetonitrile, Flow rate: 190 µL/min., Temperature: 40oC, Detection: UV
254 nm, Solutes: 1. Uracil, 2. Caffeine, 3. 2−Ethylpyridine, 4. Phenol, 5. Butyl benzoate, 6. Benzene, 7. N, N−diethylaniline, 8. Toluene, 9.
Phenylacetyl acetone, 10. Naphthalene, C18 column: Shiseido, CAPCELL PAK C18 UG120.

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CHROMATOGRAPHY, Vol.34 No.1 (2013)

Ken Hosoya

esting because EDMA packing material prepared using toluene as

uniformly sized polymer packing material to LC.
Another contribution of the uniformly sized polymer−based

porogenic solvent afforded only one peak for three isomers of xy-

packing materials is that detailed comparison of column character-lenes. Only porogen utilized for preparation of uniformly sized

istics and efficiency among various columns can be determined onpolymer particles can contribute the separation of isomers in LC

their appearances of chromatograms. This is simply because paContribution of molecularly imprinted packing materials

rameter of particle size and its distribution can be ignored. Fig. 2offered a glimpse into tiny differences in retention properties for al-

If we utilize some analytical target molecule as temple moleculekyl benzenes from benzene to pentylbenzene on the same station-in the preparation of polymers, the above mentioned “imprinting”

ary phases derived from the monomer, ethylene dimethacrylate

effect is called as molecular imprinting effect [13]. Molecularly im-(EDMA), except the porogenic solvent utilized [10]. The found dif-

printed packing material [14−18] has greatly contributed to target

ferences in retention properties were due to so−called porogen im-

selective concentration toward pharmaceutical samples or environ-printing effect [11,12], which was another contribution of polymer

mental pollutants existing with really low concentration in its ma-−based packing materials for molecular recognition in LC process.

trix. In molecular imprinting, real target molecule is usually util-

The left chromatogram (a) was obtained on the column packed

ized as the “template” molecule directly, however, I have utilizedwith EDMA polymer prepared using benzene as porogenic solvent,pseudo template molecule having similar structure or a part ofwhere retention time of benzene was slightly larger compared withstructure of the target molecules [19−28]. When tert−butyl phenolthat of on the other two chromatograms (b) and (c), while the cen-

was utilized the pseudo template for bisphenol A, Fig. 4 proved se-ter (b) is on that using toluene as porogenic solvent, where toluene

lective concentration of bisphenol A in environmental water in-

resulted in relatively larger retention time to narrower peak dis-cluding one of the natural organic matters, humic acid on the im-tance between toluene and ethylbenzene. The right one is chroma-

printed polymer cartridge [29]. The concentrated bisphenol A cantogram on EDMA by ethyl benzene as the porogenic solvent.

be easily detected using general UV detector. However, in the case

These facts mean that polymer−based packing materials canof high concentration ratio up to 1,000 times, tiny impurities suchmemorize the environment of polymerization conditions such as

as humic acid retained on the treatment device become possible

the shape of porogenic solvent in these cases. In fact, the porogenproblem for detectability of bisphenol A in HPLC analysis [30−imprinting effects realized isomer separation between xylenes as34]. Therefore I introduced new surface modification [35−38] toshown in Fig. 3 [11]. The separation (a) was done using the EDMA

molecularly imprinted polymer packing material using some func-

packing material prepared using ortho xylene as the porogenic sol-tional monomers to exclude humic acid [39−42].

vent, while separation (b) was realized the packing material pre-As shown in Fig. 5, on non−surface modified column, impurities

pared using para xylene as the porogenic solvent. It is really inter-

were detected at the peak front. The hydrophilic modification using

Fig. 2 Chromatograms of alkylbenzenes on EDMA stationary phases prepared with (a) benzene, (b) toluene, and (c) ethylbenzene as porogen.
Mobile phase, 80% aqueous methanol. Flow rate, 0.7 mL/min. Detection, UV 254 nm. Solutes: 0, benzene; 1, toluene; 2, ethylbenzene; 3,
propylbenzene; 4, butylbenzene; and 5, pentylbenzene.

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CHROMATOGRAPHY, Vol.34 No.1 (2013)

Fig. 3 Chromatograms of xylene isomers on EDMA stationary phases prepared with (a) o−xylene and (b) p−xylene as porogen.
Mobile phase, 80% aqueous methanol. Flow rate, 0.7 mL/min. Detection, UV 254 nm.

Fig. 4 Chromatograms of humic acid and bisphenol A before and after the treatment with prepared polymer packed cartridge.
Condition of HPLC evaluations: Mobile phase: 50% methanol aq. Flow rate: 1.0 mL/min Detection: UV 254 nm., Column: C18 column(Merck) 100 mm×4.6 mm (I.D.) Temperature: 30oC.

―４―CHROMATOGRAPHY, Vol.34 No.1 (2013)

Ken Hosoya

Co−continuous type separation media

GDMA (glycerol di−methacrylate)/GMMA (glycerol mono−methacrylate) much reduced the impurities, but not perfect level. Pack-

Monolithic columns have been extensively investigated foring materials modified with a functional monomer, namely MASKHPLC applications as an alternative to a packed column with silica(methacrylic acid 3−sulfopropyl potassium salt) electrostaticallyparticles conventionally used. The merits of the monolith haveexcluded impurities to realize nearly perfect exclusion of the impu-

been cited such as simpler preparation in situ (confined in mold),

rities. The exclusion of impurities greatly contributed efficiency of

the alleviation of the time−consuming packing process, and higher

ionization process of MS detection as shown in Fig. 6. The MASK

permeability enhancing the diffusion of solute molecules to the po-

modified column clearly afforded much greater SIM (selected ionrous stationary phase, and the capability of manufacturing a longmonitoring) peak compared with that on non−modified column.

column with higher efficiency (larger plate number) as well.

This phenomenon is probably due to concentrated impurities as

The monolithic column is commonly classified into two catego-

shown on lower chromatogram of Fig. 6 by UV detection. Unex-

ries, (organic) rigid polymer and silica monolith. Firstly, the poly-pectedly, MS detection with SIM mode is affected by concentrated

mer monoliths have been widely investigated for HPLC application

impurities, which is possible problem for environmental analysis.

since the early 1990s. The polymer monolith preparation has been
carried out mainly by the free radical polymerization of vinyl or

New automated pre−concentration HPLC system [43] with two

methacrylate monomers in a rather poor porogenic solvent selected

multi−channel valves as illustrated in Fig. 7 was introduced further

among alcohols, aromatic hydrocarbons, ethers or their mixture ascontribution for accurate analysis of environmental samples requir-

claimed in the patent by Svec and Frechet [45]. The recent status ofing pre−concentration [44]. This two valve system resulted in

the polymer monoliths has been reviewed by Svec [46].

much longer running time for analytical column due to avoiding di-

However, the preparation of polymer monoliths is usually car-rect charge of environmental water sample. Using surface modi-

ried out with the said poor porogenic solvents as major component

fied, molecularly imprinted, pre−concentration column with themore than 50 vol% (isometric composition) for creating a macro-

