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Colloids and Surfaces A: Physicochem. Eng. Aspects 445 (2014) 40– 47

Contents lists available at ScienceDirect

Colloids and Surfaces A: Physicochemical andEngineering Aspects

jo ur nal ho me p ag e: www.elsev ier .com/ locate /co lsur fa

oronic acid functionalized polymeric microspheresor catecholamine isolation

alan Özdemir ∗, Ays e C akır, Burcu Somtürkhemistry Department, Faculty of Science, Erciyes University, TR-38039 Kayseri, Turkey

i g h l i g h t s

3-Aminophenyl boronic acidfunctionalized-poly(3-chloro-2-hydroxypropyl methacrylate) [APBAfunctionalized-poly(HPMA-Cl)]microspheres.A new sorbent for catecholamine iso-lation.The reversible catecholamine bindingbehavior of the APBA functionalized-poly(HPMA-Cl) microspheres.

g r a p h i c a l a b s t r a c t

r t i c l e i n f o

rticle history:eceived 1 October 2013eceived in revised form 2 January 2014ccepted 6 January 2014vailable online 15 January 2014

eywords:atecholaminesoronate affinity techniqueminophenyl boronic acidolymeric microspheresuspension polymerization

a b s t r a c t

In this study, 3-aminophenyl boronic acid functionalized-poly(3-chloro-2-hydroxypropyl methacrylate)[APBA functionalized-poly(HPMA-Cl)] microspheres were prepared and characterized as a new alterna-tive sorbent for catecholamine isolation. Chloropropyl group containing poly(HPMA-Cl) microsphereswere synthesized in spherical gel form with a suspension polymerization technique, and it is followedby the APBA conjugation to the poly(HPMA-Cl) microspheres through amide bond formation. After-wards, for the characterization of the prepared microspheres, SEM images were taken and FTIR analysiswas performed. Furthermore, to determine APBA content of prepared microspheres, nitrogen analysiswas performed and the amount of APBA was calculated as 94.6 mg APBA/g microsphere. The aver-age size of the APBA functionalized-poly(HPMA-Cl) microspheres was calculated as 205 ± 35 �m. Thereversible catecholamine (dopamine, epinephrine and norepinephrine) binding behavior of the APBAfunctionalized-poly(HPMA-Cl) and poly(HPMA-Cl) microspheres was investigated in batch fashion type.In the reversible catecholamine binding experiments, the maximum equilibrium binding was obtained atpH 8.5 which is very close to the pKa of boronic acid, therefore all the experiments were carried out at thispH. While no-significant non-specific binding was observed onto the non-functionalized microspheres,

the maximum catecholamine binding capacities of the APBA functionalized-poly(HPMA-Cl) microsphereswere found as 61 mg, 7 mg and 8 mg per gram of microsphere for dopamine, epinephrine and norepi-nephrine, respectively. All three of the catecholamines were released from the microspheres with theyields higher than 90% by weight. The obtained results indicated that APBA functionalized-poly(HPMA-Cl) microspheres are promising sorbents that can be used for the efficient isolation of catecholamines.

∗ Corresponding author. Tel.: +90 352 207 66 66x33176; fax: +90 352 437 49 31.E-mail addresses: nalanoz dmr@hotmail.com, ozdemirn@erciyes.edu.tr

N. Özdemir), cakir.aysee@hotmail.com (A. C akır), burcu smtrk@hotmail.comB. Somtürk).

927-7757/$ – see front matter © 2014 Published by Elsevier B.V.ttp://dx.doi.org/10.1016/j.colsurfa.2014.01.005

© 2014 Published by Elsevier B.V.

