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Journal of Chromatography A, 1218 (2011) 7796– 7803

Contents lists available at SciVerse ScienceDirect

Journal of Chromatography A

j our na l ho me p ag e: www.elsev ier .com/ locate /chroma

nergetics of protein adsorption on amine-functionalized mesostructuredellular foam silica

ungseung Kim, Rebecca J. Desch, Stephen W. Thiel ∗, Vadim V. Guliants, Neville G. Pintochool of Energy, Environmental, Biological and Medical Engineering, University of Cincinnati, Cincinnati, OH 45221-0012, USA

r t i c l e i n f o

rticle history:eceived 18 July 2011eceived in revised form 25 August 2011ccepted 26 August 2011vailable online 3 September 2011

eywords:esostructured cellular foam (MCF) silica

minopropyl group (APTES)dsorptionnergeticslow microcalorimetry (FMC)odium sulfateysozyme

a b s t r a c t

The energetics of lysozyme adsorption on aminopropyl-grafted MCF silica (MCF-NH2) are compared tothe trends observed during lysozyme adsorption on native MCF silica using flow microcalorimetry (FMC).Surface modification on MCF silica affects adsorption energetics significantly. All thermograms consist oftwo initial exothermic peaks and one later endothermic peak, but the heat signal trends of MCF-NH2 areopposite from those observed for adsorption onto native MCF silica in salt solutions of sodium acetateand sodium sulfate. At low ionic strength (0.01 M), LYS adsorption onto MCF-NH2 was accompanied bya large exotherm followed by a desorption endotherm. With increasing ionic strength (0.1 and 3.01 M),the magnitude of the thermal signal decreased and the total process became less exothermic. Also ahigher protein loading of 14 �mol g−1 was obtained at low ionic strength in batch adsorption isothermmeasurements. Taken together, the FMC thermograms and batch adsorption isotherms reveal that MCF-NH2 has the nature of an ion exchange adsorbent, even though lysozyme and the aminopropyl ligandshave like net charges at the adsorption pH. Reduced electrostatic interaction, reduced Debye length, andincreased adsorption-site competition attenuate exothermicity at higher ionic strengths. Thermogramsfrom flow microcalorimetry (FMC) give rich insight into the mechanisms of protein adsorption. A two-

step adsorption mechanism is proposed in which negatively charged surface amino acid side chains on thelysozyme surface make an initial attachment to surface aminopropyl ligands by electrostatic interaction(low ionic strength) or van der Waals interaction (high ionic strength). Secondary attachments take placebetween protruding amino acid side chains and silanol groups on the silica surface. The reduced secondaryadsorption heat is attributed to the inhibitory effect of the enhanced steric barrier of aminopropyl groupon MCF silica.

. Introduction

Mesoporous silica surfaces can be modified with functionalroups with a charged nature (such as amine, sulfonic, and car-oxyl groups) or a hydrophobic nature (such as octyl and octadecylroups) [1]. These groups can be used to control biomoleculeoading and release in drug delivery systems [2–4], protein adsorp-ion and desorption for bioseparations [5,6], and immobilizednzyme technology for biocatalysis [7]. Amine-terminated groupsre widely used to modify mesoporous silica surfaces becausehese groups are easily grafted to the silica surface at a high sur-ace density [8]. This surface modification changes electrochemicalroperties, such as surface charge density and surface functionality,

s compared to those of native mesoporous silica [9]. Amine-erminated silica can be used as a support for enzymes immobilizedor adsorption [9] and hydrolysis reactions [10] in aqueous solution

∗ Corresponding author. Tel.: +1 513 556 4130, fax: +1 513 556 3474.E-mail address: Stephen.Thiel@UC.edu (S.W. Thiel).

021-9673/$ – see front matter © 2011 Elsevier B.V. All rights reserved.oi:10.1016/j.chroma.2011.08.083

© 2011 Elsevier B.V. All rights reserved.

as well as for esterification [11] and transesterification reactions[12] in nonaqueous solution.

Amine-terminated functional groups have been used to con-trol adsorption onto silica in a number of systems. Adsorbedfluorescent dye was observed to be released more slowly fromamine-functionalized mesoporous silica than from native meso-porous silica nanoparticles at pH 5; this difference was explainedby changes in the electrostatic interactions between the moleculeand the surface due to the amine group [13]. Aminopropyl func-tionalization was used to control protein sequestration and releaseon SBA-15 silica and MCF silica, enabling incorporation of theion-exchange properties of the aminopropyl functionality on thenegatively charged molecular sieve silica substrate at pH 7 [14].A previous study showed that silanol-rich MCF silica does nothave ion-exchange properties [15]. The adsorption capacities ofbovine serum albumin and cellulase were reported to be higher for

amine-functionalized mesoporous FDU-12 silica than for the cor-responding native silica due to stronger electrostatic interactions[16]. It has also been observed that immobilized enzymes on amine-functionalized mesoporous silica can show improved biocatalytic

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J. Kim et al. / J. Chromato

ctivity compared to those immobilized on the native mesoporousilica, indicating possible protein orientation control [17].

