Accepted Manuscript

Molecularly imprinted microspheres and nanoparticles prepared using precipi‐

tation polymerization method for selective extraction of gallic acid from

Emblica officinalis

Sushma Pardeshi, Rita Dhodapkar, Anupama Kumar

PII: S0308-8146(13)01349-6

DOI: http://dx.doi.org/10.1016/j.foodchem.2013.09.084

Reference: FOCH 14712

To appear in: Food Chemistry

Received Date: 25 April 2013

Revised Date: 30 August 2013

Accepted Date: 14 September 2013

Please cite this article as: Pardeshi, S., Dhodapkar, R., Kumar, A., Molecularly imprinted microspheres and

nanoparticles prepared using precipitation polymerization method for selective extraction of gallic acid from

Emblica officinalis, Food Chemistry (2013), doi: http://dx.doi.org/10.1016/j.foodchem.2013.09.084

This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers

we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and

review of the resulting proof before it is published in its final form. Please note that during the production process

errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

1

Molecularly imprinted microspheres and nanoparticles prepared 1

using precipitation polymerization method for selective extraction of 2

gallic acid from Emblica officinalis 3

Sushma Pardeshia, Rita Dhodapkarb*, Anupama Kumara* 4

a Chemistry Department, Visvesvaraya National Institute of Technology, Nagpur 5

440010, India 6

b Waste Water Technology Division, CSIR-National Environmental Engineering 7

Research Institute, Nagpur 440020, India 8

9

10

11

12

13

14

15

16

17

* Corresponding authors:

Tel.: +91-712-2801357, E-mail: drkumaranupama@rediffmail.com,

anupamakumar@chm.vnit.ac.in (A. Kumar),

Tel.: +91-712-2249885, E-mail: rs_dhodapkar@neeri.res.in (R. Dhodapkar)

2

Abstract 18

This paper reports the preparation of Gallic acid (GA) molecularly imprinted 19

polymers (MIPs) by the precipitation polymerization and highlights the effect of 20

porogen on particle size and specific molecular recognition properties. MIP, M-100 21

prepared in the porogen, acetonitrile and MIP, M-75 prepared in a mixture of 22

acetonitrile-toluene (75:25 v/v), resulted in the formation of microspheres with 23

approximately 4µm particle size and surface area of 96.73 m2g-1 and nanoparticles 24

(0.8 to 1000 nm) and a surface area of 345.9 m2g-1, respectively. The Langmuir-25

Freundlich isotherm study revealed that M-75 has comparatively higher number of 26

binding sites which are homogenous and has higher affinity for GA. The MIPs 27

selectively recognized GA in presence of its structural analogues. Pure GA with 28

percent recovery of 75 (±1.6) and 83.4 (±2.2) was obtained from the aqueous extract 29

of Emblica officinalis by M-100 and M-75, respectively and hot water at 60ºC served 30

as the eluting solvent. 31

Keywords: Molecularly imprinted polymer; Gallic acid; Emblica officinalis; 32

Precipitation polymerization; Microspheres. 33

34

35

36

37

38

3

1. Introduction 39

Gallic acid (GA) is a polyphenolic compound with potential health effects on 40

human health, such as antioxidative, anticancer and anti- inflammatory (Kim, 2007). 41

GA is abundantly present in herbs and fruits especia lly, in the fruits of Emblica 42

officinalis (E. officinalis). The pulpy portion of fruit contains about 1- 2 % of GA 43

(Majeed, Bhat, Jadhav, Srivastava, & Nagabhushanam, 2009). The E. officinalis is 44

native to India and also grows in tropical and subtropical regions including Pakistan, 45

Uzbekistan, Srilanka, South-East Asia, China and Malaysia. GA is cytotoxic against 46

certain cancer cells thus, inhibits their growth, without harming the normal cells 47

(Borde, Pangrikar & Tekale, 2011). It is found to possess strong in vitro and in vivo 48

anticancer efficacy against prostate, breast and lung cancer cells (Raina, 49

Rajamanickam, Deep, Singh, Agarwal, & Agarwal, 2008). Apart from its medicinal 50

importance, it is also used in production of an antibiotic trimethoprim, propyl gallate, 51

inks, and in process of tanning (Lu, Wei, & Yuan, 2007). GA has a huge demand of 52

about 8,000 tons per annum worldwide, due to its pharmaceutical and industrial 53

importance. Conventionally, GA is produced by the hydrolysis of tannic acid. 54

However, the chemical methods of GA production consume large quantities of 55

chemicals and release toxic effluents leading to environmental hazards (Lokeshwari 56

& Reddy, 2010). GA is abundantly present in the natural matrices, nevertheless, 57

selective extraction of GA is influenced by the complex nature of the sample matrix. 58

Therefore, it is necessary to develop a selective and practicable enrichment material 59

for separation and determination of GA from aqueous environment of natural 60

matrices. 61

4

Molecular imprinted polymers (MIPs) are porous materials with specific molecular 62

recognition sites for a particular target molecule (Pardeshi, Kumar, & Dhodapkar, 63

2011). A template molecule is self-assembled in a complex with the functional 64

monomers and then polymerized in presence of a cross- linker which leads to the 65

formation of a crosslinked imprinted polymer. On removal of the template from the 66

MIPs matrix, tailored molecular recognition cavities with specific binding capability 67

for the template are obtained. The ease of preparation, along with the superior 68

chemical and mechanical properties render MIPs an appealing alternative to natural 69

receptors for a variety of applications ranging from pharmaceutical analysis 70

(Esfandyari-manesh et al., 2011), food analysis (Gholivanda, Karimian, & 71

Malekzadeh, 2012), chemical sensors (Qiu, Luo, Sun, Lu, Fan & Li, 2012) and solid-72

phase extraction (SPE) (Zhang & Chen, 2013). MIPs, as selective sorbents have 73

attracted considerable attention for extraction of compounds from complex mixtures 74

of chemical species (Quesada-Molina, Claude, Garcia-Campana, Olmo-Iruela, & 75

Morin, 2012; Puoci et al., 2011). 76

Earlier attempts of GA imprinting include, preparation of MIPs by bulk 77

polymerization and suspension polymerization (Zhu, Cao, Yang, Li, Wang, & Ding, 78

2009; Pardeshi, Dhodapkar, & Kumar, 2012; Puoci, Scoma, Cirillo, Bertin, Fava, & 79