HPLC system, practically detectable peak of the target molecule,porous structure for liquid flow. Under such solvent rich condition,bisphenol A was obtained as proved in Fig. 8 using simple UV de-the van der Waals attraction of the growing polymer chains over-tection [40]. This combination of the pre−concentration column

comes their mutual steric hindrance and dominates the interaction

and the two valves HPLC system highly contributed to simple andbetween the polymer chain and the solvent [47]. This enhances theaccurate LC analyses.

segregation of polymer (phenomenally, free energy of mixing in-

Fig. 5 Comparative chromatograms of lake water obtained MASK−(MIP−TM), GDMA/GMMA− (MIP−TG), non− (MIP−TN) modified
MIPs used for the pretreatment column in the column switching HPLC system.
HPLC conditions: mobile phase, 20 mM sodium phosphate buffer (pH 7.0)−acetonitrile (70: 30 (v/v)); flow rate, 0.8 mL/min for analysis and2.5 mL/min for pre− treatment; column, Shim−pack VP−ODS (150 mm×4.6 mm); detection, UV 220 nm; temperature, 40oC concentration
volume, 50 mL.
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CHROMATOGRAPHY, Vol.34 No.1 (2013)

Fig. 6 Increased sensitivity by the removal of interference in MS detection.
LC/MS conditions were same as Table 1 except UV detection and pretreatment column.
UV detection was performed at 275 nm and for confirming the effect of MAS modification, non−surface modified MIPs were employed.

Fig. 7 Automated two valves HPLC system with pre−concentration column.

―６―CHROMATOGRAPHY, Vol.34 No.1 (2013)

Ken Hosoya

Expanded10㪪㪧㪛㪄㪈㪇㪘㫍㫇㩷㩿㪉㪎㪌㫅㫄㪀㪚㪈㪎㪄㪥㪦㪤㪈㪇୚Ꮧ㉼㪄㫅㫆㫎㪸㫊㪿㪄㪊㪇㩼㪄㪈㪚㪈㪎㪄㪥㪦㪤㪈㪇୚Ꮧ㉼㪄㫅㫆㫎㪸㫊㪿㪄㪊㪇㩼㪄㪈

8

V

BPA

0.020

Surface modified

0.015

2
0

0.010

5

10

15

20
min

25

30

㪪㪧㪛㪄㪈㪇㪘㫍㫇㩷㩿㪉㪎㪌㫅㫄㪀㪚㪈㪌㪄㪥㪦㪤㪈㪇୚Ꮧ㉼㪄㫅㫆㫎㪸㫊㪿㪄㪊㪇㩼㪄㪈㪚㪈㪌㪄㪥㪦㪤㪈㪇୚Ꮧ㉼㪄㫅㫆㫎㪸㫊㪿㪄㪊㪇㩼㪄㪈

0.025

4

0

㪪㪧㪛㪄㪈㪇㪘㫍㫇㩷㩿㪉㪎㪌㫅㫄㪀㪚㪈㪎㪄㪥㪦㪤㪈㪇୚Ꮧ㉼㪄㫅㫆㫎㪸㫊㪿㪄㪊㪇㩼㪄㪈㪚㪈㪎㪄㪥㪦㪤㪈㪇୚Ꮧ㉼㪄㫅㫆㫎㪸㫊㪿㪄㪊㪇㩼㪄㪈Non modified

Non-modified(upper)
Surface modified (lower)

6
V

0.030

㪪㪧㪛㪄㪈㪇㪘㫍㫇㩷㩿㪉㪎㪌㫅㫄㪀㪚㪈㪌㪄㪥㪦㪤㪈㪇୚Ꮧ㉼㪄㫅㫆㫎㪸㫊㪿㪄㪊㪇㩼㪄㪈㪚㪈㪌㪄㪥㪦㪤㪈㪇୚Ꮧ㉼㪄㫅㫆㫎㪸㫊㪿㪄㪊㪇㩼㪄㪈

35

40

BPA =
200ng/L

5

10

15

20
min

25

30

35

40

Fig. 8 Comparative chromatograms of Suwannee River NOM obtained with or without surface modification of MIP used for the pretreatment
column in the column switching HPLC system (left). Right figure is close up, expanded one.
HPLC conditions: mobile phase, 20 mM sodium phosphate buffer (pH 7.0)−acetonitrile (70: 30 (v/v)); flow rate, 0.8 mL/min for analysis and2.5 mL/min for pretreatment; column, Shim−pack VP−ODS (150 mmL×4.6 mm i.d.); detection, UV 275 nm; temperature, 40oC; concentration volume, 50 mL.

creases) [48] and the phase separation rapidly proceeds via spino-

provement in A term as well as C term of van Demeter equation as

dal decomposition. Therefore, the polymer monolith thus preparedsummarized in Table 1, which was obtained in capillary format ofbecomes a brittle agglomerated structure of globules with small

GDMA monolithic column. There results were the first experimen-

mechanical integrity. Such macroporous monoliths usually reveal atal prove of visco−elastic phase separation mechanism. But unfor-

coarse macroporous structure with the maze−like flow channels

tunately, the monolithic columns prepared using the above men-and insufficient population of the mesopores at the surface fortioned preparation method did not improve their column efficiency

small solute retention (according to IUPAC definition, mesopore iscompared with those on silica−based monolithic columns.

defined in size as less than 2 nm, mesopore is defined as 2−50 nm,
Another improvement in co−continuous polymer was done by

and macropore as larger than 50 nm, [49] respectively).

the use of epoxy resin type monolithic media [55]. Two monomers
My attention focuses on freezing such time−evolved bi−continu-

as depicted in Fig. 11 were simply polymerized in polyethylene

ous polymer structure induced via visco−elastic phase separation

glycol as porogenic solvent to afford nicely controlled co−continu-

by thermal or photo−initiated polymerization before the coarseningous polymers, where the morphology can be controlled by theof the structure so to optimize the monolith morphology as a HPLC

change of molecular weight of polyethylene glycol as well as po-column. In terms of this, the porogenic solvent is very important.

lymerization temperature as shown in Fig. 12. The detailed study

Recently, we reported the poly−GDMA monoliths with bi−con-revealed that the formation of polymer morphology was seriously

tinuous structures by thermally initiated free radical polymerization

affected by the change of polymerization temperature as shown inusing azo initiator [50−54]. We chose as a porogen the ultra−highFig. 13 based on expected two−step polymerization mechanism

molecular weight mono−disperse polystyrene solution in chloro-[56]. This is unimaginable from nicely controlled 3D skeletal struc-benzene. Our experiment indicated that the said polystyrene solu-

ture, The monolithic polymer did not directly contribute the im-tion presumably induced the visco−elastic phase separation afford-

provement in high column efficiency. However a kind of joke re-ing the bi−continuous structures of poly−GDMA monoliths. I pre-vealed that reverse “J” letter shape of the rod type monolithic poly-liminarily reported µ−HPLC measurement of poly−GDMA mono-

mer purified dirty water including sludge spontaneously involving

lith column. As shown in Fig. 9, much improved, 3D skeletal struc-anti−bacterial effect [57]. The driving power of liquid flow wastures of GDMA polymer were obtained using thermal radial po-

capillary action and rather large sand or sludge was excluded bylymerization. The bi−continuous (co−continuous) polymer has rea-

micron−size through pores by filtration. The adsorption towards

sonable bimodal pore size distribution as shown in Fig. 10 due toimpurities were achieved by rather hydrophobic properties of the

combination of low molecular weight solvent and ultra−high mo-polymer, while anti−bacterial action was probably due to combina-

lecular weight polymer. This method contributed the great im-tion of rigid hydrophobic part and rather hydrophilic amino− alco-―７―