The characteristics associated with a particular surface func-ionality can be influenced by solution properties such as saltoncentration, salt type, and pH. Neutral salts can modulate pro-ein adsorption and desorption in processes such as hydrophobicnteraction chromatography (HIC) and ion exchange chromatogra-hy (IEC) [18], by affecting the surface charges of both the protein19,20] and the solid surface [21]. In HIC, high salt concentrationncreases hydrophobic interactions and adsorption while low saltoncentration is used for protein elution. The opposite salt effects used for IEC [18]. Lysozyme adsorption on amine-functionalizedBA-15 depends on salt type and salt concentration, indicating spe-ific ion effects [9].

The current understanding of the fundamental interactionsetween amine-functionalized mesoporous silica and proteins dur-

ng immobilization in salt solutions is limited. This informationould be useful in designing highly efficient immobilized enzyme

ystems and controlling protein immobilization. Under non-idealolution and surface conditions, such as at high protein concen-rations [22], flow microcalorimetry (FMC) is an invaluable toolor measuring heat effects to reveal adsorption thermodynamicsnd improve the understanding of the complex phenomena of pro-ein adsorption. A previous FMC study [15] of lysozyme adsorptionn mesostructured cellular foam demonstrated that the thermody-amics of protein adsorption on native silica can be modulated byhanging the salt concentration in solution. As the salt concentra-ion increased, decreasing electrostatic interactions with reducedebye length and increasing van der Waals interactions betweenrotein and surface contributed to larger exothermic heats ofdsorption. Multiple heat events were observed in these experi-ents, suggesting that multiple points or modes of attachment are

ossible during protein adsorption on a mesoporous silica surface.In the study reported here, the thermodynamics of lysozyme

dsorption on amine-functionalized mesostructured cellular foamilica (MCF-NH2) are examined using flow microcalorimetry (FMC)nder different salt concentrations. Lysozyme adsorption ther-odynamics on the functionalized silica are compared those on

ative silica surfaces. This report demonstrates the effects of saltoncentration and surface modification for protein adsorption ther-odynamics on functionalized mesoporous silica and establishes

he ion exchange nature of MCF-NH2.

. Experimental

.1. Materials

Tetraethyl orthosilicate (TEOS, 98%), (3-aminopropyl) tri-thoxysilane (APTES, 99%) and lysozyme from chicken egg whiteLYS, 90%) were obtained from Sigma–Aldrich (St. Louis, MO). Aceticcid and hydrochloric acid were obtained from Fisher Scientificnd Pharmco-Aaper (Brookfield, CT), respectively. Sodium acetatenhydrous and sodium azide (Biotech research grade) was obtainedrom Fisher Scientific (Fair Lawn, NJ). MCF silica was synthesizedsing the procedure reported previously [15].

.2. Surface modification of MCF silica

The synthesized MCF silica material was acid-washed using.2 M HCl solution for 2 h at 80 ◦C to remove carbon residuesnd maximize the surface silanol concentration. (3-Aminopropyl)

riethoxysilane (APTES) was grafted onto the acid-washed silicaurface [8] by mixing 1 g of acid-washed silica with 5 mL of APTESn 100 mL of ethanol at 70–80 ◦C for 4–5 h; this material is referredo below as MCF-NH2. The MCF-NH2 particles were recovered by

218 (2011) 7796– 7803 7797

vacuum filtration, washed with fresh ethanol, and dried at roomtemperature. An initial APTES concentration of approximately0.27 M was selected to produce maximum coverage on the MCFsilica surface [8].

2.3. Characterization of MCF silicas

Nitrogen adsorption–desorption measurements (Tristar 3000,Micromeritics, Norcross, GA) were performed at 77 K to determinesilica pore size, pore volume and BET surface area. All sampleswere outgassed for 2 h at 150 ◦C before the measurements. The poredimensions of cell and window structure were determined from thenitrogen adsorption and desorption branches, respectively, usingthe BdB (Broekhoff-de Boer)-FHH (Frenkel-Halsey-Hill) method[23]. Surface modification was characterized by thermogravimet-ric analysis in air (TGA, SDT Q 600, TA Instruments, New Castle,Delaware) to confirm the introduction of surface functional groupsand determine their surface coverage. The surface coverage, NS, wasestimated using Eq. (1) [24] and the weight loss measured by TGA.