1. Introduction

Dopamine (DA), epinephrine (E) and norepinephrine (NE) arevery well known adrenal hormones, which are also called cate-cholamines. They are a group of biogenic amines that also containa 3,4-dihydroxy-substituted phenyl ring [1,2] and have a number

: Phy

opriuhrbMrnbiaatoatosgftfm

akabct

ctiTigaeatlttattatmraoasppi[sbta

N. Özdemir et al. / Colloids and Surfaces A

f important physiological roles in the human body [3]. For exam-le, as a monoamine neurotransmitter, dopamine plays a majorole in controlling movement, emotional responses and the abil-ty to experience pleasure and pain [4]. Catecholamines have beensed as classic biomarkers for the diagnosis of certain diseases andave also been used in the treatment of the diseases [4–8]. For thiseason, the isolation and determination of catecholamines from aiological environment (fluids or tissues) hold a great potential.any studies in the literature have been conducted to develop

eliable bioanalytical methods for the separation and determi-ation of catecholamines [1,3,9–13]. Several methods have alsoeen proposed for the extraction of catecholamines. When work-

ng with clinical samples, liquid–liquid and solid-phase extractionre the two most frequently used techniques for the extractionnd preconcentration of catecholamines. The solid-phase extrac-ion is mostly preferred, because this technique does not require notnly large volume of toxic organic solvent but also labor intensivend time-consuming double extraction procedure [14]. However,here are limited numbers of sorbent materials which are capablef the adsorption of catecholamines. Sephadex G10 [12], weak ortrong cation exchange resin [15–17], C18 matrix [18], boronateels [15,19], and alumina [9,20] are commonly used adsorbentsor the extraction of catecholamines from clinical samples. Sincehe adsorption capacity of those adsorbents is not efficient enoughor catecholamines, there is still a huge need for new adsorbent

aterials with high adsorption capacities.There is a considerable interest in the synthesis, design and

pplication of polymeric materials for the separation of differentinds of biomolecules [21–28]. Polymeric adsorbents have somedvantages, such as higher adsorption efficiency and chemical sta-ility, lower cost and being environment friendly. Their surfacehemistry and pore structure can be adjusted and thus the selec-ivity can be controlled.

Affinity chromatography is an effective technique for the purifi-ation of biomolecules. This chromatography technique utilizeshe biospecific interaction between target biomolecules and affin-ty ligand immobilized to the matrix of the chromatography.he boronate affinity chromatography, a special form of affin-ty chromatography, uses the interaction between the hydroxylroups of boronate and the cis-diol groups of biomolecules suchs nucleotides, glycoproteins, carbohydrates [29], catecholamines,tc. The principle of this technique relies on the formation of

complex (cyclic esters) between the cis-diol moieties of thearget biomolecule and the hydroxyl groups of the boronic acidigand at alkaline pHs (usually at pH: 8.5). This complex forma-ion is reversible. When the pH of the environment is adjustedo be acidic, the complex dissociates [30]. Thereby, in boronateffinity applications, cis-diol carrying biomolecules can be selec-ively adsorbed onto the desired material and desorbed based onhis pH dependent reversible complex formation. This selectivedsorption/desorption ease the isolation and enrichment of thearget cis-diol carrying biomolecules from a complex biological

ixture [31]. Many applications of boranate-affinity chromatog-aphy have been reported in the literature. This technique haslso been successfully utilized in the separation of several kindsf biomolecules including, but not limited to, sugars, nucleiccids [32], thymine glycol DNA and nucleotides [33], catecholiderophores [34] and lectins [35]. As new sorbent systems,oly(styrene-co-vinylphenylboronic acid-co-divinylbenzene) ter-olymer based microspheres [36], thermo-responsive poly(N-

sopropylacrylamide)-co-(vinylphenylboronic acid) microparticles31], and aminophenyl boronic acid modified hydrogel micro-

pheres [37] were used to study nucleotide adsorption–desorptionehavior. In a study, poly(N-ethylmethacrylamide) thermosensi-ive particles were functionalized with phenylboronic acid groupnd proposed for various applications [38]. In another study, phenyl

sicochem. Eng. Aspects 445 (2014) 40– 47 41

boronic acid chromatographic beads were prepared and used toadsorption of cell impurities from plasmid-containing lysates [39].Furthermore, in recent study, boronic acid functionalized macro-porous uniform poly(4-chloromethylstyrene-co-divinylbenzene)particles were synthesized and characterized for isolation ofantioxidant compounds from plant extracts [40].