CHROMATOGRAPHY, Vol.34 No.1 (2013)

Fig. 9 Comparison of GDMA gel morphology between GDMA gels prepared with different polymer porogen (Ps and PMMA solution inchlororbenzene) magnification＝3,000 right: PMMA Mw＝2,000,000, 3% w/v, GDMA/porogen＝35/65, v/v, polymerized at 60oC for 24 h
left: Ps Mw＝3,840,000, 3% w/v, GDMA/porogen＝35/65, v/v, polymerized at 60oC for 24 h.

Fig. 10 Mesopore and macropore data by BET and Hg intrusion combined, respectively; PS solution in chlorobenzene, Ps Mw＝3,840,000,
Ps %＝0, 1, 3 , w/v; polymerized at 60oC for 24 h with AIBN 10 mg/ml, GDMA/porogen＝35/65, v/v.
hol functional groups through disturbance of the cell membrane of

tinuous structure are sub−micron size skeleton and relatively large

bacteria.

through pores, where size exclusion chromatography proved existence of mesopores as well as macro−pores. A capillary column

The great improvement in column efficiency was effectuated byhaving 21.5 cm long realized up to 46,000 theoretical plates for al-

the use of three functional epoxy monomer namely TEPIC with thekyl benzenes in aqueous acetonitrile mobile phase. Due to rela-diamine BACM utilized in the previous section [58]. These two

tively hydrophilic characteristics, TEPIC based stationary phasemonomers afforded nicely controlled co−continuous structure also

can be utilized as a HILIC stationary phase in higher concentrationby the polymerization in polyethylene glycol as the porogen againof acetonitrile in water. Therefore, TEPIC based column can be

as shown in Fig. 14. The characteristics of TEPIC−based co−con-utilized for the separation of highly hydrophilic bio−related com-

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CHROMATOGRAPHY, Vol.34 No.1 (2013)

Ken Hosoya

Table 1. A and C term of van Deemter equation of poly−GDMA filled capillary.
Solutecolumn samples
1. GDMA/PS (Mw＝50,000, 5%) solution inchlorobenzene＝35/65, v/v
340 mm×200 µm i.d.
50% aqueous methanol
2. GDMA/PS (Mw＝3,840,000, 3%) solution inchlorobenzene＝35/65, v/v
330 mm×200 µm i.d.
50% aqueous methanol
3. GDMA/PS (Mw＝3,840,000, 3%) solution inchlorobenzene＝33/67, v/v
320 mm×200 µm i.d.
60% aqueous methanol
4. GDMA/PS (Mw＝3,840,000, 3%) solution inchlorobenzene＝33/67, v/v
320 mm×200 µm i.d.
80% aqueous methanol

Thiourea (non−retentive)

Acetophenone (retentive)

A termµm
34

C termms
223

A termµm
327

C term
ms
377

20

71

92

99

11

41

26

63

11

35

10

62

Conditions: Detection UV at 214 (entry 1) and 245 nm (entry 2,3) by off−column adaptor with a window defined 10 cm
from the outlet from the capillary; Solute, 0.1 mg/ml in methanol except thiourea (0.01 mg/ml).

Fig. 11 Monomers utilized for epoxy resin based monolithic polymers.
pounds. It was striking that relatively long capillary column filledlable by the change of polymerization condition including thewith TEPIC based co−continuous polymer could be wired on namechange of monomers, but has clear limitation of upper pore size.

card size, plastic plates as shown in Fig. 15 [59]. The “wired” col-For example, sub−millimeter size pores can’t be usually obtained

umn having 95 cm length afforded up to 150,000 plates for alkyl-

by usual phase separation mechanism. A thermoplastic co−polymer

benzenes with low column pressure drop. This wired type column

was focused to realize to create sub−millimeter size through pores.

is simply easy to use in capillary HPLC.

Poly (ethylene−co−vinyl acetate) was utilized for this purpose, after kneading process with so−called “pore template” at higher temperature, the mixture was excluded to create polymer−rod. The

Spongy type bi−continuous columns for fast concentrationpore template was washed with water to result in co−continuous

Morphology of co−continuous, monolithic polymers is control-―９―CHROMATOGRAPHY, Vol.34 No.1 (2013)

Fig. 12 Change in morphology of epoxy resin based monolithic polymers based on the feed ration listed in table under the SEM pictures.

Fig. 13 Change in morphology by the change of polymerization temperature.

spongy like polymer. Normal size column having 4.6 mm I.D canwhere as shown in Fig. 17 benzo[a]pyrene in a pyroligneous acidbe easily fabricated by insertion of the spongy type polymer rod as

was detected using general UV detection in sub−ppb level concen-

shown in Fig. 16 [60−64]. Interestingly, this spongy type column

tration in it. Exclusion of relatively hydrophilic solutes as men-showed preferable retention toward poly−aromatic hydrocarbons,tioned before nicely contributed effective concentration of aromaticwhile relatively poor retention properties for hydrophilic solutes

target molecule. In addition, the spongy type pre−concentrationsuch as alcohols, phenol, and carboxylic acids. In addition, to thiscolumn was also useful for total analysis of PAHs in river water as

retention characteristics and sub−millimeter size through pores,

shown in Fig. 18. The analysis of PAHs has been still very impor-relatively higher flow rate for concentration was employed withtant for environmental evaluation.

previously introduced automated pre−concentration HPLC system,―１０―

CHROMATOGRAPHY, Vol.34 No.1 (2013)

Ken Hosoya

Fig. 14 SEM pictures of TEPIC−based capillary columns.

12
8

3
Theoretical plate
1 Benzene䇭䇭 143900
2 Toluene 䇭䇭 䇭 149200
3 Ethylbenzene䇭䇭149900
4 Propylbenzene 149800
5 Butylbenzene 141500
6 Amylbenzene 138300
7 Hexylbenzene 134200

6

mVolt

4

4
5
6
7

2

0
0

20

40

60

Time (min)Fig. 15 Separation of alkylbenzenes using wired capillary column.
Conditions: Mobile phase: CH3CN/20 mM Sodium Phosphate buffer pH 7.0＝60/40 (v/v), Column: TEPIC − BACM Sheet column, Effective
length: 95 cm, ∆P＝6.5 MPa, u＝1.53 mm/s, Detection: 210 nm, Temperature: 20oC, Off column: 9 cm×50 mm i.d., Sample: alkylbenzenes(from benzene to hexyl benzene).

