Thiol-Epoxy Click Polymerization for Preparation of Polymeric

Subscriber access provided by OHSU WEST CAMPUS OR GRAD INST

Article

Thiol-epoxy Click Polymerization for Preparation of Polymeric Monoliths with Well-defined 3D Framework for Capillary Liquid Chromatography Hui Lin, Junjie Ou, Zhongshan Liu, Hongwei Wang, Jing Dong, and Hanfa Zou Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.5b00006 • Publication Date (Web): 13 Feb 2015 Downloaded from http://pubs.acs.org on February 18, 2015

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

Analytical Chemistry is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 25

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Analytical Chemistry

1

Thiol-epoxy Click Polymerization for Preparation of

2

Polymeric Monoliths with Well-defined 3D Framework for

3

Capillary Liquid Chromatography

4 5

Hui Lina,b, Junjie Oua,*, Zhongshan Liua,b, Hongwei Wanga,b, Jing Donga, Hanfa Zoua,*

6 7

a

8

Physics, Chinese Academy of Sciences (CAS), Dalian, 116023, China

9

b

University of Chinese Academy of Sciences, Beijing 100049, China

10

*

To whom correspondence should be addressed:

11

Dr. Junjie Ou

12

Tel: +86-411-84379576

13

Fax: +86-411-84379620

14

E-mail: [email protected]

15

Prof. Hanfa Zou

16

Tel: +86-411-84379610

17

Fax: +86-411-84379620

18

E-mail: [email protected]

Key Laboratory of Separation Science for Analytical Chemistry, Dalian Institute of Chemical

19 20 21 22 23 24 1

ACS Paragon Plus Environment


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

25

Page 2 of 25

Abstract

26

A facile approach was developed for direct preparation of organic monoliths via

27

the alkaline-catalyzed thiol-epoxy click polymerization. Two organic monoliths were

28

prepared by using tetraphenylolethane glycidyl ether as a multi-epoxy monomer, and

29

trimethylolpropane

30

tetrakis(3-mercaptopropionate) as the multi-thiol monomer, respectively, in the

31

presence of a ternary porogenic system (DMSO/PEG200/H2O). The obtained organic

32

monoliths showed high thermal, mechanical and chemical stabilites. Benefiting from

33

the step-growth polymerization process, two organic monoliths possessed

34

well-defined 3D framework microstructure, and exhibited high permeabilities and

35

column efficiencies in capillary liquid chromatography. A series of neutral, basic and

36

acidic small molecules were used to comprehensively evaluate the separation abilities

37

of these monoliths, and satisfactory chromatographic performance with column

38

efficiencies ranged from 35,500 N/m to 132,200 N/m was achieved, demonstrating

39

good separation abilities of these organic monoliths prepared via thiol-epoxy click

40

polymerization approach. Besides, multiple retention mechanisms, including

41

hydrophobic, hydrophilic and π-π conjugate interactions were observed during the

42

separation of analytes on these monoliths, which would make them promising for

43

more extensive applications in capillary liquid chromatography.

tris(3-mercaptopropionate)

44 45 46 47 48 49 50 51 52 53 54 2


and

pentaerythritol

Page 3 of 25

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

55




INTRODUCTION

56

As a state-of-the-art format of stationary phases, the monolithic columns have

57

attracted substantial interest and been extensively applied in the field of analytical

58

chemistry from sample pre-treatment to microscale/nanoscale chromatographic

59

separation.1-6 Compared to both silica-based and organic-inorganic hybrid monoliths,

60

organic monoliths emerged earlier and were easily prepared, and also possessed richer

61

chemistry as well as better pH tolerance.7,8 However, the high heterogeneity of the

62

microstructure along with the low surface area make the chromatographic

63

performance of polymeric monoliths far from the level of packed columns, or even

64

other types of monolithic columns in liquid chromatography (LC), especially for the

65

separation of small molecules.9-12 Such remarkable differences have attracted great

66

concern in the scientific community.13 So far, many new methods have been

67

developed to address this great challenge,14,15 such as various living radical

68

polymerization et al.16-18 Recently, Svec and coworkers introduced hypercrosslinking

69

technique into the post-modification process of polymeric monoliths to dramatically

70

increase

71

performance.9,19-21 By contrast, although no increment of surface area was occurred,

72

benefiting from the well-controlled skeletal structures, the monoliths prepared via

73

epoxy-amide based ring-opening polymerization reaction also exhibited significant

74

improvement of separation performance in analysis of small molecules.22-25 These

75

successful precedents greatly inspired us to search new candidates in the arsenal of

76

chemistries for convenient preparation of organic monoliths with high column

77

efficiency in capillary liquid chromatography (cLC) separation of small molecules.

their

surface

areas,

thereby

improving

the

chromatographic

78

Benefiting from the excellent specificity, unrivaled efficiency and high

79

robustness to oxygen or water, the click chemistry has revolutionized almost all fields

80

of chemistry,26-30 and particularly, emerged as a useful tool in the arena of novel LC

81

stationary phase exploration.31-34 Up to now, however, there are very few papers on

82

direct preparation of monolithic columns via click polymerization, mainly focusing on

83

the

84

alkaline-catalyzed thiol-epoxy click chemistry, despite the success in industrial and

application

of

radical-mediated

thiol-ene

click

3


chemistry.35,36

The


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 4 of 25

85

biomedical applications, is overlooked in the fabrication of macroporous monoliths