This study was aimed at the investigation of the reversiblecatecholamine binding behavior of the APBA functionalized-poly(HPMA-Cl) microspheres as a new type of sorbent. Thestudy consists of two parts. In the first part, poly(3-chloro-2-hydroxypropyl methacrylate) [poly(HPMA-Cl)] reactive micro-spheres were prepared. Afterwards, 3-aminophenyl boronic acid(APBA) was covalently bonded to the poly(HPMA-Cl) microspheresvia nucleophilic substitution reaction. APBA was used as a spe-cific ligand to interact with the catecholamines. In the second part,specific cis-diol interaction immobilization of catecholamines wasperformed with the APBA functionalized-poly(HPMA-Cl) micro-spheres and the reversible binding behavior of the target moleculeswas described. Dopamine, epinephrine and norepinephrine wereselected as model molecules. The interaction between the selectedmolecules and proposed material was investigated in batch fash-ion at room temperature. The obtained results indicated that APBAfunctionalized-poly(HPMA-Cl) microspheres are promising sor-bents that can be used for the isolation of catecholamines.

2. Materials and methods

2.1. Materials

All chemicals used in the experiments were of analytical grade.3-Chloro-2-hydroxypropyl methacrylate (HPMA-Cl), poly(vinylalcohol) (PVA), ethylene glycol dimethacrylate (EGDMA), ethylben-zene (EB), dopamine hydrochloride, (±)-epinephrine hydrochlo-ride and dl-noradrenaline hydrochloride, ethyl alcohol, 3-aminophenyl boronic acid (APBA), HCl, and NaOH were purchasedfrom Sigma–Aldrich (USA). Benzoyl peroxide (BPO) and HEPES(4-(2-hydroxyethyl)-1-piperazine ethane sulfonic acid) were pur-chased from Fluka. Sodium acetate and acetic acid were purchasedfrom Merck (Germany). Distilled water was used for preparing allthe solutions. Standard solutions of catecholamines were preparedfreshly at desired concentrations by serial dilution with buffer solu-tions.

2.2. Synthesis of the polymeric microspheres

The reactive polymeric microspheres were prepared by thesuspension polymerization technique. In a typical procedure,to prepare continuous medium, the stabilizer, PVA (0.5 g, Mr:87,000–146,000, degree of hydrolysis: 89%), was dissolved in a cer-tain amount of distilled water (50 mL) with a magnetic stirrer. Themonomer phase containing EGDMA (2.5 mL), HPMA-Cl (2.5 mL) andEB (2.5 mL) was prepared in a test tube and sonicated in a waterbath at room temperature. Then the initiator, BPO (0.1 g), was dis-solved in the organic phase. This homogeneous organic phase wasadded into the continuous medium in the reactor and heated to90 ◦C under magnetic stirring at 200 rpm. After a period of 4 h, thepolymerization was stopped and the reactor was cooled down toroom temperature. Finally, the prepared reactive poly(HPMA-Cl)microspheres were washed with excess ethyl alcohol and distilledwater, respectively, to remove unreacted chemicals. This processwas repeated 3 times.

2.2.1. Attachment of APBA onto the poly(HPMA-Cl) microspheresAfter preparation of the reactive poly(HPMA-Cl) microspheres,

3-aminophenyl boronic acid (APBA) was easily bonded onto thesepolymeric microspheres via a nucleophilic substitution reaction in