Contribution of monolithic polymer to affinity chromatogra-

lysate, while handling of particulate affinity resins might be simpli-phyfied. This presumably results in loss of valuable items (ligand as

Research on search of the target proteins using ligand immobi-

well as protein lysate). Therefore, it can be hardly utilized for very

lized affinity resin is a classic yet new method, which is gettingrare ligand such as naturally occurring compounds or toxic com-much attention for drug discovery, because the resin immobilizing

pounds. In addition, proteins are possibly denatured through thisbioactive compounds as ligand is able to capture its target proteins

column method due to relatively large volume in the column.

directly from a protein lysate prepared using possibly affected orTo avoid the disadvantages of column method, tiny amount ofgans and/or cells.
Traditionally the solid supports immobilizing ligand wereaffinity resins might be directly added into protein lysate to capture

packed into columns to capture the target proteins in fluid (affinity

the target proteins. In comparison to the column method, experi-

chromatography), but this column method tends to require rather

mental procedures using dispersed affinity resins should be compli-

large volume of materials including the gels as well as protein

cated, because collection of affinity resin is essential. Therefore,―１
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CHROMATOGRAPHY, Vol.34 No.1 (2013)

Fig. 16 Appearances of spongy type polymers and packed column. (a) Physical appearance, (b and c) SEM image, and (d) photo of column
end of the spongy monolithic column.

Fig. 17 Determination of benzo[a]pyrene using two valve HPLC system.
LC condition: Flow rate: 1.0 mL/min, Mobile phase: 80% MeCN aq., Column: ODS (150×4.6 mm i.d.), Pretreatment column: Spongymonolith (EVA D 50×4.6 mm i.d.), Temperature: 40oC, Detection: PDA (254 nm), Concentrate flow rate: 2.5 mL/min, Concentrate time: 12min.

some devisal should be required for preparation of the affinity res-ety of ligand concentration was available. As summarized in Tableins. In addition to the devisal, quantitative immobilization of ligand

2, maximum ligand concentration was 125 µmol/ml, which isto the affinity gel, simplified experimental procedures includingmuch greater than that of a typical commercially available affinity

washing and elution steps, and effective binding of target proteinsbeads. Because ethylene oxide based, relatively hydrophilic mono-

without non−specifically bound proteins will be required.

mers were utilized, non−specifically bound proteins were greatly

Monolithic type, hydrophilic affinity resins were prepared using

reduced compared with that on commercial methacrylate type af-

ethylene oxide base methacrylate monomers including functional

finity beads. The monolithic type affinity reins prepared weremonomer depicted in Fig. 19 [65−69]. By the change of ratio of thecalled as Moli−gel, which is abbreviation of monolithic affinity

functional monomer that is utilized for ligand immobilization, vari-

gel.

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CHROMATOGRAPHY, Vol.34 No.1 (2013)

Ken Hosoya

Fig. 18 Separation of PAHs.
Chromatographic conditions: mobile phase: water/acetonitrile gradient, Flow rate: 1.5 mL/min for analysis, 1 mL/min for concentration, sample: 10 mL of Kamo river water spiked with PAH samples.Analytical column: Restek Pinacle II PAH (250 mmL.×4.6 mmI.D.)
Concentration column: SPONGE (50 mmL.×4.6 mmI.D.)
Column temperature: 40oC, Detection: RF−20A
Sample concentration: 2,3,4,7,8,12,13,14,15; 10 ng/L, 5,6,9,10,11; 20 ng/L, 1,13; 100 ng/L.

Fig. 19 Preparation of monolithic type affinity resin, Moli−gel.

Interesting experimental facts were observed using Ketoprofen,

tured the target protein on commercial Toyopearl. The detailed rea-

Ibuprofen, and Aspirin as ligands using the monolithic type affinity

sons have not been elucidated yet but, these investigations strongly

resins as well as commercial Toyopearl as base resins. As summa-

suggested that micro−environment on solid support seriously af-

rized Western blot data in Fig. 20 [70−72], completely opposite re-fected the results of affinity chromatography to result in misunder-

sults in capture of one of the common target protein of the ligands,

standing of the essential. Hydrophilic affinity gel, Moli−gel was

COX−1 were observed. These unexpected results in capturing wereapplied to elucidate possible target proteins of one of algae toxins,

affected by the density of ligand as shown in Fig. 21. On Moli−gel,Microcystin LR. The results were reported in our recent paper [73].

higher ligand density captured COX−1, while lower density cap-

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CHROMATOGRAPHY, Vol.34 No.1 (2013)

Table 2. Feed ratio of monolithic type affinity gels.
Lig−m (µl) (ratio)

DEG−m (µl)

9G (µl)DEG−p (ml)

ADVN (mg)

3.5 (0.1)
17.4 (0.5)
25.9 (0.75)
34.5 (1)68.9 (2)120.8 (3.5)
172.3 (5)

15.8

390.8

750

10

Fig. 20 Capture of COX−1 using Moli−gel and Toyopearl.
Buffer: 0.25 M sucrose, 0.3 mM DDC, 25 mM Tris−HCl pH 7.5, 0.5% Tween 20, Time for capture: 4oC, 4 h, Protein solution: Buffer spiked
with COX−1 (1.4 pmol/ml), Detection: SDS−sample buffer (25oC，1000 rpm, 10分), WB: Anti−COX−1 Mouse-mono, Anti−mouse−IgGHRP.

work.

Summary
Through this contribution, “simple LC” was set up as the theme.
However, the point of this contribution is to reveal importance ofExperimental

preparation of pre−treatment procedure of samples. Modern HPLC

Packing material utilized in Fig. 1

system has been greatly improved and highly automated rapidly,

Solvents: Acetonitrile and tetrahydrofuran (THF) were of the high-however, we have to still pay attention very carefully, whether theest grade for HPLC and used as received. Water utilized for mobile

sample applied to analysis or detection is really feasible enough orphase was ultra−pure water produced in the laboratory using anot. HPLC system probably reflects us some fact based on the sam-

Yamato, AUTO STILL Model wg−22 followed by a Branstead, E−

ple injected, but we have to consider the fact obtained is reallyPURE. Cyclohexanol as porogen was also purified using the stan-what reflects inside science information of target samples. In otherdard distillation technique.

word, sample preparation and/or pre−treatment have to be reallysuitable and accurate method for modern LC analyses. After thisMaterials: Glycerol dimethacrylate (GDMA) was a gift fromsummary, detailed experimental information will be listed. I sin-

Kyoeisya Chemicals Inc., (Osaka, Japan) and another alkyl meth-cerely thank all the students and stuff for their really great help andacrylate monomers were all purchased from Wako Pure Chemical―１
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CHROMATOGRAPHY, Vol.34 No.1 (2013)

Ken Hosoya

Fig. 21 Capture of COX−1 with Moli−gel and Toyopearl having different ligand density.
Buffer: 0.25M sucrose, 0.3 mM DDC, 25 mM Tris−HCl pH 7.5, 0.5% Tween 20, Capture test: 4oC, 4 h, rat brain lysate spiked with COX−1(1.4 pmol/ml), resolution: SDS−sample buffer (4oC, 1000 rpm, 10 min), WB: Anti−COX−1 Mouse-mono, Anti−mouse−IgG−HRP, CBB
stained.
Ltd., (Osaka, Japan). All the monomers utilized for preparation of

ml of the purified and de−oxygenized water was added to initiate

stationary phases [methyl methacrylate (MMA), butyl methacrylate

the soap free polymerization. After the initiation, 7 ml of styrene(BMA), and 2−ethylhexyl methacrylate, (2−EHMA)] were purified

was added every one hour and finally 2 ml of styrene was added af-

using standard distillation techniques under reduced pressure to re-ter 7 hours from the initiation. Total amount of styrene utilized wasmove polymerization inhibitors.