86

for chromatographic application.28,37

87

Herein, a novel approach was developed for direct preparation of polymeric

88

monolithic columns via thiol-epoxy click polymerization reaction by using

89

tetraphenylolethane

glycidyl

90

trimethylolpropane

tris(3-mercaptopropionate)

91

tetrakis(3-mercaptopropionate) (PTM), as precursors (Figure 1). Systematic

92

characterizations of two organic monoliths, including SEM images, FT-IR spectra,

93

pore

94

adsorption/desorption measurement were carried out. Besides, the separation

95

capability of these polymeric monoliths was assessed comprehensively by separating

96

various samples, and high column efficiency was achieved for almost all types of

97

analytes in cLC.

size

measurement,

ether

(TPEGE)

thermal

and

a

(TPTM)

gravimetric

multi-thiol or

analysis

monomer,

pentaerythritol

and

nitrogen

98 99 100



EXPERIMENTAL SECTION Chemicals

and

Reagents.

TPEGE,

TPTM,

PTM,

101

3-glycidoxypropyltrimethoxysilane (GPTMS) and poly(ethylene glycol) (PEG, Mn =

102

200 and 10,000) were obtained from Aldrich (Milwaukee, WI, USA). Trypsin was

103

purchased from Promega (Madison, WI, USA). Bovine serum albumin (BSA), EPA

104

610 (including naphthalene, acenaphthylene, acenaphthene, fluorene, phenanthrene,

105

anthracene,

106

benzo(b)fluoranthene, benzo(k)fluoranthene, benzo(a)pyrene, dibenzo(a,h)anthracene,

107

benzo(g,h,i)perylene, indeno(1,2,3-cd)pyrene), thiourea, alkylbenzenes, trifluoroacetic

108

acid (TFA) and other standard compounds, such as polycyclic aromatic hydrocarbon

109

(PAHs), phenols, anilines and polystyrenes (Mw = 800, 4,000, 13,200, 35,000, 50,000,

110

90,000, 280,000, 900,000) were obtained from Sigma (St Louis, MO, USA).

111

Dithiothreitol (DTT) and iodoacetamide (IAA) were products of Sino-American

112

Biotechnology Corporation (Beijing, China). The fused-silica capillaries with

113

dimensions of 50 and 75 μm i.d. and 365 μm o.d. were supplied by the Refine

114

Chromatography Ltd. (Hebei, China). HPLC-grade acetonitrile (ACN) was used for

fluoranthene,

pyrene,

benzo(a)anthracene,

4


chrysene,

Page 5 of 25

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


115

preparation of mobile phases. The water used in all experiments was doubly distilled

116

and purified by a Milli-Q system (Millipore Inc., Milford, MA, USA). Ethanol,

117

tetrahydrofuran (THF), dimethyl sulphoxide (DMSO), KOH, NaOH, HCl and other

118

chemical reagents were all of analytical grade.

119

Pretreatment of Fused-silica Capillary. Prior to preparation of monolithic

120

columns, the inner wall of fused-silica capillary was pretreated and rinsed with 1.0

121

mol/L NaOH for 4 h, water for 30 min, 1.0 mol/L HCl for 14 h, and water for another

122

30 min, successively, and then dried by a nitrogen stream at room temperature. After

123

that, a solution of GPTMS/methanol (50%, v/v) was used for modification of the

124

capillary inner wall with epoxy groups. Then, the capillary was filled with

125

GPTMS/methanol solution, sealed with rubbers at both ends and submerged in a

126

water bath at 50 oC for 12 h. Finally, the capillary was rinsed with methanol to flush

127

out the residual reagents, and dried again with a stream of nitrogen gas.

128

Preparation of Monoliths via Thiol-epoxy Click Chemistry. For the

129

preparation of monoliths, a multi-epoxy monomer (TPEGE), a multi-thiol monomer

130

(TPTM or PTM) and the pore size regulator PEG 10,000 were firstly dissolved in the

131

porogenic solvents (DMSO, PEG 200 and H2O) to form a homogenous solution, then

132

a amount of catalyst KOH was added. After fully mixing and degassing by 20 min

133

sonication, the prepolymerization mixture was injected into the pretreated capillary

134

with a syringe. The filled capillary was then sealed with rubber stoppers and

135

immersed in a water bath at appropriate temperature for 4 h. After polymerization, the

136

monolithic column was rinsed with methanol to flush out the unreacted residual.

137

For the bulk material, the prepolymerization mixture was placed in centrifuge

138

tubes and reacted in a water bath at the same temperature for 4 h. After

139

polymerization, the bulk monolith was cut into smaller pieces, extracted with DMSO

140

and ethanol successively overnight in a Soxhlet apparatus and dried in a vacuum.

141

Instrumentation. The chromatographic evaluation of monolithic columns was

142

performed using an LC system equipped with an Agilent 1100 (Hewlett-Packard)

143

micropump and a UV detector (K-2501, Knauer, Germany). Data were collected at

144

214 nm or 254 nm, and processed by a chromatography workstation (Beijing Cailu 5



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

145

Scientific Instrument Ltd., Beijing, China). A 7725i injector with a 20 μL sample loop

146

was used. A T-union connector served as a splitter with one end connected to the

147

capillary monolithic column and the other end to a blank capillary (95-cm long, 50

148

μm i.d. and 365 μm o.d.). The outlet of the monolithic column was connected with a

149

Teflon tube to an empty fused-silica capillary (75 μm i.d. and 365 μm o.d.), where a

150

detection window was made by removing a 2 mm length of the polyimide coating in a

151

position of 5.5 cm from the separation monolithic column outlet.

