Analysis of ionic strength effects on the adsorption of simple amino acids

Journal of Colloid and Interface Science 443 (2015) 153–161

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Journal of Colloid and Interface Science www.elsevier.com/locate/jcis

Analysis of ionic strength effects on the adsorption of simple amino acids Damien Sebben, Phillip Pendleton ⇑ School of Pharmacy and Medical Sciences, University of South Australia, Adelaide, SA 5000, Australia

a r t i c l e

i n f o

Article history: Received 29 October 2014 Accepted 4 December 2014 Available online 13 December 2014 Keywords: Amino acids Glycine Lysine Glutamic acid Adsorption Silica Ionic strength Deconvolution ATR-IR spectroscopy

a b s t r a c t Hypothesis: Amino acid adsorption by metals and/or oxide surfaces is important in many biomedical and industrial processes, however limited information exists discussing ionic strength influences on the mechanism of adsorption. A comparison of pure water solution and added 1:1 electrolyte should highlight the effects of electrolyte on amount adsorbed. ATR spectroscopy of the adsorbed phase should demonstrate the effects on the mechanism of adsorption. Experiments: Low solution concentration adsorption isotherms for glycine, lysine and glutamic acid on Aerosil 200 silica were generated in pure water and 10 and 100 mmol/L sodium chloride solutions. A systematic study of the adsorption isotherms as well as adsorbent and adsorptive solution properties was performed. ATR-IR spectroscopy was used to analyse the adsorbed phase in solution. Findings: Glycine adsorbs primarily through electrostatic interactions; lysine also adsorbs through electrostatic interactions in a parallel conformation with the surface. Glutamic acid adsorbs via hydrogen bonding and forms intermolecular clusters around an adsorbed nucleus. ATR-IR spectrum deconvolution
shows a peak shift for glycine and lysine associated with the dad NHþ vibration, indicating interaction 3 through the amino moieties. Amount adsorbed was decreased significantly by the addition of 10 mmol/L sodium chloride and completely inhibited by the addition of 100 mmol/L sodium chloride. Ó 2014 Elsevier Inc. All rights reserved.

1. Introduction The adsorption of amino acids by metals and oxide surfaces has recently become the subject of widespread investigations due to their significance in a variety of potential applications, including solid phase protein synthesis [1] and biomedical implants and sensors [2,3]. There is also common belief that the interaction of amino acids with mineral surfaces played an important role in prebiotic evolution, due to catalytic properties of selected oxides towards peptide formation [4,5]. In recent years, literature has emerged detailing the production of porous polymer networks as controlled release drug delivery agents [6–10]. The fundamental basis of their manufacture is the adsorption of proteins or small peptide chains to the surface of inorganic oxide ‘templates’, particularly silica. The premise of the work reported here is an improved and deeper understanding of the fundamental interactions across the adsorbed phase–adsorbent interface, obtained from welldesigned analyses of the (initially) simple amino acids adsorbed from dilute aqueous solution by a well-characterised silica adsorbent. Glycine, lysine and glutamic acid (Fig. 1) have recently been classified as type 1, type 2, and type 3 amino acids, respectively, ⇑ Corresponding author. E-mail address: [email protected] (P. Pendleton). http://dx.doi.org/10.1016/j.jcis.2014.12.016 0021-9797/Ó 2014 Elsevier Inc. All rights reserved.