42 N. Özdemir et al. / Colloids and Surfaces A: Physicochem. Eng. Aspects 445 (2014) 40– 47

CH2 Cl +

NH2

B

OH

OH

CH2 N

B

OH

H

+ HClOH-

oly(HP

attirtptmaCccoampii

2

2m

ttDtSP

2

uS(Gs

2

nC

2

cam

Fig. 1. Binding process of 3-aminophenyl boronic acid on to p

basic medium. The covalent attachment procedure of APBA ontohe poly(HPMA-Cl) microspheres may be given as follows: a cer-ain amount of poly(HPMA-Cl) microspheres (1.5 g) were dispersedn an aqueous solution of APBA (15 mL) buffered at pH 12. Theesulting dispersion was stirred magnetically at 200 rpm, at roomemperature for 24 h. Then, APBA was directly bonded onto theoly(HPMA-Cl) microspheres via the chemical interaction betweenhe amino groups of the ligand and the chloropropyl groups of the

icrospheres. APBA binding process was realized in a water batht 60 ◦C. After the coupling process, the APBA-carrying poly(HPMA-l) microspheres were kept at room temperature for cooling andleaned by centrifugation–decantation. The supernatant was dis-arded and the microspheres were redispersed in a certain amountf water by ultrasonication. This centrifugation–decantation oper-tion was repeated several times to remove all unreacted excessonomers from the microsphere dispersion. After the cleaning

rocess, the prepared microspheres were allowed to dry. The bind-ng process of APBA onto the poly(HPMA-Cl) microspheres is shownn Fig. 1.

.3. Characterization of the microspheres

.3.1. Determination of the size and size distribution oficrospheres

Morphological evaluations were made by using a Scanning Elec-ron Microscope (SEM) (LEO 440 computer controlled digital). Forhis purpose, the microspheres were dried at room temperature.ried particles were photographed with appropriate magnifica-

ion in SEM by putting them on the sample disk. After receiving aEM image, particle size measurement was performed using Imageroplus 6.0 program and the average particle size was calculated.

.3.2. Structural analysis of microspheresThe chemical structure of the microspheres was evaluated by

sing Fourier Transform Infrared Spectroscopy (FTIR) (Perkin Elmerpectrum 400). In a typical procedure, the dried microspheresabout 0.1 g) were thoroughly mixed with IR-grade KBr (0.1 g, IRrade, Merck, Germany) and pressed into tablet form. The FTIRpectra of the microspheres were obtained with an FTIR system.

.3.3. Nitrogen analysisTo determine the APBA content of the polymeric microspheres,

itrogen analysis was performed using by elemental analyzer (LEO,HNS, 932).

.4. Reversible catecholamine binding studies

Effective parameters on the reversible catecholamine bindingapacity of the prepared polymeric microspheres were selecteds medium pH, initial catecholamine concentration, amount oficrospheres and incubation time. In all studies, the volume of

OH

MA-Cl) microspheres with nucleophilic substitution reaction.

the incubation medium and the amount of microspheres werekept as 5 mL (10 mM HEPES, pH: 8.5) and 0.05 g, respectively.For the derivation of reversible binding isotherm, the initial cate-cholamine concentrations were over the range of 0.5- 4 mg/mL fordopamine, 0.05- 0.2 mg/mL for epinephrine and 0.05- 0.2 mg/mLfor norepinephrine, respectively, depending on the water solubilityof catecholamines. Specific binding and release experiments werecarried out in batch fashion and were performed at room temper-ature.

Reversible catecholamine binding experiments were performedusing both poly(HPMA-Cl) (nonfunctionalized) and APBA function-alized poly(HPMA-Cl) (functionalized) microspheres. In a typicalprocedure, first of all 10 mL of a known concentration of cate-cholamine solution (in HEPES buffer) was prepared and was thendivided into two parts. The absorbance value of the first partof the catecholamine solution (5 mL) was determined at a spe-cific wavelength (the specific maximum absorbance wavelengthof dopamine, epinephrine and norepinephrine was 280 nm) witha UV-spectrophotometer (HITACHI). Afterwards, a fixed amount ofcleaned microspheres (0.05 g) were mixed with the second partof the catecholamine solution (5 mL) in a glass Erlenmeyer flask.This mixture was incubated at room temperature and slowly agi-tated with a magnetic stirrer for a certain period of time (3 h). Afterincubation, the mixture was centrifuged with a temperature con-trolled centrifuge (Nüve, NF 800R) at 5000 rpm for 3 min at 25 ◦C.After isolation of the supernatant, the absorbance value of the cate-cholamine solution was determined with a UV-spectrophotometer.