152

SEM images were obtained by using a JEOL JSM-5600 scanning electron

153

microscope (JEOL, Tokyo, Japan). FT-IR was measured with a Bruker Tensor 27

154

FT-IR spectrometer (Bruker Daltonics, Ettlingen, Germany). Pore size measurement

155

was carried out on an Autopore IV 9500 (Micromeritics, Norcross, USA). Thermal

156

gravimetric analysis was performed on a Setsys 16/18 (Setaram, Caluire, France).

157

Nitrogen adsorption/desorption measurement of dry bulk monolith was carried out on

158

a Quadrasorb SI surface area analyzer and pore size analyzer (Quantachrome,

159

Boynton Beach, USA).

160

Preparation of BSA Tryptic Digest and cLC-MS Analysis. The tryptic

161

digestion of BSA and cLC-MS/MS analysis were performed according to procedures

162

previously reported by us with minor modification.23 The sample trapping was

163

achieved with a homemade C18-particle-packed trap column (4.0 cm length × 200 μm

164

i.d.), and the subsequent separation was carried out on a thiol-epoxy click chemistry

165

based monolith (34.0 cm length × 75μm i.d.) with an integrated emitter, which was

166

prepared by directly tapering the tip from the outlet of capillary. (Detailed

167

experimental procedures are provided in the Supporting Information.)

168 169



RESULTS AND DISCUSSION

170

Preparation of Organic Monoliths via Thiol-epoxy Click Chemistry. The

171

preparation of organic monoliths was performed according to the procedure and feed

172

recipes shown in Figure 1 and Table 1, respectively. For simplifying the optimization

173

process, a nearly equimolar amount of epoxy and thiol groups was first adopted. In

174

order to search a suitable porogenic system, several candidates, such as 6


Page 6 of 25

Page 7 of 25

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


175

DMSO/ethanol,

176

DMSO/PEG 200/H2O, were examined. Finally, the ternary porogenic system,

177

DMSO/PEG 200/H2O, was selected due to the appropriate solubility and porogenic

178

ability. Since the component of porogenic system has significant influence on the

179

morphology of monoliths, the content of each solvent was carefully optimized. As

180

shown in Table 1, for monolith TPEGE-PTM, it was found that the permeability

181

significantly decreased from 11.57 to 5.40 × 10-14 m2 (which calculated according

182

to the Darcy’s Law38 of permeability B0 = FηL/(πr2ΔP), detailed statement was shown

183

in Table 1) with the content of DMSO in the porogenic mixture (DMSO/PEG

184

200/H2O/PEG 10,000/KOH) increasing from 66.0 to 68.5% (wt%) (or the content of

185

PEG 200 in the porogenic mixture decreasing from 20.0 to 17.5% (wt%)) (as columns

186

A and B). If the amount of DMSO was further increased to 77% (wt%) (or the amount

187

of PEG 200 was further decreased to 9% (wt%)), the column could not even be

188

pumped through (as column C). Thus, the DMSO acted as the microporogenic solvent

189

(good solvent) in the porogenic system, while the PEG 200 served as the

190

macroporogenic solvent (poor solvent). The H2O had a similar effect as the PEG 200

191

on the final column morphology. As seen from the columns B, D and E, the

192

permeability of the monoliths increased when the amount of H2O was increased. In

193

contrast, the PEG 10,000 had an opposite porogenic effect to the PEG 200. Increasing

194

the content of PEG 10,000 would lead to a decrease of the permeability of the

195

monoliths (as columns B, F and G). Herein, the KOH solution (0.25 mol/L) was

196

selected as the catalyst, and the amount of KOH also had significant influence on the

197

column morphology. Increasing the amount of KOH would accelerate the thiol-epoxy

198

click polymerization reaction, leading to a more compact monolith (as columns B, H

199

and I). In addition, the effect of preparation temperature was also investigated. Higher

200

preparation temperature would not only accelerate the reaction process, but also

201

improve the solubility of porogenic system, thus resulting in a decrease of

202

permeability (data not shown). Finally, the preparation temperature was determined at

203

50 oC. After comprehensive optimization of these parameters, the column B was

204

employed in the following experiments.

DMSO/1,4-butanediol,

DMSO/H2O,

7


DMSO/PEG

200

and


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 8 of 25

205

Similarly, the feed recipes of monolith TPEGE-TPTM were also optimized. The

206

monolith (TPEGE-TPTM) prepared with TPEGE (40 mg), TPTM (32.5 mg) in the

207

presence

208

(66.0/19.0/7.5/5.5/2.0, wt%, 267 mg in total) at 60 oC was employed for further

209

experiments.

of

porogenic

system

DMSO/PEG

200/H2O/PEG

10,000/KOH

210

Characterization of the Organic Monoliths. The occurrence of thiol-epoxy

211

click polymerization reaction was convincingly demonstrated by FT-IR spectra

212

(Supplementary Figure 1), as the characteristic absorption peaks of epoxy and S-H

213

groups at 725 cm-1 to 910 cm-1 and 2570 cm-1, respectively, were significantly

214

decreased and almost disappeared. Meanwhile, the remarkable changes of these peaks

215

also implied a nearly complete consumption of two functional groups, indicating the

216

high efficiency of this thiol-epoxy click polymerization reaction based approach.