Ns = Wloss

100 g adsorbent· molligand

Mligand· molmolecule

molligand· 1000 mmol

mol(1)

The functional group density, D, was calculated using Eq. (2)[25].

D = NA · NS

SBET(2)

In Eqs. (1) and (2), Wloss is the weight loss from TGAbetween 250 ◦C and 400 ◦C; Mligand is the molecular weight ofligand (58 g aminopropyl ligand−1); NA is the Avogadro constant(6.02 × 1023 mol−1); and SBET is the BET surface area of MCF silica.

2.4. Flow microcalorimetry (FMC)

Thermograms for lysozyme adsorption were collected at roomtemperature using flow microcalorimetry (Microscal FMC 3 Vi,Gilson Instruments, Westerville, OH, USA) following previouslyreported methods [15,27–29]. The sample loop volume was1.36 mL, the mobile phase flow rate was 1.9 mL h−1, the bed volumewas 0.17 mL, and the adsorbent sample mass was 37.8 ± 3.5 mg. Themobile phase consisted of aqueous sodium acetate buffer at pH 5.2with varying concentrations: 0.01 M sodium acetate, 0.1 M sodiumacetate, and 0.01 M sodium acetate with 1 M sodium sulfate. Samplesolutions were prepared by dissolving lysozyme in samples of eachmobile phase. The integral heat of adsorption was determined fromthe area under deconvoluted asymmetric Gaussian peaks using thePeakFIT software package (Systat Software Inc., San Jose, CA, USA);calibration factors were calculated for each mobile and solid phasecombination as described previously [15]. Peaks were reproduciblewith a peak area standard error of 17%.

2.5. Adsorption isotherm

A study of lysozyme adsorption was completed to investigatethe effect of mobile phase composition on adsorption behaviorusing MCF-NH2 silica for the three mobile phases used in theFMC experiments. The detailed procedure for the measurementof batch adsorption isotherms is reported in a recent publication[15]. The batch adsorption was done for 1 h, which was the sametime scale as the FMC experiment. A Type I isotherm equation was

Mathematica software package (Wolfram Research, Champaign,IL, USA). The reported precision of the regression parame-ters represent a 95% confidence interval around the parameterestimates.

7798 J. Kim et al. / J. Chromatogr. A 1218 (2011) 7796– 7803

Table 1Physical properties of MCF silica.

Sample Cell (nm) Window (nm) Pore volume(cm3 g−1)

BET surfacearea (m2 g−1)

Surfacecoverage (Ns)(mmol g−1)

Ligand density (D)(ligand nm−2)

3

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for batch adsorption of lysozyme onto MCF silica [15]: proteinloading increased with ionic strength (increasing sodium sul-fate concentration) due to attenuated electrostatic interaction and

MCF [15] 33.0 16.6 2.4

MCF-NH2 22.1 8.2 1.0

. Results and discussion

.1. Textural properties, ligand density and surface functionalityf aminopropyl-functionalized MCF silica

MCF silica combines low mass transport resistance with highdsorption capacity and controlled pore size. The open pore struc-ure is composed of relatively narrow windows and larger cellores. BdB-FHH analysis of the nitrogen adsorption–desorptionata showed that the synthesized MCF silica had a cell diame-er of 33 nm, a window diameter of 16.6 nm, a total pore volumef 2.4 cm3 g−1, and a BET surface area of 634 m2 g−1. The amino-ropyl groups incorporated during surface modification altered theilica physical properties, shown in Table 1, resulting in reducedffective pore diameters [30]. However, the pore window diameter8.2 nm) and cell pore diameter (22.1 nm) in the MCF-NH2 func-ionalized materials are large enough to accommodate lysozyme1.9 nm × 2.5 nm × 4.3 nm) [5] into the pore structure.

The incorporation of aminopropyl groups was confirmed bySC, as shown in Fig. 1. The weight loss across broad tempera-

ure ranges (such as 200–700 ◦C [31] or 300–800 ◦C [32]) has beensed to estimate the aminopropyl group coverage on mesoporousilica to be in the range of 1–2.4 ligands nm−2 [31,32]. Using thispproach, the estimated aminopropyl surface density for the MCF-H2 produced in this study is about 1.1 ligand nm−2 on MCF-NH2,onsistent with the earlier reports. However, dehydroxylation canause weight loss at temperatures in the range of 450–900 ◦C [33].revious thermal analysis of aminopropyl grafted-mesoporous sil-ca found that the main aminopropyl decomposition event occurs inhe range of 250–400 ◦C [31]. Consequently, in this study the weightoss from 250 ◦C to 400 ◦C (2.9%) was used to estimate the amino-ropyl density and coverage to be 0.5 mmol ligand g−1 adsorbent

nd 0.54 ligand nm−2.