Picci, 2012; Nicolescu, Meouche, Branger, Margaillan, Sarbu, & Donescu, 2012). 80

The drawback of the bulk polymerization is that the polymer monolith has to be 81

ground and sieved to obtain the desired sized particles. Moreover, the post processing 82

of MIPs leads to loss of useful particles. Also, the particles obtained in this manner 83

are typically irregular in size. Uniformity in size and shape of the MIPs particles is 84

5

desirable for the intended applications, particularly in chromatographic and SPE 85

applications (Beltran, Marcé, Cormack, & Borrulla, 2009). The suspension 86

polymerization requires specific reagents as the continuous and dispersing phase, 87

suspension stabilizers and a careful choice of reagents for polymer synthesis, 88

rendering the procedure tedious (Haginaka, 2008). 89

Precipitation polymerization method of MIPs synthesis is developed to ease the 90

procedure and to improve the performance of resultant MIPs. The advantages of this 91

method are: a) no post processing steps such as grinding, sieving and sedimentation, 92

b) particles with spherical shape and controlled size distribution produced (Wang, 93

Cormack, Sherrington, & Khoshdel 2007), c) a simpler and better method than 94

emulsion, suspension and multistep swelling polymerization methods as it does not 95

involve the use of stabilizers and surfactants which can contaminate the final MIPs 96

product (Hu, Pan, Zhang, Lian, & Li, 2013). 97

The present work involves the precipitation polymerization as the synthesis method 98

for GA based MIPs for developing selective sorbents with the improved morphology 99

and recognition performance. To the best of author’s knowledge, this is the first 100

attempt for preparation of MIPs microspheres and nanoparticles using precipitation 101

polymerization method for the template GA. 102

2. Materials and methods 103

GA, acrylic acid (AA), 4-hydroxy benzoic acid (4-HBA), 3,4-dihydroxy benzoic 104

acid (3,4-DBA), 3,5-dihydroxy benzoic acid (3,5-DBA), and 2,4-dihydroxy benzoic 105

acid (2,4-DBA) (Fig. S1, supplementary material) were purchased from Sigma-106

Aldrich (Buchs, Switzerland). Ethylene glycol dimethacrylate (EGDMA) was 107

6

purchased from Merck (Darmstadt, Germany), and 2,2-azoisobutyronitrile (AIBN) 108

from National Chemicals (India). AIBN was recrystalized using methanol before use. 109

All the solvents were HPLC grade and purchased from Merck (India) and used 110

without further purification. 111

2.1 Synthesis of molecularly imprinted polymers 112

The GA and functional monomer AA were dissolved in porogenic solvent in a 250 113

mL round bottom flask followed by the crosslinker EGDMA and 50 mg of the 114

initiator AIBN. The mixture was sonicated in an ultrasonicator bath (Bio-Technics, 115

India) until a clear solution was obtained. This mixture was kept at 0°C for 10 116

minutes, purged with a gentle flow of nitrogen and sealed under the nitrogen 117

atmosphere. The flask was kept in a rotamantle (Remi, India) with mild stirring. The 118

temperature was ramped from room temperature to 60 °C over a period of 2 ho urs 119

and then kept constant at this temperature for 22 hours. After polymerization, the 120

polymer particles were collected by centrifugation (Remi, India,) at relative 121

centrifugal force (RCF) 2830 g and room temperature for 15 minutes. 122

The MIPs were washed using methanol: acetic acid (80:20 v/v) in a soxhlet 123

extractor to remove the GA from its polymeric matrix. The MIPs were washed till no 124

desorption of GA was detected using UV–Visible spectrophotometer (UV-1800, 125

Shimadzu, Japan) with matched 1 cm quartz cells at 272 nm. The residual acetic acid 126

was removed from the MIPs by washing with a solution of 0.1 mmolL-1 Na2CO3 127

followed by several washings with distilled water. 128

The non- imprinted polymers (NIPs) were synthesized under identical conditions 129

except for the omission of the template; GA. NIPs particles were collected after 130

7

polymerization by centrifugation and washed with distilled water to remove the 131

unreacted precursors. Finally, all polymers were dried at 70 °C in a hot air oven and 132

stored at room temperature for further experiments and characterization. 133

The synthesis protocol was repeated for acetonitrile porogen and mixtures of 134

acetonitrile-toluene porogen in 90:10 v/v (M-90), 80:20 v/v (M-80), 75:25 v/v (M-135

75), and 50:50 v/v (M-50) (Table S1, Supplementary material). 136

2.2 Morphological characterization 137

The polymer samples were analyzed using a Scanning electron microscope (SEM, 138

JEOL JSM-6360LV). The samples were coated with a thin gold film before 139

observation in SEM. 140

The particle size analysis of the MIPs was done using a Nanosizer (Nanopartica SZ-141

100, Horiba, Japan) after uniform suspension in chloroform. 142

The FT-IR spectra of compounds were determined using a Fourier Transform 143

Infrared Spectrometer (IR-Affinity-1, Shimadzu). The samples were ground with 144

anhydrous KBr and the spectra recorded between 4000 and 400 cm-1, by averaging 45 145

scans for each spectrum. 146

The surface area, total pore volume and average pore diameter were analyzed by the 147

Brunauer–Emmett–Teller (BET) method on Micromeritics ASAP-2020. The samples 148

were degassed for 4 hours at 100 °C before analysis. 149

50 mg of the dry polymer were suspended in 1.5 mL of distilled water in a 150

microtube and shaken vigorously for 2 minutes followed by equilibration for 5 hours. 151

The final weight of the wet sample was measured after filtering out excess of the 152

8

solvent. This procedure was repeated thrice and percent swelling ratio was calculated 153

using Eq. (1). 154

% Swelling ratio = Mw - Md / Mw *100 (1) 155

Where Mw is the mass of the wet polymer and Md is the mass of the dry polymer. 156

2.3 Binding experiments 157

The binding experiments were carried out batch-wise in triplicate to study the 158

recognition performance of MIPs in the aqueous solutions using a method given 159

below. 160

2.3.1 Optimization of pH 161

The initial pH values of 20 mmolL−1 of GA in distilled water were adjusted from 162