217

Besides, the high conversion of the epoxy and thiol groups resulted in a high degree

218

of cross-linking, and further endowed the monoliths high thermal stabilities. As

219

shown in Supplementary Figure 2, no significant weight loss was observed until the

220

temperature above 300 oC, and the pyrolysis continued up to 600 oC for the monolith

221

TPEGE-PTM. Such a high heat resistance is even better than some hybrid

222

monoliths.23,39

223

The SEM images revealed that the monolithic matrices with reticular framework

224

were tightly anchored to the inner walls of the capillaries without any disconnection,

225

and the macropores of ~1 μm were homogenously separated by the narrow framework

226

(~0.5 μm) (Figure 2). Particularly, their morphologies were distinct from the

227

cauliflower-like microglobules of traditional organic monoliths prepared via free

228

radical polymerization, and were similar to those of monoliths synthesized via

229

epoxy-amine based ring-opening polymerization or controllable living radical

230

polymerizations.18,22-24 The high regularity of microstructure was also demonstrated

231

by the narrow pore size distribution (Supplementary Figure 3). Such well-controlled

232

3D framework microstructure was possibly attributed to the step-growth mechanism

233

of thiol-epoxy click polymerization. As mentioned above, the epoxy and thiol groups

234

were stoichiometric fed and almost equally consumed, indicating that a step-growth 8


Page 9 of 25

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


235

polymerization process is favored during the formation of organic monoliths.30,35

236

Distinct from the free radical polymerization, the mild step-growth polymerization

237

would induce spinodal decomposition (SD)17 during the thiol-epoxy click

238

polymerization process, resulting in high conversions of the functional groups at the

239

gel point and largely uniform, homogeneous networks,30,40 and finally formed a

240

highly ordered bicontinuous skeleton.

241

The relationship between flow rate and the back-pressure drop of monolith

242

TPEGE-PTM was also measured. As shown in Supplementary Figure 4, the good

243

linear relationship (R = 0.9968) between the back-pressure and flow rate was obtained.

244

The monolith even could undergo a high pressure over 42.0 MPa without any

245

deterioration on the framework, indicating satisfactory mechanical strength. In

246

addition, the permeability was calculated at 5.40 × 10-14 m2 (as seen the column B in

247

Table 1), indicating a good permeability of the monolith.

248

Swelling propensity (SP) is another important parameter for evaluation of the

249

stability of organic monoliths, which has a significant influence on the resolution and

250

column efficiency.41 The SP factor can be calculated with the equation SP =

251

[p(solvent)-p(water)]/p(water) by measuring the pressure drop for water and for an

252

organic solvent,42 where p is the ratio of the mobile phase pressure drop and the

253

corresponding viscosity. Closer to zero of SP factor, smaller effect of

254

shrinkage/swelling of monolith in a given solvent. Firstly, as a commonly used mobile

255

phase additive, ACN was used to test the SP of monolith TPEGE-PTM. The SP factor

256

of 0.5 indicated a high organic solvent tolerance of the monolith. Surprisingly, almost

257

no swelling effect was observed when the monolith was immersed into the weakly

258

polar solvents such as methanol and THF (the SP factors were very close to zero).

259

Although the reason for this phenomenon is still unknown, there was no doubt that

260

these organic monoliths possess better organic solvent tolerance than common

261

polymethacrylate organic monoliths.

262

Chromatographic Evaluation of the Organic Monoliths in cLC. Owing to the

263

satisfactory mechanical and chemical stabilities as well as the highly regular

264

microstructure, these organic monoliths were applied for separation of analytes in 9



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

265

cLC. As shown in Figure 3A, 5 alkylbenzenes were well-separated under the mobile

266

phase of ACN/water (60/40, v/v) according to their hydrophobicity on monolith

267

TPEGE-PTM, indicating a typical reversed-phase (RP) retention mechanism

268

(Supplementary Figure 5). It should be pointed that the monolith TPEGE-PTM

269

exhibited high column efficiencies for alkylbenzenes, and the highest one could reach

270

ca. 132,000 N/m (for benzene, Figure 3B). Comparable high column efficiencies were

271

also achieved on another monolith TPEGE-TPTM (the highest ca. 128,000 N/m for

272

propylbenzene, Supplementary Figure 6). Such high column efficiencies could be

273

ascribed to the high homogenous microstructure of these monoliths.

274

Although the nitrogen adsorption/desorption measurement implied low surface

275

areas of these monoliths (e.g. 2.25 and 1.75 m2/g for monoliths TPEGE-PTM and

276

TPEGE-TPTM, respectively), which is possibly unfavourable for improving the

277

separation performance. The size exclusion chromatography in THF afforded total

278

porosity of monolith TPEGE-PTM at 86.2%, while the volume of through-pores was

279

calculated at 57.6% (as shown in Supplementary Figure 7). This significant pore

280

volume was beneficial to the separation of small molecules. Besides, the large

281

through-pore/framework size ratio as well as the high ordered microstructure would

282

enable fast mass transfer as the convection is dominant during the separation

283

process.43-45 Meanwhile, the thin framework would also effectively reduce the

284

diffusion distance and greatly accelerate the mass transfer,13,46-51 thus leading to a

285

smaller C-term in van Deemter equation. What’s more, the high ordered

286

microstructure could also significantly limit the eddy diffusion (resulting in a smaller

287

A-term in van Deemter equation).23,43,47 Such effects were vividly reflected in the van

288

Deemter curves of alkylbenzenes on monolith TPEGE-PTM (Figure 3B and Table 2).