The overall properties of the aminopropyl-functionalized sil-ca will potentially be influenced by free silanol groups thatan be available even after the surface has reached maximum

ig. 1. TGA and DSC results for aminopropyl functionalized mesocellular foam silicaMCF-NH2). Heating rate: 10 ◦C min−1; air flow rate: 50 mL min−1.

634.3 – –401.3 0.5 0.54

functionalization [30]. Since amorphous silicas have a silanol sur-face density of 4.6–5.7 OH nm−2 regardless of origin and structuraldifferences [34], and since each grafted APTES ligand binds to upto three silanol groups [1], about 4 silanol groups per APTES ligandare expected to remain on the functionalized surface.

3.2. Effect of ionic strength and surface modification

Aminopropyl-functionalized SBA-15 (SBA-15-NH2) has beenreported to have a higher surface charge than native SBA-15 acrossa wide pH range (pH 3–10) [9,30]. The surface charge of nativemesoporous silica changes with salt concentration and salt typedue to specific cation effects rather than specific anion effects [21].Protein loading on SBA-15-NH2 is influenced by the presence of dis-solved salts; for example, at pH 9, the adsorption of lysozyme onSBA-15-NH2 was increased by increasing the ionic strength usingvarious salts [9]. These literature results indicate that both the saltconcentration and the surface modification have significant roles inmodulating the nature of the guest–host interactions that underliethe protein adsorption process on mesoporous silica.

The batch isotherms obtained in this study for lysozyme adsorp-tion on MCF-NH2 silica at pH 5.2 in different ionic strengthsolutions, modulated by sodium sulfate, are presented in Fig. 2.The curves were obtained from non-linear regression using aType I isotherm (r2 ≥ 0.95); regression parameters are presentedin Table 2. The highest protein loading was obtained at the lowestionic strength (0.01 M); the protein loading at an ionic strengthof 0.1 M was higher than that observed at an ionic strength of3.01 M. In a recent paper the authors reported the opposite trend

enhanced van der Waals interaction. Independently measured

Fig. 2. Isotherms for adsorption of lysozyme onto aminopropyl functionalizedmesocellular foam silica (MCF-NH2) at pH 5.2, 25 ◦C, 150 rpm, and 1 h adsorptionperiod. Initial protein concentration: 139–1048 �M; (1) ionic strength: 0.01 M; (2)ionic strength: 0.1 M; (3) ionic strength: 3.01 M.

J. Kim et al. / J. Chromatogr. A 1

Table 2Type I isotherm parameters for the adsorption of lysozyme on MCF-NH2 silica at pH5.2 and 25 ◦C as a function of ionic strength. Adsorption period: 1 h; initial proteinconcentration: 139–1048 �M. Values are presented as mean ± standard error.

Ionic strength (M) nm (�mol g−1) K (mM−1) r2

0.01 SA 61. ± 18. 0.0012 ± 0.0006 0.9890.1 SA 19.8 ± 1.4 0.0033 ± 0.0001 0.996

S

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3.01 (0.01 M SA + 1 M SS) 19.7 ± 6.0 0.0019 ± 0.0011 0.959

A = sodium acetate; SS = sodium sulfate.

ysozyme loadings for the mobile and stationary phases usedn this study at the FMC sample concentration of 10 mg mL−1

YS are shown in Table 3. These loading ranged from 16 ± 1 to2 ± 2 mg mL−1. For comparison, the monolayer LYS capacity on

sulfopropyl agarose adsorbent at 0.02 M NaCl was reported toe 74 ± 2 mg mL−1 [35]; the less repulsive adsorption environmentreated by the negatively charged sulfopropyl group could con-ribute to the higher loading on the agarose adsorbent.

Flow microcalorimetry was used to measure the effect of ionictrength on the enthalpy of protein adsorption. The thermogramsor lysozyme adsorption on MCF-NH2 silica at the same buffernd sodium sulfate concentrations used for the isotherm measure-ents are shown in Figs. 3–5. In each thermogram, an exotherm

egan when the protein sample reached the packed bed 10–15 minfter the protein injection and continued until about 70 min whenresh mobile phase began to replace the protein sample in the bed.rom 70 min to 110 min, an endotherm developed as partial proteinesorption occurred.