2.0 to 8.0 using HCl and NaOH and 100 mg of the MIPs were dispersed in 10 mL of 163

each pH solution. The mixtures were shaken in water bath shaker at 25 +5 °C for 6 164

hours. The solutions were centrifuged at (RCF = 2830 g, room temperature) for four 165

minutes and the supernatant was filtered into 10 mL volumetric flask and analyzed for 166

GA using UV–Visible spectrophotometer at 268 nm. 167

2.3.2 Dose and time optimization 168

The dose of MIPs were varied in the range 15 mg to 100 mg and equilibrated in 10 169

mL solutions of 20 mmolL−1 GA at the optimized pH using a water bath shaker for 6 170

hours. The solutions were analyzed for GA using UV–Visible spectrophotometer at 171

268 nm after centrifugation. 172

The contact time was optimized by equilibrating the optimized dose of polymer, 173

9

with 20 mmolL-1 of GA solutions at optimized pH for fixed time periods from 30 174

minutes up to ten hours. The solutions were analyzed for GA after centrifugation after 175

each time interval using UV–Visible spectrophotometer at 268 nm. 176

2.3.3 Binding isotherm 177

A series of GA standard solutions ranging from 1 to 20 mmolL-1 were prepared at 178

the optimized pH. 10 mL aliquots of each standard solution was mixed with the 179

optimized dose of the polymer and shaken for the determined optimum time period. 180

The solutions were analyzed for GA after centrifugation using UV–Visible 181

spectrophotometer at 268 nm. 182

The binding capacity, B (µmolg-1) of imprinted and non-imprinted polymer was 183

calculated using Eq. (2) 184

B = (Cinitial-Cfinal)* Vsolution/ W (2) 185

Where, Cinital and Cfinal are the initial and final concentration of the GA solution, 186

respectively. Vsolution is the volume of GA solution and W is the weight of polymer. 187

2.4 Selectivity study 188

The MIPs and NIPs were equilibrated with an aqueous mixture of 20 mmolL−1 of 189

GA and the structural analogues, 4-HBA, 2,4-DBA, 3,4-DBA, and 3,5-DBA at the 190

optimized pH value for the optimum time period. The residual concentration of GA 191

and 4-HBA, 2,4-DBA, 3,4-DBA, and 3,5-DBA in the solution were analyzed by High 192

performance liquid chromatography (HPLC) after centrifugation. 193

HPLC analysis 194

10

The HPLC analysis was performed using a High performance liquid 195

chromatography from Waters equipped with a binary pump (515 water HPLC pump) 196

and UV-visible detector (Waters 2489). The analytical column used was ODS-SP C18 197

(5 µm, 4.6×250 mm) from Merck. The mobile phase consisted of water: acetonitrile: 198

acetic acid (90:10:0.2 v/v), at flow rate of 1 mL min−1. The injection volume was 10 199

µL and the UV detector wavelength was set at 273 nm. 200

Each measurement was performed in triplicate and the data expressed as means of 201

relative standard deviations (RSD). The intermediate precision (inter-day precision) 202

and repeatability (intra-day precision) was evaluated by injecting the GA sample six 203

times on the same day and six times over different days. Accuracy was evaluated by 204

means of recovery experiments carried out by adding standard solutions of the GA (1 205

to 6 mgmL-1) to the E. officinalis extract and the percent recoveries were calculated. 206

2.6 Application of MIPs to the real sample of E. officinalis 207

2.6.1 Preparation of E. officinalis extract 208

The fresh fruits of E. officinalis were purchased from local market and washed 209

thoroughly with water to remove the foreign matter and dust from outer surface. The 210

cleaned raw material was then commuted to reduce its size. The commuted raw 211

material was ground and extracted with the methanol. The resulting dark brown 212

solution was filtered to separate out the solid residue. The methanol was evaporated 213

in a vacuum rotary evaporator to obtain a dry powder of E. officinalis. The powder 214

was stored in a dry container containing pouch of silica gel and used for the further 215

11

experiments. 1.8 g of this powder was dissolved in 50 mL of distilled water for the 216

GA extraction experiments. 217

2.6.2 Selective extraction of GA from E. officinalis 218

The GA was selectively extracted from aqueous extract of E. officinalis using the 219

best obtained MIPs as the sorbent. The MIPs were equilibrated with the 10 mL of E. 220

officinalis extract in a conical flask for the determined optimum time period. The 221

MIPs were washed with 10 mL aliquot of the acetonitrile-water (50:50 v/v) for 30 222

minutes. The GA was eluted using 10 mL of water at 60 °C. After each step, the MIPs 223

were centrifuged for 10 min (RCF = 2830 g, room temperature) and the eluent 224

analyzed for GA by HPLC. 225

3. Results and discussion 226

3.1 Synthesis and characterization of MIPs 227

The monomer AA was selected using the density functional theory (DFT) based 228

quantum chemical computational modeling in our previous study (Pardeshi, Patrikar, 229

Dhodapkar, & Kumar, 2012). The results of computational modeling confirmed 230

formation of hydrogen bonding interactions between GA and AA. The mole ratio of 231

GA to the functional monomer was also optimized. The carboxylic functional group 232

of acidic functional monomers is considered as an excellent hydrogen bond donor 233

acceptor group which can participate in the formation of hydrogen bonding 234

interactions with the template. 235

In the present investigation, initially five types of MIPs were synthesized using 236

varying composition of acetonitrile-toluene mixture (Table S1, Supplementary 237

12

material) to observe the effects of solvent composition on the morphology of MIPs. 238

The MIPs were characterized using SEM and particle size analyzer. The SEM 239

micrographs of MIPs are presented in the Fig. 1. 240

The SEM image of M-100 shows uniformly sized, discrete and nearly monodispersed 241

particles of sizes around 4µm (Fig.1 a), whereas that of M-90 shows uniformly sized 242

MIPs microspheres of particle size 2 µm (Fig.1 b). Irregular shaped and agglomerated 243

particles (Fig.1 c) with 1µm size are observed in the SEM images of M-80. Fig. 1 d) 244

and Fig. 1 e) depicts the SEM images of M-75 and M-50 respectively. The 245

morphology of these MIPs is highly agglomerated and irregularly shaped with 246

particle size in nanometers. 247

It is observed that when the quantities of toluene in the acetonitrile-toluene mixture 248

were low (0 or 10 %), the MIPs were formed in the form of large beads with 249

uniformity and monodispersity of the beads also maintained. However, as the 250

percentage of toluene increased in the solvent mixture (20-50%), the particle shape 251

changed from globular to irregular and the polydispersity also increased. In general, 252

to obtain larger microspheres, matching the solubility parameter of the developing 253

polymer network to that of the porogenic solvents is particularly important (Lai, 254