289

The values of A, B, C-terms were much smaller than those of other traditional organic

290

monoliths in LC, and even comparable to those of silica-based and hybrid monoliths

291

or some particulate-packed columns in capillary electrochromatography (CEC).52,53 It

292

is well known that the eddy diffusion term is the main contributor of the band

293

broadening, especially in the separation of small molecules.54,55 Herein, the relatively

294

small values of A-term ranged from 0.981 to 2.619, implying the limitation of eddy 10


Page 10 of 25

Page 11 of 25

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


295

diffusion. Such small A-terms were comparable to those of columns packed with

296

core-shell and totally porous particles, whose typical values were 0.9 and 1.5,

297

respectively.56 Furthermore, the small values of C-term also indicated a better

298

communication between the stationary phase and analytes (the mass transfer was

299

faster as the molecules were transported almost solely by convection instead of

300

diffusion). Thus, ascribing to the combination of these effects, high column

301

efficiencies were achieved on these organic monoliths in cLC separation of small

302

molecules.

303

It should be mentioned that the elution order of thiourea and toluene on monolith

304

TPEGE-PTM was reversed when the ACN content in mobile phase was higher than

305

85% (v/v) (Supplementary Figure 8), exhibiting a hydrophilic interaction

306

chromatography (HILIC) retention mechanism. This phenomenon may be attributed

307

to the generation of hydroxyl groups on the surface of the matrix via thiol-epoxy click

308

reaction (Figure 1). The multiple retention mechanisms of these monoliths could

309

provide more choices of separation modes in analysis of some polar compounds, and

310

is beneficial to more extensive cLC application.

311

Applications of the Organic Monoliths in cLC. For an overall assessment of

312

the separation capability of these polymeric monoliths in analysis of small molecules,

313

various kinds of analytes including neutral and polar, basic and acidic, simple and

314

complicated mixtures were separated on monolith TPEGE-PTM. As shown in

315

Supplementary Figure 9, 5 polycyclic aromatic hydrocarbons (PAHs) were

316

well-separated with the mobile phase of ACN/water (75/25, v/v), and column

317

efficiencies were in the range of 100,800-112,000 N/m. Particularly, the elution order

318

of 4,4’-dimethylbiphenyl and acenaphthene was reversed by varying the ACN content

319

in mobile phase (Supplementary Figure 10). It is implied that these monoliths could

320

provide π-π conjugate interaction besides the RP retention mechanism in separation of

321

analytes with π electron, attributing to the existence of a lot of benzene rings (as

322

shown in Figure 1).

323

Figure 4A showed the separation chromatogram of six phenols, and the column

324

efficiencies were in the range of 86,600-107,700 N/m. The baseline-separation of the 11



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

325

two positional isomers (hydroquinone and pyrocatechol) demonstrated the possibly

326

potential of these polymeric monoliths in analysis of some positional isomers.

327

Generally, it is difficult to separate the basic compounds on C18 silica particles

328

packed LC columns as the phenomenon of peak tailing is emerged. However, such

329

phenomenon was not observed on the monolith TPEGE-PTM. As shown in Figure 4B,

330

5 anilines were baseline-separated with good peak shapes, achieving column

331

efficiencies in the range of 58,900-114,100 N/m. Additional, satisfactory

332

chromatographic performance was also obtained in the analysis of five alkaline drugs,

333

and the column efficiencies were ranged from 35,500 to 71,300 N/m (Figure 4C),

334

demonstrating the potential applications of these organic monoliths in pharmaceutical

335

research.

336

Satisfactory separation of acidic compounds was also achieved, as 5 benzoic acid

337

analogues were well-separated with column efficiencies in the range of

338

81,300-107,700 N/m (Figure 4D). Compared to the monoliths synthesized via

339

epoxy-amide based ring-opening polymerization, these thiol-epoxy click chemistry

340

based monoliths are more suitable for the separation of charged analytes, as they were

341

lack of ionizable groups on the surface, and the electrostatic interaction between the

342

analytes and stationary phase was not occurred. It is worth noting that no significant

343

decrease of column efficiency or clear column deterioration was observed on

344

monolith TPEGE-PTM even after continuously using for several weeks, even under

345

the acidic mobile phase, demonstrating high chemical stability of the monoliths.

346

Additionally, the separation of highly complicated samples was also performed

347

on monolith TPEGE-PTM. As illustrated in Figure 5, favorable separation of EPA 610

348

was obtained with ten baseline-separated ones and six partly-separated ones, and a

349

column capacity of 47 was calculated. Furthermore, the BSA digest was also used to

350

evaluate the separation ability of monolith TPEGE-PTM, and an acceptable

351

performance was achieved (Supplementary Figure 11), 48 unique peptides were

352

positively identified with protein sequence coverage of 50.58%. All results

353

convincingly demonstrated the great capability of these thiol-epoxy click chemistry

354

based organic monoliths in highly efficient separation of small molecules. 12


Page 12 of 25

Page 13 of 25

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

355




CONCLUSIONS

356

In summary, a facile approach was firstly developed for direct preparation of

357

organic monoliths via thiol-epoxy click polymerization reaction. Ascribing to the high

358

conversion of the epoxy and thiol functional groups, the resulting organic monoliths

359

exhibited

360

polymethacrylate(acrylate) organic monoliths prepared via free radical polymerization.