To determine catecholamine binding capacities of the preparedpolymeric microspheres, Eq. (1) was used.

Q =[

Ai − Af

Ai

]×

[Ci × V

M

](1)

where Q is the binding capacity (mg/g), Ai is the initial absorbancevalue of the incubation medium at a specific wavelength, and Af isthe final absorbance value of the incubation medium at the samewavelength. Ci is the initial concentration of the catecholamine(mg/mL), V is the volume of the incubation solution (mL) and Mis the amount of the dry microsphere (g).

Besides the binding capacity, another important feature ofthe prepared microspheres is the efficiency of catecholaminerelease retained by the microspheres. After completion of thecatecholamine binding process and determination of bindingcapacities, the polymeric microspheres were washed with distilledwater. Then these microspheres were dispersed into the 10 mLof desorption medium (pH: 5.0) in a glass Erlenmeyer flask andthis mixture was slowly agitated with a magnetic stirrer for 1 hat room temperature. The microspheres were centrifuged with a

temperature controlled centrifuge at 5000 rpm for 3 min at 25 ◦C.The supernatant was isolated and the absorbance value of therelease solution was determined with a UV-spectrophotometer.The release ratio of the catecholamines (%, w/w) was calculated by

N. Özdemir et al. / Colloids and Surfaces A: Phy

cm

s

Hcimc(C(

iurt

2

50t0rc

3

3

aSapS

Fig. 2. SEM image of APBA functionalized-poly(HPMA-Cl) microspheres.

onsidering the retained amount of catecholamine by polymericicrosphere via specific cis-diol interaction immobilization.To determine the release ratio of the prepared polymeric micro-

pheres, Eq. (2) was used.

Mg1 =(

Ai − Af

Ai

)× C × V

Mg2 = V × (C∗)

release% =(

Mg2

Mg1

)× 100

(2)

ere, release% is the release ratio. Mg1 is the amount of specificallyaptured catecholamines. Ai is the initial absorbance value of thencubation medium. Af is the absorbance value of the incubation

edium after the binding process. C is the initial concentration ofatecholamines (mg/mL). V is the volume of the incubation solutionmL). Mg2 is the amount of the catecholamines in release solution.* is the concentration of catecholamines after the binding processmg/mL).

Each parameter was studied in triplicate and the binding capac-ties and release% values of the microspheres were determinednder optimum conditions. The binding capacities and releaseatios of the synthesized microspheres are given as the average ofhe results.

.4.1. Binding isothermsEquilibrium binding isotherms were obtained by incubating

mL of catecholamine solutions at different concentrations with.05 g microspheres at room temperature. The initial concentra-ions of catecholamines were 0.5–4 mg/mL, 0.05–0.2 mg/mL and.05–0.2 mg/mL for dopamin, epinephrine and norepinephrine,espectively, depending on the water solubility of the cate-holamines.

. Results and discussion

.1. The size and size distribution of microspheres

The morphology and size distribution of the microspheres werenalyzed by scanning electron microscopy (SEM). According to the

EM images, the particles exhibited well-formed spherical shapes expected (Fig. 2). The average size of the APBA functionalized-oly(HPMA-Cl) microspheres was calculated as 205 ± 35 �m. TheEM images of the microspheres are shown in Fig. 2.

sicochem. Eng. Aspects 445 (2014) 40– 47 43

3.2. Structural analysis of microspheres

The chemical structure of the APBA functionalized-poly(HPMA-Cl) microspheres was characterized by using Fourier TransformInfrared Spectroscopy (FTIR). The characteristic absorption peaks ofthe APBA functionalized-poly(HPMA-Cl) microspheres can be seenin the FTIR spectrum. The broad adsorption band at 3344.77 cm−1

refers to the O H vibration absorption band resulting from 2-hydroxypropyl methacrylate and boronic acid. The band at around3000 cm−1 (2953 cm−1) is due to the phenyl groups of the ligand. Inaddition, a ring vibration peak at 1250 cm−1 is also observed. Thetwo absorption peaks at 706.65 and 751.28 cm−1 are because of agroup at meta position.