50 ml. After the completion of 24 hours’ polymerization, the emul-

Styrene monomer for the preparation of the seed particle utilized

sion system was purified using a centrifugation technique at 5,000

for following multi−step swelling and polymerization method, wasrpm for 30 minutes. The yield was up to 61% and the seed styrene

washed using 5% sodium hydroxide solution followed by saturated

particle was re−dispersed into pure water (5.52×10−2 g/ml) to be

sodium chloride solution and dried over calcium chloride followed

utilized as a seed dispersion.

by the distillation in vacuum. Benzoyl peroxide (BPO) as a radical
initiator was purified using re−precipitation technique from the

Preparation of stationary phases through multi−step swellingchloroform solution into methanol to remove aqueous stabilizer.

and polymerization method: To the suspension of seed particle

Potassium peroxysulfide as a water−soluble initiator was also puri-(1.44 ml), was added micro−emulsion of the activating solvent pre-fied using re−crystallization technique.

pared from dibutyl phthalate (0.405 ml), sodium dodecylsulfate(0.028 g) and the water 10 ml by sonifier. The swelling was com-

Preparation of the seed particle: To completely de−oxygenizedpleted in 4 hours at room temperature.

water (300 ml) through helium bubbling and boiling, 0.39 g of so-

The micro−emulsion was prepared by sonification from thedium chloride and 6 ml of the purified styrene were added. The

porogen, cyclohexanol (5 ml), BPO (0.10 g) and water (25 ml)

suspended system was heated up to 75°
C under argon atmosphere.

containing polyvinyl alcohol (dp＝500, 0.48 g). This micro−emul-

Then, aqueous solution of 0.27 g of potassium peroxysulfide in 50

sion was added into the swollen system prepared above and the―１５―swelling was continued for 5 hours. The further swelling was car-nal particle size was 5.5 µm in diameter, and CV values of the pre-ried out using the micro−emulsion of 5 ml of monomers (GDMA

pared particles were about 5%.

＋monomer) and 25 ml of water containing polyvinyl alcohol (dpChromatography: The polymeric stationary phases were packed＝500, 0.48 g) and completed in 5 hours at room temperature.
The polymerization was carried out at 70°
C for 24 hours. After

into stainless steel column (150 mm×4.6 mm i.d.) by slurry

the polymerization, the prepared particles were washed using

method using a mixture of methanol, 2−propanol, and glycerol aswater, methanol, THF, and acetone and dried for calculation of theslurry medium. HPLC was performed using a Shimadzu−LC 4 A

chemical yields. The prepared stationary phases were packed into

pump equipped with a Rheodyne 7125 valve loop injector, a Shi-semi−micro size, stainless steel columns (2 mm ID×150 mm) by

madzu SPD−2A UV detector, and a Shimadzu C−R4A integrator.

slurry method using mixtures of water, acetonitrile, and 2−propanol, and the composition of slurry solvent is varied depending on

Packing material utilized in Fig. 4

the properties of polymer packing materials.

Materials: Monomers, Ethylene glycol dimethacrylate (EDMA) as

Semi−micro HPLC system: NANOSPACE S−1 (Shiseido Co.,the cross−linking agent, and 4−vinylpyridine as the functional

Tokyo, Japan) was used for evaluation of stationary phasesmonomer, both from Wako Chemicals (Osaka, Japan) were effec-

equipped by UV detector..

tively purified by vacuum distillation techniques to remove polymerization inhibitor. Template molecules, p−t−butylphenol andPacking materials utilized in Fig. 2 & 3

bisphenol A were purchased from Nacalai Tesque (Kyoto Japan)

Materials: Ethylene dimethacrylate (EDMA) was purchased fromand used as received. A polymerization radical initiator, 2,2’−azo-Tokyo Kasei Kogyo Co., Ltd. (Tokyo, Japan) and purified by a dis-bis−(2,4−dimethyl− valeronitrile) (ADVN) was purchased fromtillation technique to remove radical inhibitors before use. All other

Wako Chemicals (Kyoto Japan) and purified using a standard puri-materials for the preparation of polymer stationary phases werefication method. A solvent realizing porous structure (porogenic

used without further purification. o−Xylene and p−xylene as poro-solvent), toluene from Nacalai Tesque was the highest grade andgens, and 2,2’−azobis−(2,4−dimethylvaleronitrile) as a radical in-

used as received.

itiator, were of the highest grade available from Wako Pure Chemical Industries, Ltd. (Kyoto, Japan). Other materials were of the

Preparation of the molecular imprinting polymer: To prepare

highest grade available from Nacalai Tesque (Kyoto, Japan).

polymer−based separation devices, we utilized two−step swellingand polymerization method, which afforded uniformly sized polymer particles, utilizing polystyrene seed particles as shape tem-Preparation of Uniformly Sized Polymer Stationary Phases.
Uniformly sized polystyrene seed particles were prepared by anplate. The polystyrene seed particles were prepared through anemulsifier−free emulsion polymerization method. The diameter of

emulsifier free emulsion polymerization, which has been reported

the polystyrene seed particles was ca. 1 µm. Uniformly sized,[9].

macroporous, cross−linked polymer stationary phases were pre-The two−step swelling and polymerization method easily af-

pared by a multistep swelling and polymerization method as fol-

forded uniformly sized polymer particles with following feed ratio;lows. The polystyrene seed particles (7.0×10−2 g/mL, 7.8×10−1

EDMA: 10.0 ml, 4−vinylpyridine as functional monomer: 0.992mL) were admixed with a microemulsion prepared from dibutyl

ml, toluene: 10.0 ml, p−t−butylphenol: 0.173 g, ADVN: 0.7 g.

phthalate (2.5×10−1 mL), sodium dodecyl sulfate (3.5×10−2 g),(EDMA/4−vinylpyridine/t−butylphenol＝46/4/1, in mole ratio).