361

Besides, benefiting from the relatively ordered phase-separating process resulted by

362

the step-growth polymerization, the obtained organic monoliths possessed

363

well-defined 3D framework microstructure, which were remarkably distinct from

364

those of organic monoliths synthesized via free radical polymerization. Such highly

365

regular microstructure granted these organic monoliths not only favorable mechanical

366

stability, but also high separation efficiency in cLC analysis of small molecules.

367

Satisfactory chromatographic performances were achieved in the separation of

368

various kinds of analytes, including alkylbenzens, PAHs, phenols, basic and acidic

369

compounds, EPA 610 as well as BSA digest. These results demonstrated the great

370

potential of this thiol-epoxy click chemistry based approach in preparation of

371

polymeric monoliths with high separation capability and efficiency. Further

372

investigation and application of this novel approach were under exploration and will

373

be reported soon.

better

thermal

and

chemical

stabilities

than

traditional

374 375



376

Corresponding Author

377

*E-mail: [email protected] (H.F. Zou); [email protected] (J.J. Ou).

AUTHOR INFORMATION

378 379



380

Financial support is gratefully acknowledged from the China State Key Basic

381

Research Program Grant (2013CB-911203, 2012CB910601), the National Natural

382

Sciences Foundation of China (21235006), the Creative Research Group Project of

383

NSFC (21321064), and the Knowledge Innovation program of DICP to H. Zou as

384

well as the National Natural Sciences Foundation of China (No. 21175133) to J. Ou.

ACKNOWLEDGEMENTS

13



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

385



386

Additional information as noted in text. This material is available free of charge via

387

the Internet at http://pubs.acs.org.

SUPPORTING INFORMATION AVAILABLE

388 389 390 391 392 393 394 395 396 397 398 399 400 401 402 403 404 405 406 407 408 409 410 411 412 413 414 415 416 417 418 419 420 421 422 423 424 425



REFERENCES (1) Huang, X.; Yuan, D. Crit. Rev. Anal. Chem. 2012, 42, 38-49. (2) Wu, R.; Hu, L. G.; Wang, F. J.; Ye, M. L.; Zou, H. J. Chromatogr. A 2008, 1184, 369-392. (3) Desmet, G.; Eeltink, S. Anal. Chem. 2013, 85, 543-556. (4) Chester, T. L. Anal. Chem. 2013, 85, 579-589. (5) Namera, A.; Nakamoto, A.; Saito, T.; Miyazaki, S. J. Sep. Sci. 2011, 34, 901-924. (6) Nema, T.; Chan, E. C. Y.; Ho, P. C. J. Pharm. Biomed. Anal. 2014, 87, 130-141. (7) Wu, M.; Wu, R. a.; Wang, F.; Ren, L.; Dong, J.; Liu, Z.; Zou, H. Anal. Chem. 2009, 81,

3529-3536. (8) Liu, K.; Aggarwal, P.; Lawson, J. S.; Tolley, H. D.; Lee, M. L. J. Sep. Sci. 2013, 36, 2767-2781. (9) Maya, F.; Svec, F. Polymer 2014, 55, 340-346. (10) Stassen, C.; Desmet, G.; Broeckhoven, K.; Van Lokeren, L.; Eeltink, S. J. Chromatogr. A 2014, 1325, 115-120. (11) Aggarwal, P.; Asthana, V.; Lawson, J. S.; Tolley, H. D.; Wheeler, D. R.; Mazzeo, B. A.; Lee, M. L. J. Chromatogr. A 2014, 1334, 20-29. (12) Nischang, I.; Teasdale, I.; Brueggemann, O. Anal. Bioanal. Chem. 2011, 400, 2289-2304. (13) Nischang, I. J. Chromatogr. A 2013, 1287, 39-58. (14) Svec, F. J. Chromatogr. A 2010, 1217, 902-924. (15) Arrua, R. D.; Causon, T. J.; Hilder, E. F. Analyst 2012, 137, 5179-5189. (16) Peters, E. C.; Svec, F.; Frechet, J. M. J.; Viklund, C.; Irgum, K. Macromolecules 1999, 32, 6377-6379. (17) Kanamori, K.; Nakanishi, K.; Hanada, T. Adv. Mater. 2006, 18, 2407-2411. (18) Hasegawa, G.; Kanamori, K.; Ishizuka, N.; Nakanishi, K. ACS Appl. Mater. Interfaces 2012, 4, 2343-2347. (19) Urban, J.; Svec, F.; Frechet, J. M. J. Anal. Chem. 2010, 82, 1621-1623. (20) Lv, Y.; Lin, Z.; Svec, F. Anal. Chem. 2012, 84, 8457-8460. (21) Maya, F.; Svec, F. J. Chromatogr. A 2013, 1317, 32-38. (22) Hosoya, K.; Hira, N.; Yamamoto, K.; Nishimura, M.; Tanaka, N. Anal. Chem. 2006, 78, 5729-5735. (23) Lin, H.; Ou, J.; Zhang, Z.; Dong, J.; Zou, H. Chem. Commun. 2013, 49, 231-233. (24) Lin, H.; Ou, J.; Tang, S.; Zhang, Z.; Dong, J.; Liu, Z.; Zou, H. J. Chromatogr. A 2013, 1301, 131-138. (25) Lin, H.; Zhang, Z.; Dong, J.; Liu, Z.; Ou, J.; Zou, H. J. Sep. Sci. 2013, 36, 2819-2825. (26) Fu, R.; Fu, G.-D. Polym. Chem. 2011, 2, 465-475. (27) Sumerlin, B. S.; Vogt, A. P. Macromolecules 2010, 43, 1-13. 14