owing to their predominantly neutral, basic, and acidic natures on dissolution in pure aqueous solutions [11]. Amino acids were chosen from each group in order to gain an understanding of the role of the amino and carboxylic acid moieties on adsorption. The amino groups of lysine contain a similar chain length separation as the carboxylic acid groups of glutamic acid. Several recent studies pertaining to amino acid adsorption have focussed on silica adsorbents [1,2,12–15]. Silica is a standard, welldefined material that has undergone extensive characterisation, resulting in large amounts of relevant literature. Relatively high surface area solids in the form of (ordered) porous silica have also been studied, however, pores introduce an additional variable to adsorption over and above external surface contributions. These are yet to be correctly separated for a fundamental analysis of the solution–adsorbent interface. De Stefano et al. described the effect of dissolved ions on the dissociation and speciation of amino acids in natural waters [11]. Their work highlights the need for ionic strength to be considered when dealing with amino acid adsorption from aqueous solution. Vlasova and Golovkova demonstrated solution pH effects on amino acid adsorption by silica in both pure water and 10 and 100 mmol/L salt solutions [14], however they discounted salt solution effects on the silica surface chemistry and did not offer an explanation as to the mechanism by which ionic strength affects adsorption. Gao et al. analysed the adsorption of amino acids by

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ASIS Scientific, Australia) was used for all borate buffer preparations. Standard solutions of NaOH and HCl (as both 100 and 10 mmol/L solutions, ex. ASIS Scientific, Australia) were used as received. Fresh 10 mmol/L trinitrobenzenesulfonic acid (TNBSA) solution was prepared prior to use by appropriate dilution of a 10% solution (ex. Sigma Aldrich, Australia). Ultra-high purity gaseous nitrogen and helium (99.999% ex. BOC gases, Australia) were used in the nitrogen adsorption analyses.

Glycine

Lysine

Glutamic acid 2.2. Silica surface characterisation

Fig. 1. Molecular structures of glycine, lysine and glutamic acid.

mesoporous silica as a function of pH and ionic strength [3]. Their ionic strength data focussed solely on arginine, and found that increases in sodium chloride concentration caused a decrease in the amount adsorbed. The vast majority of previous amino acid solution adsorption isotherms have been generated with relatively high equilibrium solution concentrations, ranging as high as 400 mmol/L [1]. Such concentrations lead to amounts adsorbed where all possible specific surface sites are completely covered, impairing detailed interpretation of the intermolecular interactions at the adsorbed phase–solid interface. Very low equilibrium solution concentrations create additional difficulties, primarily in accurate solution concentration determination, and thus problems in material balance calculations (as amount adsorbed). ATR-IR spectroscopy has been used to study the behaviour of amino acids in solution [16–18] and as an adsorbed phase [15,19–23]. This technique enables speciation of the amino acid in solution by revealing the functional groups present, and is favourable for studying interactions between adsorbent and adsorbate. Deconvolution of infrared spectra for the well-defined vibration energies of selected functional groups is a powerful tool to identify the appearance and/or disappearance of peaks obscured within broad neighbouring peaks [18,22]. These analyses enable visualisation of peaks that otherwise may have been ignored, as well as close monitoring of slight changes within functional group peaks. Our work presents in-depth investigation of the possible adsorbed phase–adsorbate interactions and concludes possible overall adsorption mechanisms between three amino acids (glycine, lysine and glutamic acid) and Aerosil 200 silica at the silica–solution interface. Detailed physical and chemical characterisation of the silica surface properties serves to enhance the interpretation of the adsorption interactions and overall mechanism. The effect of solvated ions on adsorption is analysed by the comparison of adsorption isotherms generated in pure water as well as in the presence of 10 and 100 mmol/L sodium chloride. The effect on amino acid and silica surface speciation due to the presence of solvated ions is calculated via Pitzer’s method and used in conjunction with the isotherm data and silica surface conditions to gain an understanding of the adsorption process. ATR-IR spectroscopy of the adsorbed phase and subsequent spectrum deconvolution analysis provides further insight into the mechanism of adsorption. 2. Materials and methods 2.1. Materials Aerosil 200 amorphous silica (ex. Degussa, Germany) was used as received for all adsorption experiments. Glycine, lysine and glutamic acid of purity >99% (ex. Sigma Aldrich, Australia) were used as received and to prepare stock solutions in Milli-Q water (18 MX cm), 10 and 100 mmol/L sodium chloride solutions. Sodium chloride (99.99% SuprapurÒ ex. Merck, Australia) was used without further purification. Sodium tetraborate decahydrate (ex.