2,2’−azobis (2,4−dimethylvaleronitrile) (5.0×10−2

The polymerization was carried out at 50°
C for 24 h.

g), and distilledwater (40 mL) by sonication. This suspension was stirred at 125

The prepared polymer particles were dispersed into methanol

rpm until oil droplets of the added emulsion were completely ab-

and the supernatant was discarded after sedimentation of the poly-sorbed on the seed particles at room temperature. A suspension ofmer particles. This procedure was repeated three times in methanol

cross−linking agent (5.0 mL), porogen (5.0 mL), poly (vinyl alco-

and twice in tetrahydrofuran (THF), and then the polymer particles

−1

g),

were filtered with a membrane filter and dried at room temperatureand distilled water (45 mL) was added to the swollen polystyrene

to determine the chemical yields. The chemical yields were almost

seed particles. This suspension was stirred at 125 rpm for 2 h atquantitative. The polymer particles had 10.4 µm in diameter withroom temperature. The polymerization was carried out at 50°
C forexcellent size uniformity.

hol) (DP) 500, saponification degree − 96 mol%) (7.2×10

24 h under argon atmosphere. The resulting polymer particles were
washed with methanol and tetrahydrofuran by a repeated sedimen-

Concentration of bisphenol A: We prepared water solution oftation− redispersion process and the yield was quantitative. The fi-bisphenol A including excess of humic acids as contaminant. The―１
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CHROMATOGRAPHY, Vol.34 No.1 (2013)

Ken Hosoya

water solution contained bisphenol A (2 ppm), while humic acids

while the solution was stirred at 125 rpm. Completion of the firstwas saturated in the water solution.

step swelling was determined by the vanishing point of oil dropletsin added micro−emulsion using an optical microscope.

First, the prepared polymer particles (0.5 g) were packed into a

A dispersion of 3 ml of toluene (porogenic solvent), 0.34 mL ofglass cartridge having a syringe shape followed by water flow as a

4−VP, 0.06 g of TBP or 0.04 g of MBP, 0.15 g of ADVN and 0.06

pre−treatment of the polymer adsorbent layer.
Second, the prepared water solution was pumped into the glass

g of sodium dodecylsulfate into 35 ml of water containing 0.45 g ofcartridge packed with the polymer adsorbents continuously (about

poly (vinyl alcohol) (degree of polymerization, DP＝2000; saponi-

300 ml) followed by pure water (10 ml). Third, methanol was

fication value＝86.5−89 mol%) as dispersion stabilizer was added

pumped into the glass cartridge to recover the adsorbed bisphenol

to the dispersion of swollen seed particles. This second step swel-

A. Finally, the concentration of bisphenol A recovered from theling was carried out at room temperature with stirring at 125 rpm.
After the second step swelling was completed, the other disper-

glass cartridge was determined by HPLC with C18 column.

sion of 3 ml of EDMA, 0.06 g of sodium dodecylsulfate into 35 ml
Packing materials utilized in Fig. 5, 6, and 8of water containing 0.45 g of poly (vinyl alcohol) was added to theMaterials:

dispersion of the swollen particles. This swelling step was carried

Monomers, ethylene glycol dimethacrylate (EDMA) as a cross−out for 6 h at room temperature while the solution was stirred at

linking agent, and 4−vinylpyridine (4−VP) as the functional mono-

125 rpm. For the polymerization of swollen particles, the aqueous

mer, both from Wako Pure Chemicals (Osaka, Japan) were effec-

dispersion was stirred at 50°
C for 24 h under argon atmosphere.

tively purified by vacuum distillation techniques to remove polym-

The polymer particles obtained were washed with water, methanol,erization inhibitor. Glycerol dimethacrylate (GDMA) and glycerol

and tetrahydrofuran to remove the porogenic solvent, templatemonomethacrylate (GMMA) were purchased from Kyoeisya

molecules other impurities. The feed ratio was as follows, EDMA−

Chemical (Osaka, Japan) and used without further purification. The

4−VP−template, 40: 8: 1 in mole ratio.
Some of obtained MIPs were surface modified as described intemplate molecule, p−tert−butyphenol (TBP) was purchased fromfollowing sections.

Nacalai Tesque (Kyoto, Japan) and 4,4’−methylenebisphenol(MBP) and butyl methacrylate (BMA) were purchased from Wako

Surface modification methods:

Pure Chemicals. A polymerization initiator, 2,2’−azobis−(2,4−di-

The non−modified polymer particles prepared using the multi−

methyvaleronitrile) (ADVN) and benzoil peroxide were purchased

step swelling and polymerization method (0.8 g) (base polymer

from Wako Pure Chemicals.
A solvent realizing porous structure (porogenic solvent), toluene

particles) were dispersed in 50 ml of acetone and the hydrophilicfrom Nacalai Tesque was of the highest grade. All chemicals formonomers of a mixture of GMMA and GDMA, same as describedpreparing HPLC mobile phase, sodium dihydrogen phosphate,previous section and ADVN were added (5% in weight ration of

disodium hydrogenphosphate and acetonitrile were purchased frommonomers) and polymerized at refluxing temperature of acetone.

Wako Pure Chemicals. Water for preparing BPA standard solution

The polymerization was continued for 24 h and the obtained parti-

was obtained from Milli−Q water purification system of Millipore

cles were washed with acetone and water in order.
The base polymer particles were dispersed in 50 ml of methanol(Bedford, MA, U.S.A).

and the ionic monomer of methacrylic acid 3−sulfopropyl potassium salt (MASK) and benzoyl peroxide (BPO) were added (5% in

Preparation of the molecularly imprinted polymer:
Uniformly sized polystyrene seed particles were prepared by aweight ratio to monomers) and polymerized at refluxing tempera-

typical emulsifier−free emulsion polymerization method and puri-ture of methanol. The polymerization was continued for 24 h andfied by a centrifugation method. The size of seed particles was

the obtained particles were washed with methanol and 1N HCl andaround 1 µm in diameter with excellent size mono−dispersity.

water in order.

Preparation of uniformly sized macro−porous polymer particle
Column packing method:by a multi−step swelling and polymerization method was carried

The prepared particles were packed into a stainless steel column

out as follows. In the first step, 0.162 ml of aqueous dispersion of−1

ml/ml) was ad-(30 mm×4.6 mm i.d.) by slurry techniques to evaluate their char-mixed with micro−emulsion of dibutyl phthalate (activating sol-acters. We mainly utilized mixture of water, isopropanol, andvent), 0.04 g of sodium dodecylsulfate, and 10 ml of distilled water

methanol as packing medium.

by sonication.

Chromatographic measurement

the purified polystyrene seed particles (2.23×１0

HPLC measurement was carried out with the LC−VP HPLC sys-

This first step swelling was carried out at room temperature―１
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CHROMATOGRAPHY, Vol.34 No.1 (2013)used as received.

tem from Shimadzu (Kyoto, Japan) consisted of a LC−10 Avp sol-

1) 2,2’−azobisisobutyronitrile, AIBN, Mw＝164.21, 98%, Pur-vent delivery pump, CTO−10 Avp column oven, FCV−12 AH twochased from Nacalai Tesque, Kyoto, Japan.