Yang, & Niessner, 2007). The solubility parameter of toluene is 8.9 MPa while, the 255

solubility parameter of EGDMA is 18.2 MPa. When the concentration of toluene in 256

the system increased the mismatch between the solubility parameters of components 257

in the system also increased resulting in the formation of irregular MIPs 258

nanoparticles. This is also expressed in the earlier investigations on MIPs, wherein 259

divinylbenzene (DVB) was used as a crosslinker for MIPs preparation by 260

13

precipitation polymerization (Wang, Liu, Rong & Fu, 2011). The size of MIPs 261

particles increased with the increase in the percentage of toluene in the solvent 262

mixture. The DVB has solubility parameter of 8.5 MPa which is nearly same with the 263

solubility parameter of toluene. 264

Fig. 2 shows the results of particle size analysis of MIPs along with the particle 265

diameter and mean particle diameter of MIPs. The effect of increase in the percentage 266

of toluene on the morphology of MIPs is also reflected in the results of particle size 267

analysis. The particle size decreased with the increase in the toluene percentage due 268

to the different solubility parameters of components as discussed above. 269

In the further experiments, the MIPs were equilibrated with acetonitrile for two 270

hours and the eluent fraction was injected in HPLC in order to check any template 271

bleeding from the MIPs. No traces of GA were observed in the eluent (data not 272

shown). 273

The specific binding capacities (BMIP-BNIP) of MIPs were determined by 274

equilibrating them with GA solution and presented in Table S1 (Supplementary 275

material). The MIP-100, MIP-90 and MIP-80 exhibited almost similar binding and no 276

significant influence of solvent variation was observed on the binding performance of 277

these three MIPs. However, the specific binding capacity of M-75 was found to be 278

increased. M-50 on suspension in the GA solution resulted in a turbid solution which 279

did not settle even on rigorous centrifugation and filtration of the solution. Further 280

studies were carried out by using M-100 and M-75 as they exhibited higher specific 281

binding capacity. 282

3.2 FT-IR characterization of M-100 and M-75 283

14

The FT-IR spectra of the M-100 before and after removal of template GA are 284

presented in Figure 3 (a) and (b), respectively. A broad band at 3600 cm-1 due to –OH 285

stretching vibration of monomer AA was seen in the FT-IR spectra of M-100 before 286

removal of GA from its matrix (Figure 3 (a)). The –OH stretching vibration band of 287

monomer AA appeared at 3630 cm-1 in the spectra of M-100 after GA removal 288

(Figure 3 (b)). 289

Appearance of a broad band at lower vibrational frequency, as seen in Fig 3 (a) 290

indicated that the template GA formed hydrogen bonding interaction with monomer 291

AA (Kemp, 1991). This band is shifted to a higher wavenumber (at 3630 cm-1) after 292

removal of GA in M-100 (Figure 3(b)). 293

The peak at 3631 cm-1 in Fig. 3 (c) corresponds to the –OH stretching of monomer 294

AA in the FT-IR spectra of N-100. The shifting of vibrational frequency to the lower 295

wavenumber was not observed for the –OH stretching band in the spectra of N-100 296

due to the absence of GA in its matrix. 297

A conspicuous band at 1576 cm-1 in the spectra of M-100 (Figure 3 (a)) is ascribed 298

to –C=C- aromatic ring stretching vibration of GA. This band disappeared after 299

removal of GA in M-100 (Figure 3 (b)) and was not observed in spectra of N-100 due 300

to absence of GA (Figure 3 (c)). 301

Similarly, the FT-IR spectra of the M-75 before and after removal of GA are 302

presented in Figure 3 (d) and (e), respectively. A broad band at 3594 cm-1 is attributed 303

to the –OH stretching of monomer AA (Figure 3 (d)). This band shifted to 3630 cm-1 304

in the spectra of M-75 after GA removal (Figure 3 (e)) which indicates the formation 305

15

of hydrogen bond initially between GA and AA in M-75. The –OH stretching band of 306

monomer appeared at 3621 cm-1 in N-75 (Figure 3(f)). 307

The –C=C- aromatic ring stretching vibration of GA was seen at 1574 cm-1 in 308

Figure 3 (d), which disappeared after removal of GA in M-75 (Figure 3 (e)). It was 309

also not observed in the spectra of N-100 due to absence of GA (Figure 3 (f)). 310

The other important bands observed in the spectra of MIPs before and after GA 311

removal and in the spectra of NIPs were: carbonyl stretching (1730 cm-1), -C-O 312

stretching (~1155 cm-1) and symmetric and asymmetric stretching of C-H due to the 313

methyl and methylene groups existing in the polymer network (doublet peak at ~2986 314

and ~2955 cm-1, ~1455 cm-1, ~1388 cm-1 ~950 cm-1 and ~880 cm-1). This indicated a 315

similarity in the MIPs and NIPs backbone structure due to incorporation of 316

crosslinker EGDMA. 317

3.3 BET surface area characterization and effect of swelling in MIPs 318

BET surface area characterization showed that both types of MIPs exhibited a well 319

developed pores structure in dry state. M-100 has micropore volume 3.35 m3g-1, 320

micropore area 77.58 m2g-1, surface area 96.73 m2g-1, and average pore diameter 2.64 321

nm. M-75 has micropore volume 9.91 m3g-1, micropore area 221.3 m2g-1, surface area 322

345.9 m2g-1, and average pore diameter 6.14 nm. The typical adsorption-desorption 323

hysteresis of MIPs (Fig. S2, supplementary material) indicated a type IV behavior, 324

indicating the mesoporosity. 325

M-75 had higher surface area and pore volume indicating formation of a porous 326

matrix. The presence of toluene during synthesis of M-75 is responsible for higher 327

porosity as it acts as porogen here in the acetonitrile-toluene solvent mixture. Toluene 328

16

prevents the growing polymeric chains in the solution from collapsing and also delays 329

the process of phase separation resulting in MIPs with well developed pore structure. 330

On the other hand, acetonitrile is responsible for precipitating the polymeric chains 331

once they reach a certain critical mass. The bigger pore diameter of M-75 can provide 332