Each thermogram was deconvoluted into two exothermic peaks,ttributed to adsorption, and an endothermic peak, attributed toesorption, as shown in Figs. 3–5. Heats of adsorption and des-rption onto MCF and MCF-NH2 silica are compared in Table 3,o highlight the effects of surface functionalization on adsorp-ion energy. Previous calorimetric protein adsorption studiesevealed that an exothermic adsorption event occurs when attrac-ive forces between protein and surface dominate; repulsiveateral protein–protein interactions, protein restructuring, and sol-ent release from the surface can cause endothermic adsorption22,36]. Although complex thermograms are sometimes attributedo protein conformation changes [27,37], lysozyme is not proneo conformational change [36]; no restructuring endotherm isbserved. A previous study of lysozyme adsorption on mesoporousilica attributed multiple exothermic peaks to a primary adsorp-ion event followed by reorientation or secondary attachment [15].he second exothermic peak was greater than the initial peak forll cases of lysozyme adsorption onto MCF-NH2 silica, indicatinghat stronger interactions occurred as a result of reorientation or

econdary adsorption. The aminopropyl groups extend from theilica surface, offering a site for the initial adsorption. The sharpnitial exotherm indicates that the lysozyme has easy access tohe initial adsorption site, presumably the amine termination of

able 3eat of adsorption and desorption of lysozyme on MCF-NH2 and MCF [15] silicas at pH 5.2.9 mL h−1; MCF-NH2 sample size: 37.8 ± 3.5 mg, MCF sample size: 25.2 ± 2.9 mg; temper

Ionic strength (M) Protein loading Exothermic peaks (kJ mo

(�mol g−1) (mg mL−1) �HI �HII �HI +

MCF-NH2

0.01 M SA 14.2 ± 1.0 22.3 ± 1.6 −22.6 −37.3 −600.1 M SA 10.9 ± 0.8 17.1 ± 1.3 −4.6 −15.6 −20.0.01 M SA + 1 M SS (3.01 M) 10.2 ± 0.7 16.0 ± 1.1 −6.1 −8.6 −14.

MCF [15]0.01 M SA 10.2 ± 0.7 16.0 ± 1.1 −10.1 −11.7 −21.0.01 M SA + 1 M SS (3.01 M) 27.6 ± 0.8 43.4 ± 1.3 −25.3 −24.0 −49.

HTotal = �HI +�HII + �HIII .A = sodium acetate; SS = sodium sulfate.

218 (2011) 7796– 7803 7799

the surface ligands. Protruding positively charged residues on thelysozyme surface might then be able to penetrate through the lig-and steric barrier to form strong secondary attachments to silanolgroups on the MCF silica surface. The broad peak of the secondexotherm indicates restricted access to the sterically hindered sec-ondary adsorption site.

The largest heat of adsorption on MCF-NH2 silica was observedat 0.01 M ionic strength, with similarly patterned but attenuatedthermograms observed in 0.1 M and 3.01 M ionic strength buffers.The magnitude of the heat of adsorption was approximately fourtimes greater in 0.01 M ionic strength (0.01 M acetate buffer) thanin 3.01 M ionic strength (1.0 M sodium sulfate in 0.01 M sodiumacetate buffer) and the adsorption process became less exothermicwith increasing ionic strength. Together, these trends indicated thatthe attractive adsorption interactions weaken with increasing ionicstrength. The FMC results are consistent with the trend of decreasedprotein loading at high ionic strength.

The energetic trends of MCF-NH2 with increasing ionic strengthare contrasted to native MCF silica in Fig. 6. Heat signals were nor-malized to the amount of protein adsorbed at equilibrium to allowdirect energetic comparison between MCF-NH2 and MCF. Althoughthe MCF-NH2 window pores are smaller than those of MCF (8.2 vs.16.6 nm), both window sizes allowed lysozyme penetration intothe pore structure. At 0.01 M ionic strength, MCF showed a sharpinitial exotherm followed by a small, flat secondary exotherm anda small endotherm. The MCF-NH2 exotherm was broader with anenhanced second exotherm and larger endotherm. At 3.01 M ionicstrength, MCF had a broad and strong merged primary and sec-ondary exotherm with a negligible endotherm, while MCF-NH2had an attenuated initial exotherm followed by a small, broad sec-ondary exotherm and a weak endotherm.