2.2.1. Nitrogen adsorption An automated manometric gas adsorption apparatus was used to determine the specific surface area of the Aerosil 200 silica [24]. Sample degassing was performed at 473 K and a background pressure of 1  104 Pa for 8 h. The liquid nitrogen level for the measurements was held constant ± 0.2 mm using a modified level-control system [25]. The dead-volume of the adsorption system was defined via helium expansion at 77 K. Upon completion of the adsorption measurements, the samples were weighed to determine any mass change resulting from the adsorption or degassing. Equilibration time varied from 20 to 25 min for each experimental data point along the adsorption and desorption branches of the isotherm. 2.2.2. Water adsorption Water adsorption experiments were conducted at 298 ± 0.02 K using a Belsorp-MaxÒ adsorption instrument equipped with a vapour adsorption kit. The adsorption temperature was controlled using a NeslabÒ refrigerated bath circulator. HPLC-grade water, used as the adsorptive, was degassed using a 5-times freeze–thaw method to ensure it was free of any dissolved gases. Before each experiment the Aerosil 200 silica was heated at 473 K for 8 h and degassed under vacuum (1  104 Pa). 2.2.3. Zeta potential The Aerosil 200 zeta potential was analysed by preparing 0.1% w/v silica suspensions over the range 1.5 < pH < 3.5. The silica was suspended in Milli-Q water and the pH was adjusted using either 10 or 100 mmol/L HCl or NaOH solutions and measured using a drop-type pH meter (ex. Hanna Instruments, Australia). The pH detector was calibrated using standard solutions of pH 4.0, 7.0 and 10.0. Measurements were made using a Malvern Zetasizer Nano-ZS zeta potential analyser. 2.3. Amino acid adsorption methodology 2.3.1. Amino acid quantification Amino acid quantification was accomplished using a modified version of a UV–Vis method for primary amine analysis [26]. Method calibration required the addition of pre-determined amounts of amino acid stock solutions to plastic cuvettes which were then made to 1900 lL with pH 8.5 borate buffer for glycine and lysine, or pH 10.0 borate buffer for glutamic acid. An aliquot of TNBSA solution (100 lL) was added to each cuvette then agitated to achieve thorough mixing, and allowed to react for 3 min. Each solution concentration was defined by the absorbance at 420 nm using a Varian CaryÒ 50 UV–Vis spectrophotometer immediately upon completion of the reaction time. Adsorption samples were analysed in a similar manner, whereby 1000 lL of supernatant was added to a cuvette, followed by 900 lL of buffer and 100 lL of TNBSA. 2.3.2. Solution adsorption analyses Amino acid solutions were prepared as stock concentrations (2000 mg/L) in either Milli-Q water or in aqueous sodium chloride