GA molecules more steric maneuverability within the pore and this leads to a higher 333

binding of template. 334

The percent swelling ratio of M-100 and N-100 in water was 6% and 8% 335

respectively. Similarly, the percent swelling ratio of M-75 and N-100 in water was 5% 336

and 7% respectively. The swelling of MIPs polymeric matrix may modify the shape 337

of imprinted cavities and thus the binding capacity and performance of MIPs. A 338

moderate swelling in MIPs is advantageous for the GA extraction protocol. 339

3.4 Binding performance of M-100 and M-75 in aqueous condition 340

3.4.1 Study of the effect of pH 341

The influence of pH on the binding capacity of GA onto imprinted and non-342

imprinted polymers was studied [Fig. S3 (a) and (b) for M-75 and M-100, 343

respectively, Supplementary material]. The binding capacity of MIPs was higher in 344

the range of pH 3 to 4.5 with the maximum at 3.5. At pH >5 the binding by MIPs and 345

NIPs became almost equal leading to complete loss of imprinting efficiency. The 346

observed trend in the rebinding capacity of MIPs can be attributed to the existence of 347

different forms of GA at different pH. GA is protonated in acidic pH, thus, the affinity 348

of polymer for GA decreased in the acidic pH. Whereas, in the basic pH, GA is 349

present in its anion form (sodium salt of GA). There is a mismatch between the 350

17

imprinted sites and sodium salt of GA. At both the higher and lower pH, GA is not 351

available for formation of hydrogen bonding interactions with the AA. Thus, the 352

binding capacity of the polymer for GA was greatly reduced in both the conditions as 353

acid–base equilibrium or hydrogen bond interaction is subject to changes in pH 354

(Urraca, Carbajo, Torralvo, Gonzalez-Vazquez, Orellana & Moreno-Bondi, 2008). 355

The pKa of GA and AA is 4.7 and 4.35, respectively and both of them are present in 356

their neutral form at this pH range around 3 to 4.5 Thus, they can form hydrogen 357

bonding interactions with each other leading to a higher binding capacity of the MIPs. 358

3.4.2 Dose and Time optimization study 359

The results of dose optimization experiments are presented in Fig. S3 (c) 360

(Supplementary material) which shows that the binding capacity of MIPs increased 361

with the increase in dose and became constant after 50 mg of dose for both the M-100 362

and M-75. The further experiments were carried out using this as the optimum dose. 363

The dynamic curves for the sorption of GA onto the polymers at various time 364

intervals were determined [Fig. S3 (d), Supplementary material]. The curves of MIPs 365

indicated that the sorption of GA increased rapidly with time and then reached 366

equilibrium. The sorption process could be divided into a rapid step up to 180 367

minutes and subsequent slow one. The total contact time required to reach 368

equilibrium was 240 minutes. This may be due to the imprinting cavities or 369

recognition sites present scattered in the crosslinked polymeric matrix. During the 370

binding, the imprinting cavities present near the surface are quickly occupied by the 371

template till the surface is saturated, resulting in a high rate of binding. Further, the 372

rate of binding decreases, as a result of diffusion of template inside the polymer 373

18

network. Generally, the equilibration times from 12 to 20 hours are reported. 374

Moreover, MIPs contain a heterogeneous distribution of binding sites i.e. high and 375

low affinity sites which range in affinity and selectivity. The low affinity binding sites 376

have slow binding kinetics. The surface imprinting of MIPs coated on substrates or 377

adsorbents with high surface area can circumvent the problem of slower binding 378

kinetics (Yang, Jiao, Zhou, Chen, & Jiang, 2013). 379

3.4.3 Binding isotherm 380

The binding performance of MIPs was studied [Fig. S4 (a) and (b) for M-75 and M-381

100 respectively, Supplementary material]. The binding isotherm of both MIPs 382

displayed the curved trend which is an indication of presence of both higher and 383

lower affinity of binding sites in MIPs. The data was fitted in to Langmuir-Freundlich 384

isotherm (LFI) model as this is well suited to model both saturated and unsaturated 385

regions of binding isotherm. LFI (Eq.3) is a hybrid model of Langmuir and 386

Freundlich isotherms models. It describes a specific relationship between the 387

equilibrium concentration of bound (B) and free analyte (F) in the solution with three 388

fitting coefficients i.e. Nt, a and m. Where, ‘Nt’ is the total number of binding sites, ‘a’ 389

is the affinity distribution related to the average binding affinity (K) towards K = a1/m, 390

and ‘m’ is the heterogeneity index. 391

B= Nt aFm/(1+aFm) (3) 392

The isotherm was fitted to the LFI model using a log plot [Fig. S4 (c) and (d) for M-393

75 and M-100 respectively, Supplementary material] followed by calculation of 394

binding parameters (Nt, a, m and K) using the solver function in Microsoft Excel 395

19

(Umpleby, Baxter, Chen, Shah, & Shimizu, 2001). The binding parameters are 396

presented in Table 1. 397

The R2 values for fitting of curves indicated good fitting of data. In general, MIPs 398

exhibited higher number of binding sites (N t), greater affinity distribution of binding 399

sites (a) and higher binding affinity (K) than the corresponding NIPs. The values of 400

‘m’ indicated formation of more heterogeneous binding sites in case of MIPs. The 401

observed trend can be attributed to the presence of GA in the synthesis procedure of 402

MIPs. GA is responsible for creation of binding pockets of definite shape and 403

selectivity in the MIPs; however it is also responsible for heterogeneous distribution 404

of binding sites (m). M-75 has comparatively higher number of binding sites (Nt) and 405

affinity (a) for GA and the values of ‘m’ indicated homogenous binding sites in M-75. 406

Thus, the binding sites of similar nature formed in the M-75 are responsible for its 407

better binding performance. 408

M-100 had much lower surface area (96.73 m2g-1) than M-75 (345.9 m2g-1), and 409

comparatively exhibited good binding capabilities. This is due to the fact that the 410

recognition ability of MIPs is due to formation of binding cavities in the MIP network 411

and it is independent of surface area of MIPs (Spivak, 2005). 412

3.5 Selectivity study of MIPs 413

The complete confirmation of the imprinting performance involves the study of the 414

selectivity of the MIPs. This was accomplished by determination of binding capacity 415

of MIPs in presence of other structural analogues of the GA. The mixture of GA and 416

other structural analogues was equilibrated with polymers and analyzed by HPLC and 417

the chromatograms are presented in the Fig.S5 (supplementary material). The 418

20

retention time of GA and structural analogues were determined by comparing with the 419

retention time of their standard. Fig.S5 clearly shows the decrease in the intensity of 420