The difference between lysozyme adsorption onto MCF silicaand MCF-NH2 silica is attributed to significant changes in the silicasurface charge and chemistry, demonstrating that surface modi-fication affects protein adsorption thermodynamics. It has beenreported that terminated surface functional groups have signifi-cant effects on protein adsorption, and that an amine-terminatedsurface shows less protein adsorption affinity than surfaces withhydrophobic termination [38]. Aminopropyl grafting changes theelectrochemical properties of the mesoporous silica surface result-ing in positive zeta-potentials and positive surface charge densitiesover a broad range of solution pH (pH 3–7) [9,30]. It was suggestedthat aminopropyl-grafted mesoporous silica has the dual charac-teristics of size exclusion and ion exchange adsorbents [14]. In ionexchange applications, low salt concentration is used for selectiveprotein retention on adsorbents and high salt concentration is usedmainly for protein elution [18]. The lysozyme adsorption capacity

and enthalpy of adsorption demonstrate that MCF-NH2 behavesas an ion-exchanger, showing stronger attraction, less desorption,and higher lysozyme loading in a lower ionic strength buffer, andattenuated interactions and loading at higher ionic strength.

; feed protein concentration: 10 mg mL−1; sample loop volume: 1.36 mL; flow rate:ature: 25 ◦C. Values are presented as mean ± standard error.

l−1) Endothermic peak (kJ mol−1) Net heat of adsorption (kJ mol−1)

�HII �HIII �HTotal

. ± 10. 37.3 −22.6 ± 3.82 ± 3.4 4.1 −16.0 ± 2.77 ± 2.5 8.2 −6.5 ± 1.1

8 ± 3.7 4.5 −17.3 ± 2.93 ± 8.4 0 −49.3 ± 8.4

7800 J. Kim et al. / J. Chromatogr. A 1218 (2011) 7796– 7803

Fig. 3. Deconvoluted thermograms for lysozyme adsorption and desorption on MCF-NH2 at pH 5.2 in 0.01 M sodium acetate. Sample loop volume: 1.36 mL; flow rate:1.9 mL h−1; adsorbent sample size: 37.8 ± 3.5 mg; temperature: 25 ◦C; feed protein concentration: 10 mg mL−1. Curves shown are for (1) experimental data (black line); (2)total peak fit (purple line); (3) 1st exothermic peak fit (red line); (4) 2nd exothermic peak fit (blue line); (5) endothermic peak fit (olive line). (For interpretation of thereferences to color in this figure legend, the reader is referred to the web version of the article.)

Fig. 4. Deconvoluted thermograms for lysozyme adsorption and desorption on MCF-NH2 at pH 5.2 in 0.1 M sodium acetate. Sample loop volume: 1.36 mL; flow rate:1.9 mL h−1; adsorbent sample size: 37.8 ± 3.5 mg; temperature: 25 ◦C; feed protein concentration: 10 mg mL−1. Curves shown are for (1) experimental data (black line); (2)total peak fit (purple line); (3) 1st exothermic peak fit (red line); (4) 2nd exothermic peak fit (blue line); (5) endothermic peak fit (olive line). (For interpretation of thereferences to color in this figure legend, the reader is referred to the web version of the article.)

Fig. 5. Deconvoluted thermograms for lysozyme adsorption and desorption on MCF-NH2 at pH 5.2 with 1.0 M sodium sulfate in 0.1 M sodium acetate. Sample loop volume:1.36 mL; flow rate: 1.9 mL h−1; adsorbent sample size: 37.8 ± 3.5 mg; temperature: 25 ◦C; feed protein concentration: 10 mg mL−1. curves shown are for (1) experimentaldata (black line); (2) total peak fit (purple line); (3) 1st exothermic peak fit (red line); (4) 2nd exothermic peak fit (blue line); (5) endothermic peak fit (olive line). (Forinterpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)

J. Kim et al. / J. Chromatogr. A 1218 (2011) 7796– 7803 7801

Fig. 6. Comparison of thermograms for lysozyme adsorption and desorption on MCF silica and MCF-NH2 silica at pH 5.2. Sample loop volume: 1.36 mL; flow rate: 1.9 mL h−1;adsorbent sample size (MCF-NH2 silica): 37.8 ± 3.5 mg; adsorbent sample size (MCF silica): 25.2 ± 2.9 mg; temperature: 25 ◦C; feed protein concentration: 10 mg mL−1. (a)0 howno sion o

atcapLtqteaahed

Wpraitrai

K

wstTtalstolea

scope for attachment. In addition, as the Debye length decreases,secondary attachment, which requires positive amino acid sidechains to move toward the silica silanol groups by electrostaticinteraction, becomes less likely.

Table 4Relative dielectric constant, εr and Debye length, k0

−1, as functions of ionic strength.