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solutions (10 or 100 mmol/L). Subsequent dilutions of these solutions to prepare initial amino acid concentrations for the adsorption measurements were made using either Milli-Q water or the appropriate sodium chloride solution. Dilution with the latter ensured no changes in the sodium chloride concentration prior to adsorption. The amino acid concentration was then adjusted (equivalent to 0–250 mg/L) using the appropriate solvent system. The mixture was briefly sonicated to suspend the silica (5.0 ± 0.2 mg), before being placed on a rotating wheel for 24 h at 40 rpm. Following the completion of the adsorption process, samples were centrifuged at 4000 rpm for 5 min before 1500 lL of supernatant was transferred to an Eppendorf tube and centrifuged for a further 5 min at 13,000 rpm at constant 298 K. Experiments were also performed with no silica present to determine any adsorption by the containers and transfer tubes; solution concentration remained unchanged within initial solution concentration uncertainty limits. All adsorption experiments were performed using polypropylene centrifuge tubes which were washed and dried to remove any soluble release agents and/or adsorbed molecules. 2.3.3. ATR-FIR analyses Adsorption samples were prepared by weighing approximately 500 mg of Aerosil 200 silica powder and predetermined amounts of amino acid into 50 mL polypropylene centrifuge tubes. The silica–amino acid mixture was suspended in 7 mL of Milli-Q water for pure solvent analyses, and 10 mmol/L sodium chloride solution for ionic strength analyses. The concentrations of glycine and lysine were 400 and 200 mmol/L, respectively. The solubility limit for glutamic acid in water and the sensitivity limitation of the detector used in this work prevented us from obtaining meaningful infrared spectra of the adsorbed phase. Detector limitations created the need for higher solution concentrations for IR analyses compared to adsorption analyses. The centrifuge tubes were then sonicated and placed on a rotating wheel at 40 rpm for 24 h. Upon completion of the adsorption process, a 2 mL aliquot of sample solution was centrifuged at 4000 rpm for 5 min, then the supernatant was transferred to an Eppendorf tube and centrifuged for a further 10 min at 13,000 rpm and 298 K before being passed through a 0.45 lm filter. A 1 mL aliquot of the filtrate was dispensed onto the ATR crystal for spectral analysis. Following this, the ATR crystal was washed thoroughly before 1 mL of adsorption solution was transferred to the ATR crystal and allowed to settle for 20 min before the spectrum was recorded. FTIR spectra were collected on a Shimadzu 8400S spectrometer fitted with an MCT detector, using 100 scans and a resolution of 4 cm1. A Pike HATR apparatus mounted with a trough-type ZnSe crystal was used for all ATR experiments. Subsequently, the spectra of water, silica, and filtrate were subtracted from the sample spectrum to reveal the spectrum of the adsorbed amino acid.

3.1.2. Water adsorption Water vapour adsorption is an excellent probe of hydrophilic surface sites on inorganic oxides [28]. The monolayer equivalent water coverage was analysed as a linear BET plot over the pressure range 0.05 6 P/P0 6 0.35. A value of Vm as 10.05 cm3(STP)/g, was calculated, equivalent to 28.47 m2/g or 14.83% of the available (N2 equivalent) surface area covered by specifically adsorbed water. The accepted cross-sectional area of adsorbed water is 0.105 nm2/molecule [30]; a close-packed monolayer of water would be equivalent to 9.52 molecules/nm2. The experimental B-point value gives the equivalent of 1.40 water molecules adsorbed per nm2 with the same fractional coverage results. 3.1.3. Zeta potential analysis The Aerosil 200 zeta potential, f, was analysed through the range 1.5 < pH < 3.5. The experimental data were fitted with a cubic polynomial, indicating the point of zero charge (PZC) as 2.66. The standard deviation in the data decreases as the solution pH approached the pHPZC, promoting confidence in the value identified. Adsorption experiments were conducted at pH values P3.9, suggesting that for all experiments, the surface contained an overall negative charge. Surface charge on silica in aqueous suspensions is due to pH-dependent protonation or de-protonation of surface hydroxyl groups [31]. The surface of silica consists primarily of siloxane (SiAOASi) and silanol (SiAOH) groups, with the ratio of silanol to siloxane depending on the degree of hydration, the physical age (since initial synthesis), and thermal treatment of the silica [32]. Two types of silanol group are prevalent on silica surfaces, referred to as Q3 and Q2 silanols [33,34]: Q3 silanol groups („SiAOH) exist as single or isolated surface sites, having a lone hydroxyl group protruding into the surrounding medium; Q2 silanol groups (@SiA(OH)2) are regarded as geminal surface groups with two hydroxyl groups protruding into the surrounding medium. According to Zhuravlev, vicinal or ‘bridged’ silanol groups also exist due to hydrogen bonding between adjacent single or geminal silanol groups [35]. Examples of single (a), geminal (b) and vicinal (c) silanol groups on a silica surface are shown in Scheme 1. Silanol groups are readily deprotonated in aqueous solution, resulting in the characteristic negatively charged silica surface at solution pH > pHPZC. A study by Ong et al. showed that the two different silanol groups have pKa values at 4.5 and 8.5, assigned to the Q3 and Q2 groups, respectively [36]. It should be noted that the pHPZC differs from the pKa values of the silanol groups; the pHPZC is the point at which the charges become unbalanced, i.e. deprotonation exceeds protonation, whereas the pKa is the point at which 50% of a particular species has become deprotonated. Ong et al. also suggest these Q2 and Q3 sites co-exist in the ratio Q3:Q2  1:4; this ratio should be considered when interpreting site speciation calculations. The water adsorption isotherm analysis suggested 15% of the available surface was covered with hydrophilic sites, interpreted as