GA peak as compared to the peaks of other structural analogues for MIPs. NIPs 421

exhibited binding for GA and structural analogues due to the unspecific interactions. 422

The specific binding capacities of MIPs and NIPs are presented in Table 2. 423

4-HBA lacks the phenolic –OH groups in its structure and due to its smaller size 424

than GA, it may easily permeate into the binding sites of MIPs. Thus 4-HBA 425

exhibited considerable higher binding by all the MIPs but lesser than GA. 2,4-DBA, 426

3,4-DBA and 3,5-DBA are the polyphenolic carboxylic acids similar to GA but 427

lacking in one –OH group in their structure (Fig.S1 supplementary material). The 428

MIPs exhibited binding for these compounds up to some extent due to the presence of 429

carboxylic group in these compounds. The formation of di-hydrogen bond between 430

the carboxylic acid groups of the GA and AA is the main interaction responsible of 431

the recognition of the GA (Pardeshi, Patrikar, Dhodapkar, & Kumar, 2012). However, 432

the variation in the molecular size due to lacking of one –OH groups in the structural 433

analogues hinders the accessibility of the imprinting sites. Moreover, the nature of the 434

binding of structural analogues was unspecific because the same binding percent were 435

obtained in the NIP too. The MIPs exhibited higher specific binding for GA due to the 436

presence of imprinting cavities with fixed size, shape and stereochemistry for GA 437

molecule. It was also observed that both M-100 and M-75 were selective for GA. 438

3.6 Application of MIP to real complex samples 439

An external calibration curve for GA was constructed with R2 is equal to 0.999 and 440

the retention time of GA was found to be 4.7 minutes for the GA standard. The E. 441

21

officinalis extract was injected and a prominent peak of GA was well matched at the 442

retention time 4.7 minutes in the chromatogram. The origina l pH of aqueous E. 443

officinalis extract was 3.4 thus no pH adjustments were required in the GA extraction 444

experiments. The % RSD for intermediate precision (inter-day precision) and 445

repeatability (intra-day precision) was found to be 0.85% and 2.3% respectively. 446

Accuracy of the method was assessed by spiking the extract with the GA. Recovery 447

values were obtained between 96% to 98%, which indicated a satisfying accuracy of 448

the method. 449

The HPLC chromatograms are shown in Fig. 4 (a- i) and (b- i). A 10 mL of the 450

spiked aqueous E. officinalis extract was equilibrated with MIPs in the optimized 451

conditions. The HPLC chromatograms of the extract after equilibration with MIPs are 452

presented in Fig. 4 (a-ii) for M-100 and Fig.4 (b- ii) for M-75 respectively. A 453

considerable decrease in the peak intensity of GA can be observed after equilibration 454

with MIPs due to the retention of GA onto the MIPs in both M-100 and M-75. This 455

decrease in GA peak was more prominent in M-75. 456

MIPs were collected and a washing step was performed in order to remove the other 457

components absorbed by MIPs as a consequence of unspecific interactions. The MIPs 458

were washed with acetonitrile –water (50:50 v/v) for 30 min and analyzed. No peak 459

of GA was detected in the washing fraction (data not shown). Finally, selective 460

elution of pure GA from the MIPs was achieved by treatment with hot water at 60 °C. 461

Generally, methanol is used in the elution step (Karasova, Lehotay, Sadecka, Skacani, 462

& Lachova, 2005); however hot water treatment was preferred in order to avoid use 463

of toxic solvent and to develop a green approach. During the treatment with hot 464

22

water, water molecules compete with the template for interactions with functional 465

groups in the imprinted cavities, thus allowing GA release (Puoci et al., 2012). Fig. 4 466

(a-iii) and Fig. 4 (b-iii) reports the chromatogram of the eluting fraction, showing the 467

prominent peak for GA. It can be observed that the peaks of other components were 468

not appeared in the chromatogram, thus indicating a successful selective extraction of 469

GA from E. officinalis. The selective recovery of pure GA was calculated to be 75% 470

(±1.6) and 83.4 (±2.2) for M-100 and M-75 respectively. These results revealed that 471

MIPs could specifically extract GA from the complex sample matrix of E. officinalis 472

and establish the application potential of MIPs as a selective sorption material. 473

Earlier work on GA imprinted MIPs indicated that, GA was extracted from a 474

methanolic extract of Cornus officinalis containing 0.392 mgL-1 of GA, by the MIPs 475

prepared by bulk polymerization (Zhu et al. 2009). A six hours equilibration resulted 476

in 40.5% uptake of GA and further elution of pure GA from the MIP s matrix was not 477

reported. Further, the uptake of GA from an ethanolic green tea extract was reported 478

by Nicolescu et al. (2012) using the MIPs synthesized by suspension polymerization. 479

A 50 mg of MIPs equilibration with the 5 mL of ethanolic green tea extract containing 480

36 gL-1 of GA for 20 hours resulted in 8.33% GA uptake. These reports indicated 481

lower percent of GA enrichment from the real samples. Similarly, MIPs prepared by 482

bulk polymerization were applied to selectively extract GA from the olive mill 483

wastewaters. 500 mg of MIPs was equilibrated with 10 mL sample spiked with 170 484

mgL-1 of GA for six hours and 80% of pure GA was eluted from the MIPs matrix in 485

aqueous condition (Puoci et al. 2012). 486

23

Synthesis of MIPs by bulk polymerization is considered simple however, it is 487

comparatively uneconomical method due to the loss of yield in post processing steps 488

of grinding, sieving and sedimentation. Moreover, the process is tedious and time 489

consuming. Suspension polymerization yields ready to use MIPs and obviates the 490

post processing steps. However, this method is considered complex (Haginaka, 2008) 491

and the use of suspension stabilizers increases the cost of MIPs production. 492

Herein in the present work, we report high percent of selective extraction of GA 493

from E. officinalis (75% and 83.4 % for M-100 and M-75 respectively). The present 494

method depicts a simple and cost effective method of MIPs preparation by the 495

precipitation polymerization. The ready to use MIPs are obtained without use of 496

stabilizers or emulsifiers in synthesis for the direct application, circumventing the 497

time consuming post processing steps and material loss. 498

4. Conclusions 499

GA imprinted microspheres and nanoparticles were successfully synthesized using 500

the precipitation polymerization method. The morphology study by SEM and particle 501

size analysis of MIPs indicated influence of toluene on the particle size. The results 502

have shown that effect of toluene on the particle size of MIPs depends on the type of 503

crosslinker used and its solubility parameter. Thus, to obtain the desired particle size 504

in MIPs, matching the solubility parameter of solvent mixture and crosslinkers is 505

important. The influence of toluene was also seen on the surface areas of MIPs, 506

toluene influenced the porosity in the MIPs resulting in the higher surface area. The 507