.01 M sodium acetate; (b) 1.0 M sodium sulfate in 0.01 M sodium acetate. Curves sf the references to color in this figure legend, the reader is referred to the web ver

Because lysozyme and MCF-NH2 both carry a net positive charget pH 5.2 [9,39], one might expect electrostatic repulsion interac-ions to be dominant. The initial exotherms observed at all saltoncentrations showed that there are significant attractive inter-ctions between lysozyme and MCF-NH2 despite the shared netositive charge on both the surface and the protein. Shibata andenhoff [40] observed the adsorption of positively charged pro-eins (lysozyme and chymotrypsinogen A) onto positively chargeduartz-NH2 using total internal reflectance fluorescence spec-roscopy. The adsorption of proteins was attributed to combinedffects of electrostatic interactions, van der Waals interactionsnd hydrophobic interactions. Measurements of attenuated inter-ctions between protein and the surface were attributed toydrophobic interactions, which are typically associated withndotherms, acting against the exothermic electrostatic and vaner Waals forces during protein adsorption [40].

Double layer electrostatic interactions and Lifshitz-van deraals interactions are the main contributions to the total

rotein–surface interaction [41–43]. The authors previouslyeported [15] that electrostatic and van der Waals interactionsre both important for lysozyme adsorption on MCF silica at lowonic strength. At higher salt concentrations, electrostatic interac-ions decrease and van der Waals interactions increase significantlyesulting in enhanced exotherms. In this study, electrostatic inter-ctions were attenuated at higher ionic strengths. The Debye lengthn each salt solution is calculated with the following equation:

−10 =

√kBTε0εr

2NAe2I(3)

here kB is the Boltzmann constant; ε0 is the permittivity of freepace; T is the temperature, 298 K; NA is the Avogadro constant; e ishe charge of an electron; and I is the ionic strength of the solution.he relative dielectric constants, ε0, [44] in different salt concen-rations were estimated from the reported values. Each parameternd calculated Debye lengths are summarized in Table 4. The Debyeengths at 0.1 M and 3.01 M decreased from the value at low ionictrength by about 68% and 99%, respectively, indicating that elec-rostatic interactions cannot play a major role in protein adsorption

n MCF-NH2 silica at higher ionic strengths. The effective charge ofysozyme decreases in higher sodium sulfate solution due to pref-rential binding of sulfate anions onto positively charged aminocid groups [39]. The reduced surface charge of lysozyme can

are for (1) MCF silica (black line); (2) MCF-NH2 silica (red line). (For interpretationf the article.)

also reduce electrostatic interactions between lysozyme and MCFsilica surface. Although electrostatic repulsion between the like-charged protein and MCF surfaces might be expected to be thedominant interaction, the initial exotherm derived from attrac-tive interactions, especially in normal buffer (no salt), suggeststhat aminopropyl groups change the orientation of the adsorbingprotein to interact with negatively charged groups on the proteinsurface.

The lysozyme surface is anisotropic at pH 5.2, with 18 surfaceresidues (Arg, His, and Lys) that can become positively chargedand 4 surface residues (Asp 18, Asp 87, Asp 119 and Glu 7) thatcan become negatively charged [45]. Lysozyme has two bindingsites, region A (1 Lys, 5 Arg, 33 Lys, 114 Arg, 128 Arg) and regionB (1 Lys, 14 Arg, 15 His, 68 Arg) [46]. These binding sites preferen-tially adsorb onto negative surfaces at pH 5.2, but other bindingsites involving Asp (side chain pKa = 3.65) and Glu (side chainpKa = 4.25) [20,47] could coordinate with positively charged surfaceligands and create the observed exotherms. The negatively chargedresidues are located in close proximity in the lysozyme molecule[46], suggesting that the orientation of the protein while approach-ing and adsorbing onto the MCF-NH2 silica is crucial. At higher ionicstrengths, the Debye length (k0

−1) is shorter [19,45], significantlyreducing electrostatic interactions (either repulsive or attractive)[45]. At high Debye lengths (≥3.0 nm), the protein can be pulledinto a favorable orientation while approaching the pore surface,allowing coordination of negatively charged amino acids on theprotein and positively charged ligand groups while reducing repul-sion between positively charged surface amino acids and positivelycharged ligand groups. As the Debye length decreases, proteinmolecules increasingly approach the pore surface in random ori-entations, weakening electrostatic interactions and reducing the

Ionic strength (M) εr k0−1 (Å)

0.01 78 300.1 77 9.53.01 58 1.5

7 gr. A

dtH

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802 J. Kim et al. / J. Chromato

Short-range van der Waals interactions become the dominantriving force at higher ionic strengths [42]. The energy change dueo van der Waals interactions (�Uvdw) can be estimated using theamaker equation [48]:

Uvdw(h) = −H

6

[1h

+ 1h + 2

+ ln(

h

h + 2

)](4)