Page 14 of 25

Page 15 of 25

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

426 427 428 429 430 431 432 433 434 435 436 437 438 439 440 441 442 443 444 445 446 447 448 449 450 451 452 453 454 455 456 457 458 459 460 461 462 463 464 465 466 467 468 469


(28) Hoyle, C. E.; Lowe, A. B.; Bowman, C. N. Chem. Soc. Rev. 2010, 39, 1355-1387. (29) Chatani, S.; Sheridan, R. J.; Podgorski, M.; Nair, D. P.; Bowman, C. N. Chem. Mater. 2013, 25, 3897-3901. (30) Nair, D. P.; Podgórski, M.; Chatani, S.; Gong, T.; Xi, W.; Fenoli, C. R.; Bowman, C. N. Chem. Mater. 2014, 26, 724-744. (31) Chu, C.; Liu, R. Chem. Soc. Rev. 2011, 40, 2177-2188. (32) Lubda, D.; Lindner, W. J. Chromatogr. A 2004, 1036, 135-143. (33) Kacprzak, K. M.; Maier, N. M.; Lindner, W. Tetrahedron Lett. 2006, 47, 8721-8726. (34) Lv, Y.; Lin, Z.; Svec, F. Analyst 2012, 137, 4114-4118. (35) Alves, F.; Nischang, I. Chem. Eur. J. 2013, 19, 17310-17313. (36) Liu, Z.; Ou, J.; Lin, H.; Liu, Z.; Wang, H.; Dong, J.; Zou, H. Chem. Commun. 2014, 50, 9288-9290. (37) De, S.; Khan, A. Chem. Commun. 2012, 48, 3130-3132. (38) Stanelle, R. D.; Sander, L. C.; Marcus, R. K. J. Chromatogr. A 2005, 1100, 68-75. (39) Zhang, Z.; Wang, F.; Dong, J.; Lin, H.; Ou, J.; Zou, H. RSC Adv. 2013, 3, 22160-22167. (40) Northrop, B. H.; Coffey, R. N. J. Am. Chem. Soc. 2012, 134, 13804-13817. (41) Nevejans, F.; Verzele, M. J. Chromatogr. 1985, 350, 145-150. (42) Koeck, R.; Bakry, R.; Tessadri, R.; Bonn, G. K. Analyst 2013, 138, 5089-5098. (43) Peters, E. C.; Svec, F.; Frechet, J. M. J. Adv. Mater. 1999, 11, 1169-1181. (44) Jandera, P. J. Chromatogr. A 2013, 1313, 37-53. (45) Guiochon, G. J. Chromatogr. A 2007, 1168, 101-168. (46) Aggarwal, P.; Tolley, H. D.; Lee, M. L. J. Chromatogr. A 2012, 1219, 1-14. (47) Colon, L. A.; Li, L.; Adv. Chromatogr. 2008; Vol. 46; pp 391-421. (48) Siouffi, A. M. J. Chromatogr. A 2003, 1000, 801-818. (49) Hormann, K.; Muellner, T.; Bruns, S.; Hoeltzel, A.; Tallarek, U. J. Chromatogr. A 2012, 1222, 46-58. (50) Gritti, F.; Guiochon, G. J. Chromatogr. A 2009, 1216, 4752-4767. (51) Gritti, F.; Guiochon, G. J. Chromatogr. A 2012, 1221, 2-40. (52) Eeltink, S.; Decrop, W. M. C.; Rozing, G. P.; Schoenmakers, P. J.; Kok, W. T. J. Sep. Sci. 2004, 27, 1431-1440. (53) Yan, L. J.; Zhang, Q. H.; Feng, Y. Q.; Zhang, W. B.; Li, T.; Zhang, L. H.; Zhang, Y. K. J. Chromatogr. A 2006, 1121, 92-98. (54) Ishizuka, N.; Minakuchi, H.; Nakanishi, K.; Soga, N.; Nagayama, H.; Hosoya, K.; Tanaka, N. Anal. Chem. 2000, 72, 1275-1280. (55) Gritti, F.; Guiochon, G. Anal. Chem. 2013, 85, 3017-3035. (56) Guiochon, G.; Gritti, F. J. Chromatogr. A 2011, 1218, 1915-1938.

15



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

470

Page 16 of 25

Table 1. Effects of synthesis parameters on the formation of TPEGE-PTM monolithsa Monolith

DMSOb (wt%)

PEG200c (wt%)

H2Od (wt%)

PEG10000e (wt%)

KOHf,g (wt%)

A

66.0

20.0

8.0

4.0

2.0

11.57

B

68.5

17.5

8.0

4.0

2.0

5.40

C

77.0

9.0

8.0

4.0

2.0

Too hard to pump through

D

70.0

18.0

6.0

4.0

2.0


E

67.5

17.0

9.5

4.0

2.0

19.00

F

70.0

18.0

8.0

2.0

2.0

19.58

G

67.5

17.0

8.0

5.5

2.0


H

68. 5

17.5

9.0

4.0

1.0

15.54

I

68.5

17.5

7.0

4.0

3.0


Optical microscope image

Permeabilityh (× 10-14 m2)

471

a

472

respectively. Reaction temperature: 50 oC.