Langmuir Freundlich isotherm model described that the formation of more 508

homogenous binding sites in M-75 was responsible for its better binding 509

24

performance. The MIPs were found selective for GA in presence of other structural 510

analogues. Although higher binding with unspecific nature was observed for 511

structural analogues due to interaction between the analyte and monomer carboxylic 512

groups, the shape and stereochemistry of molecule is important for specific 513

recognition by binding site. The MIPs were applied for selective extraction of GA for 514

complex aqueous matrix of herb E. officinalis. MIPs have shown good performance 515

with superior recovery for GA when hot water was used as an eluting solvent. The M-516

100 has morphology suitable for the chromatography and SPE application while M-517

75 had better recognition performance. Water is used in the overall process of GA 518

extraction thus the present method is an eco-friendly approach for selective extraction 519

of GA from herb with the higher recovery. 520

521

522

523

524

525

526

527

528

529

25

Acknowledgement 530

The authors are grateful to Dr. Sachin Mandavgane, Department of Chemical 531

Engineering, V.N.I.T., for giving permission to assess the HPLC. 532

The authors thank the anonymous reviewers for their constructive and valuable 533

suggestions which helped in improving the quality of this paper. 534

535

536

537

538

539

540

541

542

543

544

545

546

547

26

References 548

Beltran, A., Marcé, R.M., Cormack, P.A.G., & Borrulla F. (2009). Synthesis by 549

precipitation polymerisation of molecularly imprinted polymer microspheres for 550

the selective extraction of carbamazepine and oxcarbazepine from human urine. 551

Journal of Chromatography A, 1216, 2248–2253. 552

Borde, V., Pangrikar, P., & Tekale, S. (2011). Gallic acid in Ayurvedic herbs and 553

formulations. Recent Research in Science and Technology, 3, 51-54. 554

Cirillo, G., Curcio, M., Parisi, O., Puoci, F., Iemma, F., Spizzirri, U., Restuccia, D., & 555

Picci, N. (2011). Molecularly imprinted polymers for the selective extraction of 556

glycyrrhizic acid from liquorice roots. Food Chemistry, 125, 1058–1063. 557

Esfandyari-Manesh, M., Javanbakht, M., Atyabi, F., Mohammadi, A., Mohammadi, 558

S., Akbari-Adergani, B., & Dinarvand, R. (2011). Dipyridamole recognition and 559

controlled release by uniformly sized molecularly imprinted nanospheres. 560

Material Science and Engineering C, 31, 1692-1699. 561

Gholivanda, M., Karimian, N., & Malekzadeh, G. (2012). Computational design and 562

synthesis of a high selective molecularly imprinted polymer for voltammetric 563

sensing of propazine in food samples. Talanta, 89, 513– 520. 564

Haginaka, J. (2008). Monodispersed, molecularly imprinted polymers as affinity-565

based chromatography media, Journal of Chromatography B, 866, 3–13. 566

Hu, Y., Pan, J., Zhang, K., Lian, H., & Li, G., (2013). Novel applications of 567

molecularly imprinted polymers in sample preparation. Trends in Analytical 568

Chemistry, 43, 37-52. 569

27

Karasova, G., Lehotay, J., Sadecka, J., Skacani, I., & Lachova, M, (2005). Selective 570

extraction of derivates of p-hydroxybenzoic acid from plant material by using a 571

molecularly imprinted polymer, Journal of Separation Science, 28, 2468–2476. 572

Kemp, W. (1991). Organic Spectroscopy, (3rd ed.) Macmillan, Hampshire. (Chapter 573

2). 574

Kim, Y. (2007). Antimelanogenic and antioxidant properties of gallic acid, Biological 575

& Pharmaceutical Bulletin, 30, 1052—1055. 576

Lai, J., Yang, M., & Niessner R., (2007). Molecularly imprinted microspheres and 577

nanospheres for di(2-ethylhexyl) phthalate prepared by precipitation 578

polymerization. Analytical and Bioanalytical Chemistry, 389, 405-412. 579

Lokeshwari, N., & Reddy, S. (2010). Microbiological production of gallic acid by a 580

mutant strain of Aspergillus oryzae using cashew husk. Pharmacophore. 1, 112-581

122. 582

Lu, J.J., Wei, Y., & Yuan, Q. (2007). Preparative separation of gallic acid from 583

Chinese traditional medicine by high-speed counter-current chromatography and 584

followed by preparative liquid chromatography. Separation and Purification 585

Technology, 55, 40–43. 586

Majeed, M., Bhat, B., Jadhav, A., Srivastava, J., & Nagabhushanam, K. (2009). 587

Ascorbic acid and Tannins from Emblica officinalis gaertn. Fruits-a revisit, 588

Journal of Agriculture and Food Chemistry, 57, 220-225. 589

Nicolescu, T., Meouche, W., Branger, C., Margailillan, A., Sarbu, A., & Donescu, D. 590

(2012) Tailor made polymer beads for gallic acid recognition and separation, 591

Journal of Polymer Research, 19, 1-12. 592

28

Pardeshi, S., Dhodapkar, R., & Kumar, A., (2012). Studies on molecular recognition 593

abilities of gallic acid imprinted polymer prepared using molecular imprinting 594

technique, Adsorption Science and Technology, 30, 23-34. 595

Pardeshi, S., Kumar, A., & Dhodapkar, R. (2011). Molecular Imprinting: Mimicking 596

molecular receptors for antioxidants. Material Science Forum. 675; 677, 515-520. 597