−position flow changeover valve, FCV−13 AL six−port flow selec-

2)tion valve, SIL−10 Avp automatic injector, Rheodyne 7725 manual

2,2’−azobis(2,4−dimethyl)valeronitrile,

ADVN,

Mw＝

248.37, 95%, Purchased from Wako Pure Chemical, Osaka, Japan.

injector (Cotati, CA) with 100 µL loop, SCL−10A system controller and a CLASS−VP work station software. A Coulochem II, electrochemical detector (ECD) was purchased from ESA (Chelmsford,

Porogenic solvent:MA, USA).

1) Toluene
Purchased from Nacalai Tesque and used after distillation from

Small hydrocarbons including BPA were analyzed by HPLC tocalcium hydride at bp (110.6°
C).

compare the retention times on respective MIPs.

2) Polymer porogenic solution

HPLC conditions for small hydrocarbons were as follows, Mo-

Polymer porogenic solution was prepared by dissolving polysty-

bile phase: water−acetonitrile＝55/45 (v/v), Flow rate: 0.3 mL/

rene powder in chlorobenzene.

min, Detection: UV 220 nm, Temp.: 40 deg., Column: packed withi) Chlorobenzene, 99% was purchase d from Nacalai Tesque andprepared MIPs (150 mm×4.6 mmi.d.).

was distilled at 35°
C, 20 mmHg before use.

Chromatographic applications:Surface modified MIPs were applied as pretreatment columns toii) Polystyrene, standard monodisperse samples were used. Mw=

actual determination of BPA with the column switching HPLC. To

50,000, 600,000, 3,840,000, Mw/Mn＝1.04 (Tohso Co., Japan),accomplish suppression of BPA contamination and determine theadded to chlorobenzene at 1% − 5% (w/v) and left at room tem-

BPA concentration in water samples, a special technique was re-perature for 24 h without any mechanical stirring for complete dis-quired. A column switching HPLC with a pump injection systemsolution

was one of the solutions. The pump delivered 50 mL of BPA stan-Preparation of polymerization solution:

dard solutions or environmental water samples and the BPA was

An initiator, 10 mg was weighted in 20 ml beaker with a plastic

concentrated on the pretreatment column. Then mobile phase wasspoon. Next, 2 ml of the porogenic solvent was added and stirred.

delivered a via six−port switching valve and then the concentrated

GDMA was filtered through 0.2 µm PTFE filter (DISMIC−25JP;

BPA was directed to the analytical column and detected by the de-

ADVANTEC TOYO, Japan) mounted to a syringe (5 ml; B. Braun

tector after the separation on the analytical column.

Melsungen AG), then added to the porogenic solvent and stirred

HPLC conditions employed for column switching HPLC were as

gently. Then, the mixture was poured into a test tube (10 mm inner

follows; mobile phase: 20 mM (sodium) phosphate buffer (pH＝7)/

diameter and 10 cm long). Then, the solution was bubbled with ar-acetonitrile＝65/35 (v/v), rinsing solvent: 20% (v/v) of acetonitrile

gon gas through for 10 min. All operations described above wereaqueous solution, flow rate for analysis: 0.8 mL/min, flow rate forcarried out at room temperature.

pretreatment: 2.5 mL/min, concentrated volume: 50 mL, analyticalcolumn: Shim−pack VP−ODS (150 mm L×4.6 mm I.D.), tem-

Capillary preparation:operature: 40 C, electrochemical detection: at＋0.35/＋0.55 V (ana-Commercial fused silica capillary coated with polyimide of 250

lytical cell, CH1/CH2, 1 µAF.S.) and ＋0.6 V (guard cell), UV de-or 200 µm ID was sampled with about 1.8 m long. Then, the capil-

tection: at 220 or 275 nm. For the reference, LCMS analysis waslary wound in a coil was filled with 1 N sodium hydroxide and im-compare to HPLC column switching analysis.

mersed in a bath at 60°
C for 1 h, and washed several times repeatedly with water and acetone flown through manually by a syringe.

Polymers prepared in Fig. 9The capillary inner wall was not specially treated for increasing in-Materials:

terfacial adhesion. GDMA/toluene (35/65, v/v) solution was filledin the capillary wound in a coil with a syringe pump (Harvard Apparatus Model 11). The pumping was continued until the solution

Crosslinking agentsof about 100 times of the capillary volume (0.57-0.88 ml) was

GDMA (glycerol dimethacrylate,
Mw＝pumped through. Then, the filled capillary coil was sealed with a

227, was provided by Kyoeisha Chemical, GP−101P, and used as it

packaging film (PARA Film, American National Can) at the bothwas.

ends and immersed in an oil bath kept at 60°
C for polymerization.

CH2 ＝C(CH3 )OCOCH2CH(OH)CH2OCO(CH3 )C＝CH2 ,The coil was picked up after 24 h and connected to LC pump (Shimadzu LC 6 A) for washing with tetrahydrofuran (THF) and thenInitiator:

methanol, for 24 h, respectively. The capillary of test length was

The following initiators for free radical polymerization were

―１
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CHROMATOGRAPHY, Vol.34 No.1 (2013)

Ken Hosoya

NOLOGIES, USA)..

cut out with a ceramic cutter.

Solvents: ultra−pure water was obtained through Milli−Q GPA
Measurements:

system, while methanol, acetonitrile, and tetrahydrofuran (THF)

Poly−GDMA prepared in a test tube

were purified by suitable distillation techniques.

Test tubes frozen in liquid nitrogen were broken manually to ex-

Chromatographic solutes; all the alkylbenzenes as well as alkyl

tract gel samples. The gels were immersed in THF for 24 h andphenyl ketones were commercially available and used as received.

dried in a draft for another 24 h. Then those were dried at 60°
C forPolystyrene standard samples were purchased from Showa Denko,

another 24 h and then in vacuum for 60 min.

Co., (Tokyo Japan) as size exclusion makers.
Equipment; A constant temperature oven DNE 400 (Yamato Co.)was utilized as polymerization reactor. High performance liquid

Observation with SEM:
SEM apparatus was a Hitachi S−3000N. The observation ofchromatograph was consisted of a LC−20 AT chromatographic

polymers was made after gold was vapor deposited on them. The

pump (Shimadzu), DGU−20 A as an on−line degasifier, and CE−

magnification was 400 to 5,000.

2075 UV detector (Jasco) equipped with RHEODYNE 7725 injector (RHEODYNE). Scanning electron micrograph was obtained using a Hitachi S−510. Pore size distribution was performed on a Mi-

Pore measurement:

cromeritics PORESIZEZR 9320.

BET (Brunauer, Emmett, Teller’s equation) apparatus, GEMINI
II (Micrometritics, USA) was used for mesopore measurement. Hg

Surface modification of inner wall of the capillary; the 100 µm

intrusion apparatus (Micrometrics, USA) was partly used for macroi.d. capillary was washed with 1 N NaOH aqueous solution andpore measurement.

kept at 70 oC for 30 min, followed by washing with pure water. The
capillary was washed with 1N HCl and kept at 70 oC for 30 min.