3.3. Nitrogen analysis

To determine the APBA content of the prepared microspheres,nitrogen analysis was performed using by elemental analyzer (LEO,CHNS-932). The amount of APBA was calculated as 94.6 mg APBA/gmicrosphere.

3.4. Reversible catecholamine binding studies

The specific cis-diol interaction immobilization capacity andrelease percentage values of the APBA functionalized-poly(HPMA-Cl) microspheres were used to evaluate their sorbent efficiency.Each parameter was studied in triplicate, and the binding capac-ities and release% values of the microspheres were determinedunder optimum conditions. The binding capacities and releaseratios of the synthesized microspheres are given as the average ofthe results.

3.4.1. Effect of initial catecholamine concentration on the bindingcapacity

To determine the effect of the initial catecholamine concentra-tion of the incubation solution, the initial concentrations of theincubation solutions were selected in the range of 0.5–4 mg/mL,0.05–0.2 mg/mL and 0.05–0.2 mg/mL for dopamine, epinephrineand norepinephrine, respectively. The specific cis-diol interactionimmobilization experiments were conducted at the pH of 8.5 andthe amount of microspheres was kept constant as 0.05 g. As shownin Fig. 3a and b, the specific catecholamine binding capacity of theAPBA functionalized-poly(HPMA-Cl) microspheres increases withthe increase in the initial concentration of incubation solution forall the three of catecholamines. The increase in adsorption capac-ities with the increase in the initial catecholamine concentrationis due to the increase in the driving force of the concentrationgradient as expected. While no-significant non-specific bindingwas observed onto the poly(HPMA-Cl) (non-functionalized) micro-spheres, the maximum equilibrium binding capacities of the APBAfunctionalized-poly(HPMA-Cl) microspheres were found as 61 mg,7 mg, and 8 mg per gram of microsphere for dopamine, epinephrineand norepinephrine, respectively. As mentioned before, because ofsolubility problem, initial concentrations of epinephrine and nor-epinephrine were significantly lower than initial concentration ofdopamine. Therefore, the binding capacities of epinephrine andnorepinephrine were found lower than that of dopamine.

3.4.2. Reversible binding kinetics on microspheresThe binding kinetic curve of the microspheres was obtained for

dopamine, epinephrine and nor epinephrine. As can also be seenfrom Fig. 4, adsorption increased with time and the maximumadsorption was accomplished in about 180 min which is almost thesame for dopamine, epinephrine and norepinephrine. It can also be

44 N. Özdemir et al. / Colloids and Surfaces A: Physicochem. Eng. Aspects 445 (2014) 40– 47

0

10

20

30

40

50

60

70

543210

Initial dopamine concentration (mg/mL)

Q (

mg

dop

am

ine/

g

mic

rosp

her

e) APBA functionalized

poly(HPMA-Cl)

poly(HPAM-Cl)

a)

0

2

4

6

8

10

0,250,20,150,10,050

Initial cat echolam ine concentrat ion

(mg/mL)

Q (

mg C

ate

chola

min

e/g

mi c

rosp

her

e)

Epineph rine

poly(HPMA-Cl)

Norepinephrine

poly(HPMA-Cl)

Epinephrine APBA

function alized

poly(HPMA-Cl)

Norepineph rine APBA

function alized

poly(HPMA-Cl)

b)

F merici acity

c

st

3

titcacewes

Fm

ig. 3. The effect of initial catecholamine concentration on binding capacity of polyncubation time: 3 h). (a) The effect of initial dopamine concentration on binding caponcentration on binding capacity of polymeric microspheres.

een from Fig. 4 that the dynamic adsorption increased rapidly inhe first 3 h, and showed very slight changes after the first 3 h.