473

b

474

porogenic solvents; d weight percentage of H2O in the porogenic solvents; e weight percentage of

475

PEG 10,000 in the porogenic solvents;

476

solvents.

477

g

The concentration of the catalyst KOH solution: 0.25 mol/L.

478

h

Permeability, B0 = FηL/(πr2ΔP), F: linear velocity of mobile phase; η: dynamic viscosity of

479

mobile phase; L: effective column length; r: inner radius of column; ΔP: pressure drop across the

480

column38, measured at room temperature.

The amounts of TPEGE, PTM and porogenic system were kept at 40, 33 and 262 mg,

Weight percentage of DMSO in the porogenic solvents; c weight percentage of PEG 200 in the

f

weight percentage of KOH solution in the porogenic

481 482 483 484 485 16


Page 17 of 25

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


486

Table 2. van Deemter coefficients measured with alkylbenzenes for monolith

487

TPEGE-PTM in cLC. Compounds

Eddy dispersion, A (μm)

Longitudinal diffusion, B (×103 μm2/s)

Mass transfer resistance, C (×10−3 s)

Benzene

2.619

1.700

4.240

Toluene

1.857

1.848

5.550

Ethylbenzene

1.715

1.749

6.280

Propylbenzene

1.534

1.753

6.510

Butylbenzene

0.981

1.808

7.100

488 489 490 491 492 493 494 495 496 497 498 499 500 501 502 503 504 505 506 507 508 17



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

509

Page 18 of 25

Figure captions

510 511

Figure 1. Schematic preparation of the polymeric monoliths via thiol-epoxy click

512

polymerization.

513 514

Figure 2. The cross-section of the polymeric monolithic columns prepared via

515

thiol-epoxy click polymerization. Magnification: 1000×(upper), 5000×(down).

516 517

Figure 3. Separation of (A) alkylbenzenes and (B) dependence of plate heights of

518

them on the linear velocity of mobile phase on monolith TPEGE-PTM in cLC.

519

Experimental conditions: column dimension, 28.0 cm × 75 μm i.d.; flow rate for (A),

520

240 nL/min; mobile phase, ACN/water (60/40, v/v); injection volume, 2.5 μL in split

521

mode; detection wavelength, 214 nm.

522 523

Figure 4. Separations of (A) phenols, (B) anilines, (C) alkaline drugs and (D) benzoic

524

acids on monolith TPEGE-PTM in cLC.

525

Experimental conditions: column dimension, 28.0 cm × 75 μm i.d.; flow rates for (A),

526

200 nL/min, for (B, C and D), 220 nL/min; mobile phases for (A), ACN/water (45/55,

527

v/v), for (B) ACN/water (containing 0.1% TFA) (55/45, v/v), for (C), ACN/water

528

(containing 0.1% TFA) (40/60, v/v), for (D) ACN/water (containing 0.1% TFA)

529

(45/55, v/v); injection volume, 2.5 μL in split mode; detection wavelength, 214 nm.

530 531

Figure 5. Separation of EPA 610 on monolith TPEGE-PTM in cLC.

532

Analytes: (1) naphthalene, (2) acenaphthylene, (3) acenaphthene, (4) fluorene, (5)

533

phenanthrene, (6) anthracene, (7) fluoranthene, (8) pyrene, (9) benzo(a)anthracene,

534

(10)

535

benzo(a)pyrene, (14) dibenzo(a,h)anthracene, (15) benzo(g,h,i)perylene, (16)

536

indeno(1,2,3-cd)pyrene;

537

Experimental conditions: column dimension, 28.0 cm × 75 μm i.d.; gradient, 40 %

538

water to 70% ACN in 20 min, then to 85% ACN in 15 min and keeps 85% ACN for

chrysene,

(11)

benzo(b)fluoranthene,

(12)

benzo(k)fluoranthene,

18


(13)

Page 19 of 25

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


539

another 35 min; flow rate, 240 nL/min; injection volume, 2.5 μL in split mode;

540

detection wavelength, 254 nm.

541 542 543 544 545 546 547 548 549 550 551 552 553 554 555 556 557 558 559 560 561 562 563 564 565 566 567 568 19



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

569

Figure 1.

570

571 572 573 574 575 576 577 578 579 580 581 582 583 584 585 586 587 588 589 590 591 592 593 20


Page 20 of 25

Page 21 of 25

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

594


Figure 2.

595

596 597 598 599 600 601 602 603 604 605 606 607 608 609 610 21



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

611

Figure 3.

612 613

A

B

614 615 616 617 618 619 620 621 622 623 624 625 626 627 628 629 630 631 632 633 634 635 636 637 638 639 640 22


Page 22 of 25

Page 23 of 25

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

641


Figure 4.

642 643

A

B

C

D

644 645 646 647 648 649 650 651 652 653 654 655 656 657 658 659 660 661 662 663 664 665 666 667 668 669 670 23



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

671

Figure 5.

672

673 674 675 676 677 678 679 680 681 682 683 684 685 686 687 688 689 690 691 24


Page 24 of 25

Page 25 of 25

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


692

693 694

TOC

695 696 697 698

25


Thiol-Epoxy Click Polymerization for Preparation of Polymeric

Recommend Documents