Polymer preparation described in Fig. 11 & 12:

followed by washing with pure water and acetone. After complete

4−[(4−aminocyclohexyl) methyl] cyclohexylamine (BACM)

removal of acetone by air, tetrahydrofuran and 3−aminoproplyl-

0.52 g (2.47 mmol) was dissolved in poly−ethylene glycol (PEG),

triethoxysilane (1:1 v/v) were flowed through the capillary andthen 2.33 g (6.84 mmol) of 2−[(4−{1−methyl−1−[4−(2−oxiranyl-

kept at 80 oC for 24 hours. After the reaction, the modified capil-

methoxy) phenyl] ethyl} phenoxy) methyl]oxirane (BADE) waslary was washed with ethanol repeatedly.

added into this solution with stirring. This polymerization mixture

Preparation of capillary column: TEPIC (1.60 g) and BACM

was poured into a glass test tube having 12 mm inner diameter. The(0.37 g) were completely dissolved in PEG #200 (7.00 g). This so-polymerization took place in oil bath at the prescribed temperaturelution was injected into the modified capillary by a syringe. Polym-

for 3 hours. The structures of monomers utilized and the amount oferization reaction was carried out at 80 oC for 12 hrs. The resulting

monomers as well as porogen were shown in Fig. 11. The resulting

capillary was washed out by water and methanol and dried inpolymers were removed from the test tube to be washed with watervacuo for 5 hrs.

followed by acetone, then dried at 60oC for 24 hours. We have pre-Measurement: Monolithic bulk material was prepared in test tube

pared three types of polymers with different molecular weight PEG

as well as preparation of capillary column. Pore size distribution

and polymerization temperature.

measured by mercury intrusion method. The surface area measured
by BET.

Polymer monolith in Fig. 14:
Preparation of Spongy monolith in Fig. 16:

Monomers: 4−[(4−aminocyclohexyl) methyl] cyclohexylamine(BACM) was purchased from Tokyo Kasei Co., (Tokyo, Japan)

The spongy−monolith was prepared as follows: polyolefin chips

and utilized as received. Tris−(2,3−epoxypropyl)−isocyanurate(consist of polyethylene and polyvinyl acetate: EVA resin) and(TEPIC) (racemic and chiral) was kindly donated by Nissanpore templates (water−soluble compounds: multiple alcohol) were

Chemical Co., (Tokyo, Japan) and utilized without further purifica-

melted at 130 oC and stirred to combine. The combined materialtion.

was extruded in columnar style at 130 oC. Then, the columnar ma-

Other materials: polyethylene glycol #200 (PEG 200) and #300

terial was cooled in water immediately and the stick shaped mate-(PEG 300) were purchased from Nacalai Tesque (Kyoto, Japan)

rial was obtained. After cooling, the stick material was washedand utilized as porogenic solvents. 3−Aminopropyltriethoxysilane

with water using ultrasonication to remove the water−soluble com-

was purchased from Nacalai Tesque and used as surface modifying

pounds. Here, the water−soluble compounds worked as pore tem-agent of capillary inner wall, while 100 µm i.d.×375 µm o.d.

plates. The porosity of obtained spongy−monolith was 74% and its

fused silica capillary was purchased from POLYMICRO TECH-

diameter of cross section was globally 4.7 mm.

―１
９―CHROMATOGRAPHY, Vol.34 No.1 (2013)

For packing of spongy−monolith to stainless column, we utilized

mura Chemical CO., LTD. (Wakayama, Japan). Toyopearl; AF−

the empty column having 4.6 mm−i.d. The diameter of spongy−

Amino−650 M (Toyopearl) was purchased from Tosoh Biosciencemonolith (4.7 mm) was larger than that of inter diameter of column(Pennsylvania, USA). The certified surface density of amino(4.6 mm). Therefore, we carried out the packing with taking advan-groups is as high as 92 µmol/ml. Affigel 102 (Affigel) was pur-tage of elastic characterization of spongy−monolith. The proce-chased from Bio−Rad. The certified surface density of amino

dures of packing are shown as follows: One end of spongy−mono-groups is as high as 17 µmol/ml. 76% w/w phenol/EtOH, (phenol/

lith was compressed with thermal shrinkage tube at 120 oC. After

EtOH) was purchased from Applied Biosystems (Tokyo, Japan).

cooling, the shrinkage tube was removed and the diameter of theDevices:end of spongy−monolith was become smaller than 4.6 mm. Then,UV−mini 1240 UV−VIS spectrophotometer (SHIMADZU,

the spongy−monolith was macerated into water and the shrunk end

Kyoto, Japan), E−1010 Ion Spattering Apparatus (HITACHI), andof spongy−monolith was set into empty column. Finally, the exces-

Miniscope TM−1000 (HITACHI) were used.
Preparation of Moli−gel (monolithic type affinity gel) and calcu-

sive part of spongy−monolith was cut down and the column end

lation of theoretical amino group density:

module was connected. Here, the shrunk part of spongy−monolith

A standard Moli−gel was prepared. In this case, Lig−m 186.4

was completely cut down and only normal part (4.7 mm) could be

mg (172.3 µl), DEG−m 16.6 mg (15.8 µl), 9G 435.7 mg (390.8 µl),

packed into column.

DEG−p 844.7 mg (750 µl), and ADVN 10 mg were admixed in a
Preparation method of Moli−gel:vial at once and polymerized at 60 oC for 24 hr. The resulting poly-

Reagents:

mer was washed and comminuted, and then de−BOC reaction was

Solvents and reagents were utilized without further purification

carried out. Moli−gel with free amino group was then obtained.

unless it was particularly mentioned. Monomers and porogen were

To calculate the theoretical amino group density about above

structurally illustrated in our previous report.

prepared Moli−gel, 20 µl of Moli−gel (water dispersion) was

Nacalai Tesque, INC (Kyoto, Japan), Wako Pure Chemical In-

washed by 20% aqueous ethanol and dried. (3.92 mg/20 µl). The

dustries, Ltd (Osaka, Japan), Tokyo Chemical Industry Co., LTD.

amino group density of Moli−gel was measured by titration and(Tokyo, Japan), Bio−Rad Laboratories (Tokyo, Japan) were abbre-

ninhydrin method using above mentioned processes. In the case of

viated to be shown simply as Nacalai, Wako, TCI, and Bio−Rad inninhydrin method, resulting colored supernatant was diluted 8 or

the following experimental description.

16 times by 20% aqueous ethanol.

Trifluoro acetic acid (TFA) and ninhydrin were purchased from
Nacalai. 2,2’−Oxydiethanol (DEG−p), 2,2’−azobis (2,4−dimethyl-

References:

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chased from TCI. N −Methyl−2−pyrrolidinone dehydrated (dry−[5]NMP) was purchased from Kanto Chemical Co., INC., (Tokyo, Japan). 1−Ethyl−3−(3−dimethylaminopropyl)carbodiimide (water−solublecarbodiimide:

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[9]

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