Here h is the ratio of distance between the protein and surfacend the radius of the protein (2.1 nm for lysozyme). The Hamakeronstant (H) is estimated to be 1.27 × 10−20 J at 298 K [15]. In thisodel, the maximum attraction will occur at the closest possible

istance between the protein and surface, typically taken to be.1 nm [48]. The corresponding van der Waals interaction energy

s thus estimated to be −18 kJ mol−1, 3–5 times the heat of therst exotherm (�HI) in the highest ionic strength system studiedere (when electrostatic interactions are minimal). Interestingly,he dispersion energy matches the total enthalpy of adsorption�HI + �HII) well. The steric barrier of silica surface ligands pre-ents or retards the closest approach of the protein to the silicaurface. Additionally, salt ions can form hydrogen bonds with lig-nd groups on the silica surface, suppressing protein binding inigh ionic strength solutions [49] by competing for the silica surfacemine groups.

.3. Adsorption mechanism

The proposed mechanism [15] for lysozyme adsorption ontoative MCF silica is a random adsorption driven by the combined

nteractions between electrostatic and van der Waals interactionst low salt concentration and primarily by van der Waals interac-ions at high salt concentration. This primary adsorption is followedy a reorientation of the protein relative to the silica surface accom-anied by additional electrostatic interactions or van der Waals

nteractions between surface amino acid groups on the lysozymeurface and silanol groups on the silica surface. The adsorption isollowed by desorption except at high salt concentration. Lysozymedsorption onto native MCF silica increases with increasing ionictrength because van der Waals interactions become much strongers electrostatic interactions descrease with decreased Debye lengtht high ionic strength. Likewise, the adsorption of lysozyme ontoCF-NH2 silica begins with an electrostatic interaction between

he positively charged amine group grafted to the silica surface and negative group on the lysozyme surface. Once the lysozyme haseen brought into proximity with the surface, it can form secondaryttachments with silanol groups or interact with the surface viaan der Waals interactions. With increasing salt concentration, thelectrostatic interactions decrease, the Debye length decreases, andan der Waals interactions tends to increase but might be limitedue to the steric barrier posed by the aminopropyl groups on theCF silica. In addition, competition between salt ions and surface

igand groups may cause the lysozyme to adsorb onto the surfacen more random orientations, resulting in decreased adsorptionnthalpy. Secondary adsorption is driven by van der Waals interac-ions between the protein and the surface; however, as before theurface functional ligands may acts as a steric barrier to inhibit thelosest approach of protein to surface for enhanced van der Waalsnteractions.

This study has shown that a simple silica surface functional-zation reversed the lysozyme adsorption energetic and capacityrends in the presence of varying sodium sulfate concentra-ions. Partially replacing silanol groups with aminopropyl groups

hanged silica surface properties in terms of charge and chemistryo impact protein adsorption thermodynamics. Unlike native sil-ca, MCF-NH2 functions as an ion exchanger. The interplay of ionictrength and surface modification was demonstrated with batch

[

[[

1218 (2011) 7796– 7803

adsorption isotherms and calorimetric data for protein adsorptionon functionalized mesoporous silica.

4. Conclusions

The adsorption energetics of lysozyme on amine-functionalizedmesoporous silica are reported here. FMC thermograms were usedto identify mechanisms of protein adsorption as a function ofionic strength and surface conditions. Surface modification bygrafting aminopropyl groups on MCF (MCF-NH2) silica affectedadsorption energetics significantly as compared to that of acid-washed MCF silica. Although MCF-NH2 and lysozyme had likecharges at pH 5.2, attractive interactions were the main drivingforce for protein adsorption, demonstrating that the interactionsbetween specific amino acid groups and the surface ligand werean important factor for protein adsorption energetics in thisstudy.

The total enthalpy of adsorption decreases with increasing ionicstrength for lysozyme adsorption on MCF-NH2 silica. Lower ionicstrength results in stronger exothermic peaks and correspondinglystronger endothermic desorption events. At high ionic strength, theshort Debye length means that the net protein charge is less impor-tant than interactions with specific sites on the external proteinsurface. The trend observed in the adsorption isotherm is consistentwith calorimetric results; higher protein loading was observed atlower ionic strength. Thermograms and batch adsorption isothermsconfirmed that MCF-NH2 silica has ion exchange properties asproposed in a previous study [14]. Attenuated exothermic peaksobserved with increasing salt concentration were attributed todecreasing electrostatic interaction and inhibiting van der Waalsinteractions due to steric barrier of surface functional groups(aminopropyl groups) on MCF silica.

Acknowledgments

The authors are grateful for financial support from the NanoscaleInterdisciplinary Research Teams (NIRT) program sponsored byNational Science Foundation (CTS-0403897) (JSK), the Universityof Cincinnati University Research Council (JSK) and NSF IGERT pro-gram (RJD).

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