Pardeshi, S., Patrikar, R., Dhodapkar, R., & Kumar, A. (2012).Validation of 598

computational approach to study monomer selectivity towards the template gallic 599

acid for rational molecularly imprinted polymer design. Journal of Molecular 600

Modeling, 18, 4797–4810. 601

Puoci, F., Scoma, A., Cirillo, G., Bertin, L., Fava, F., & Picci, N. (2012). Selective 602

extraction and purification of gallic acid from actual site olive mill wastewaters 603

by means of molecularly imprinted microparticles. Chemical Engineering 604

Journal, 529–535, 198–199. 605

Qiu, H., Luo, C., Sun, M., Lu, F., Fan, L., & Li, X. (2012). A novel 606

chemiluminescence sensor for determination of quercetin based on molecularly 607

imprinted polymeric microspheres. Food Chemistry, 134, 469–473. 608

Quesada-Molina, C., Claude, B., Garcia-Campana, A., Olmo-Iruela, M., & Morin, P. 609

(2012). Convenient solid phase extraction of cephalosporins in milk using a 610

molecularly imprinted polymer. Food Chemistry, 135, 775–779. 611

Raina, K., Rajamanickam, S., Deep, G., Singh, M., Agarwal, R., & Agarwal, C. 612

(2008). Chemopreventive effects of oral gallic acid feeding on tumor growth and 613

progression in TRAMP mice. Molecular Cancer Therapeutics, 7, 1258-1267. 614

29

Spivak, D., (2005). Optimization, evaluation, and characterization of molecularly 615

imprinted polymers. Advanced Drug Delivery Reviews. 57, 1779– 1794. 616

Umpleby, R., Baxter S., Chen, Y., Shah, R., & Shimizu K. (2001). Characterization 617

of Molecularly Imprinted Polymers with the Langmuir-Freundlich Isotherm. 618

Analytical Chemistry, 73, 4584-4591. 619

Urraca, J., Carbajo, M., Torralvo, M., González-Vázquez, J, .Orellana, G, & Moreno-620

Bondi M., (2008). Effect of the template and functional monomer on the textural 621

properties of molecularly imprinted polymers. Biosensors and Bioelectronics, 24, 622

155-161. 623

Wang, J., Cormack, P.A., Sherrington, D.C. & Khoshdel E. (2007). Synthesis and 624

characterization of micrometer-sized molecularly imprinted spherical polymer 625

particulates prepared via precipitation polymerization. Pure and Applied 626

Chemistry, 79, 1505-1519. 627

Wang,Y., Liu, Q., Rong, F., & Fu, D. (2011). A facile method for grafting of bisphenol 628

A imprinted polymer shells onto poly(divinylbenzene) microspheres through 629

precipitation polymerization, Applied Surface Science, 257, 6704–6710. 630

Yang, W., Jiao F., Zhou, L., Chen X., Jiang, X. (2013). Molecularly imprinted 631

polymers coated on multi-walled carbonnanotubes through a simple indirect 632

method for the determination of2,4-dichlorophenoxyacetic acid in environmental 633

water, Applied Surface Science, In press 634

Zhang, W., & Chen, Z. (2013). Preparation of micropipette tip-based molecularly 635

imprinted monolith for selective micro-solid phase extraction of berberine in 636

plasma and urine samples, Talanta, 103, 103-109. 637

30

Zhu, X., Cao, Q., Yang, X., Li, F., Wang, G., & Ding, Z. (2009). Preparation and 638

recognition mechanism of gallic acid imprinted polymers, Helvetica Chimica 639

Acta, 92, 78-87. 640

641

642

643

644

645

646

647

648

649

650

651

652

653

654

655

656

657

658

659

660

661

31

Figure captions 662

Fig.1. SEM micrographs of a) M-100, b) M-90, c) M-80, d) M-75, e) M-50. 663

Fig.2. Particle size distributions of M-100, M-90, M-80, M-75, and M-50. 664

Fig.3. The FT-IR spectra of a) M-100 before removal of GA, b) M-100 after removal 665

of GA, c) N-100, d) M-75 before removal of GA, e) M-75 after removal of GA, f) N-666

75. 667

Fig. 4. a) HPLC chromatogram i) Initial sample of E. officinalis extract, ii) E. 668

officinalis extract after equilibration with M-100, iii) chromatogram of pure GA 669

obtained after elution with hot water from M-100 matrix; 670

Fig. 4. b) HPLC chromatogram i) Initial sample of E. officinalis extract, ii) E. 671

officinalis extract after equilibration with M-75, iii) chromatogram of pure GA 672

obtained after elution with hot water from M-75 matrix. 673

Table 1

Fitting parameters for Langmuir-Freundlich isotherm model for imprinted and non-imprinted

polymers

Langmuir-Freundlich isotherm model

Polymer Nt (μmolg-1

) a (M-1

) K (mmol-1

L) m R2

M100 92.14 (±2.1) 30.46 (±3.4) 82.03 (±3.2) 0.7752 0.9892

N100 45.52 (±1.8) 13.20 (±2.1) 22.93 (±2.8) 0.8236 0.9903

M75 105.75 (±1.4) 44.41 (±2.4) 68.86 (±2.3) 0.8963 0.9882

N75 58.23 (±2.2) 10.15 (±2.3) 11.91 (±2.4) 0.9352 0.9874

Table(s)

Table 2

Binding capacities of polymers (µmolg-1

) and specific binding (BMIP-BNIP) of MIPs for the GA

and its structural analogues

Compounds B (µmolg-1

) B (µmolg-1

)

M-100 N-100 Specific

binding M-75 N-75

Specific

binding

GA 837.7 403.1 434.6 1191.8 576.3 615.5

4-HBA 723.5 612.2 111.3 968.2 713.1 255.1

3,4-DBA 682.7 596.2 86.5 833.1 632.4 200.7

3,4-DBA 645.2 555.2 90 813.2 610.2 203

2,4-DBA 583.4 493.2 90.2 741.8 632 109.8

Fig. 1

Figure(s)

Fig. 2

Fig. 3

Fig.4

1

Research Highlights

MIPs for gallic acid (GA) were prepared by the precipitation polymerization

method

The porogen influenced the structural appearance and binding properties of

MIPs.

MIPs particles were in the form of microspheres and nanoparticles.

MIPs selectively extracted GA from aqueous extracts of Emblica officinalis.

Recoveries of GA in Emblica officinalis were 75 % and 83.4 % for M-100 and

M-75 respectively.

*Highlights (for review)