.4.3. Effect of pH on the binding capacityIn catecholamine binding experiments, pH is the most impor-

ant parameter to affect the reversible binding procedure. Becauset determines the efficiency of complex formation betweenhe APBA functionalized-poly(HPMA-Cl) microspheres and cate-holamines. In these experiments, the incubation medium pH wasdjusted to be in the range 4–9. The initial catecholamine con-entration was 4 mg/mL, 0.2 mg/mL and 0.2 mg/mL for dopamine,

pinephrine and norepinephrine, respectively, depending on theater solubility of the catecholamines, as mentioned before. The

ffect of pH on specific binding capacities of the catecholamines ishown in Fig. 5.

0

10

20

30

40

50

60

1501209060300

Time (min)

Q (

mg

Ca

tech

ola

min

e/g

mi c

ro

sph

ere)

ig. 4. The effect of time on the reversible binding capacity of polymeric microspheres (cicrospheres: 0.05 g; pH: 8.5; incubation volume: 5 mL).

microspheres (pH: 8.5, amount of microspheres: 0.05 g, incubation volume: 5 mL,of polymeric microspheres. (b) The effect of initial epinephrine and norepinephrine

As seen from Fig. 5a and b, for all three of catecholamines,no-significant non-specific binding was observed onto theploy(HPMA-Cl) (non-functionalized) microspheres at all pH val-ues. When used APBA functionalized poly(HPMA-Cl) microspheres,relatively lower catecholamine binding capacities were observedfor dopamine, epinephrine and norepinephrine at pH values lowerthan 8.5 which is an expected result. As stated before, mostboronate derivatives form a complex (cyclic ester) with cis-diolcontaining molecules, such as catecholamines, in the pH range of8–9. The complex formation depends on the acidity and basicity ofthe reaction medium, also, the interaction is reversible [28,37,38].In basic conditions, (especially at pH: 8.5) catecholamines were

bound onto the APBA functionalized-poly(HPMA-Cl) microspheresvia cyclic ester formation between the cis-diol moiety of thecatecholamines and the hydroxyl groups of boronic acid. In con-trast, this cyclic ester can be hydrolyzed and dissociated in acidic

240210180

Dopam ine

Epinephrine

Nor epinephr ine

atecholamine concentration: 4 mg DA/mL, 0.2 mg E/mL and 0.2 NE/mL, amount of

N. Özdemir et al. / Colloids and Surfaces A: Physicochem. Eng. Aspects 445 (2014) 40– 47 45

0

10

20

30

40

50

60

109876543

pH

Q (

mg C

ate

chola

min

e/g

mic

ros p

her

e) APBA functionalized-

poly(HPMA-Cl)

poly(HPMA-Cl)

a)

0

2

4

6

8

10

12

109876543

pH

Q (

mg

Cate

chola

min

e/g

mic

rosp

her

e)

Epinephrine

[poly(HPMA-Cl)]

Norepinephrine

[poly(HPMA-Cl)]

Epineph rine [APBA

function alized

poly(HPMA-Cl)]

Norepinephrine [APBA

function alized

poly(HPMA-Cl)]

b)

Fig. 5. The effect of pH on the reversible catecholamine binding capacity of polymeric microspheres (catecholamine concentration: 4 mg DA/mL, 0.2 mg E/mL and 0.2 NE/mL,amount of microsphere: 0.05 g, time: 3 h). (a) The effect of pH on dopamine binding capacity of polymeric microspheres. (b) The effect of pH on epinephrine and norepinephrineb

cenc

rtigila

3

omt

Fe

inding capacity of polymeric microspheres.

onditions. The pH-dependent chemistry of boronic acids is anxcellent property for molecular recognition. It makes this tech-ique ideal for the specific capture and separation of cis-diolontaining compounds.

Although the major interaction in boronate affinity chromatog-aphy is the esterification of the hydroxyl group of boronate withhe cis-diol moiety of biomolecules, secondary interactions are alsonvolved in this technique [29]. These interactions include hydro-en bonds forming between the hydroxyl groups, hydrophobicnteractions due to the phenyl ring present in the aromatic boronateigand, and ionic interactions between the negative charges of thective tetrahedral boronate, etc.

.4.4. Effect of microsphere amount on the binding capacity

In this part of the study, the effect of microsphere amount

n binding capacity was investigated. For this purpose, theicrosphere amount was varied from 0.01 to 0.1 g and the ini-

ial catecholamine concentration was chosen to be 4 mg/mL for

0

20

40

60

80

100

0,080,060,040,020

Amoun t of micr osphere

Q (

mg C

ate

chola

min

e/g

mic

rosp

her

e)

ig. 6. The effect of microsphere amount on the reversible binding capacity of the catepinephrine and norepinephrine, pH: 8.5, time: 3 h).

dopamine, and 0.2 mg/mL for epinephrine and norepinephrine. ThepH of the adsorption medium was adjusted to be 8.5. The effect ofadsorbent amount on adsorption is shown in Fig. 6. As seen here,the equilibrium adsorption exhibited a dramatic decrease with theincrease of the microsphere amount.

3.5. Release experiments

The binding process was conducted while following the pro-cedure described in Section 2.4. After binding equilibrium wasreached, the specific captured catecholamines were released foran hour in a shaker at room temperature. In the release studies,1 M NaCl (pH: 5.0, 10 mL) aqueous solutions were used as release

solution. In this part, the initial catecholamine concentration in theincubation medium was selected as 4 mg/mL for dopamine, and0.2 mg/mL for epinephrine and norepinephrine, respectively. Theresults are shown in Table 1.

0,120,1

(g)

Dopamine

Epinephrine

Norepinephrine

cholamines (catecholamine concentration: 4 mg/mL for dopamine, 0.2 mg/mL for

46 N. Özdemir et al. / Colloids and Surfaces A: Phy

Table 1Release yields obtained from the APBA functionalized-poly(HPMA-Cl) microspheresloaded catecholamines.

Catecholamines Release yields (wt.% of retainedcatecholamines by the microspheres)

Dopamine 96

bsy

4

pcstltoabeiimimt

C

A

vra

R

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

Epinephrine 91Norepinephrine 94

The results showed that all three of the catecholamines coulde released from the APBA functionalized-poly(HPMA-Cl) micro-pheres for up to 90% (w/w), which is a reasonably high releaseield.

. Conclusion

APBA functionalized-poly(HPMA-Cl) microspheres were pro-osed as a new alternative sorbent for the separation ofatecholamines. Reactive chloropropyl group containing micro-pheres were prepared with the suspension polymerizationechnique, and then 3-aminophenyl boronic acid (APBA) was cova-ently bound onto the synthesized microspheres. It was observedhat the catecholamine binding capacity increases with the increasef the initial catecholamine content in solution at the pH of 8.5,nd the maximum equilibrium binding capacities were found toe 61 mg, 7 mg, and 8 mg per gram of adsorbent for dopamine,pinephrine and norepinephrine, respectively. Release studiesndicate that the specifically captured catecholamines can eas-ly be released for up to 90% (w/w) from the functionalized

icrospheres in acidic solutions (pH: 5.0). All these reversible bind-ng results demonstrated that, the APBA-carrying poly(HPMA-Cl)

icrospheres can be a promising sorbent for catecholamine isola-ion.

onflict of interest

All the authors declare no conflict of interest.

cknowledgements

This study was supported by the Research Fund of Erciyes Uni-ersity, Project Number FBY-10-3411. We confirm that we haveead the Journal’s position on issues involved in ethical publicationnd affirm that this report is consistent with those guidelines.

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