3. Results and discussion 3.1. Aerosil 200 surface characterisation 3.1.1. Nitrogen adsorption Nitrogen adsorption at 77 K and subsequent BET analysis yielded a specific surface area of 192.5 ± 1.4 m2/g, which agrees with the manufacturer’s value of 200.0 ± 25.0 m2/g. The absence of hysteresis suggests a Type II isotherm, indicative of adsorption of a gas by a non-porous solid [27,28] and consistent with the expected physical properties of Aerosil 200. Amino acid adsorption would then be equivalent to open surface interactions only, with no porosity contributions that could lead to enhanced condensation [29].

(a) H O Si

(b)

(c)

H

H

O

O Si

H

H

O

O

Si

Si

Bulk Solution

Surface

Scheme 1. Pictorial representation of hydroxyl groups present on a hydrophilic silica surface [33–35]: (a) Q3 or isolated groups; (b) Q2 or geminal groups; and (c) vicinal or hydrogen-bonded groups.

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sites such as these Q2, Q3 and vicinal hydroxyl groups. It is proposed that these sites would have specifically or tightly bound water molecules adsorbed when the particles are suspended for aqueous solution adsorption studies. The remaining 85% of the surface must consist of siloxane type structure, interpreted as relatively hydrophobic from a water vapour perspective. For the analysis of our solution adsorption isotherms, without further independent evidence, we have accepted the hydroxyl site ratio of Q3:Q2 = 1:4, analogous to Fisk et al. [34]. The unadjusted pH-dependent speciation of the silica surface is summarised in Table 1. The amino acid molecules to be investigated consist of pH-dependent ionised groups; appropriately charged molecules would be expected to preferentially displace the tightly-bound water from the ionised surface sites. 3.2. Amino acid adsorption Initial amino acid concentrations ranged up to 3.35 mmol/L for glycine, 1.71 mmol/L for lysine, and 1.70 mmol/L for glutamic acid. The pH of each solution was not adjusted, thereby minimising the influence of dissolved ions on the dissociation behaviour of the dissolved amino acids and silica surface groups. The percentage species dissociated for each solution investigated is summarised in Table 1, with the influence of dissolved electrolyte on the species dissociated values calculated via Pitzer’s method [37]. At the dissolved pH, zwitterions of glycine and glutamic acid predominate, but single-protonated lysine exists as a 3:2 ratio with its zwitterion counterpart. The amount of amino acid adsorbed from aqueous solution or salt solution was determined using material balance calculations. Uncertainty in the amount adsorbed was determined by propagating the uncertainty in individual measurements, volumes and masses of materials for each data point.

Table 1 Calculated percentage species in solution or on silica surface at initial adsorption conditions. Effects of sodium chloride evaluated using Pitzer’s method [37]. Speciesa

Pure water

10 mmol/L NaCl

100 mmol/L NaCl

Glycine (pH = 5.3) GlyH+2 GlyH± Gly

0.12 99.88 0.00

0.12 99.88 0.00

0.12 99.88 0.00

Silica (pH = 5.3) SiOH, Si(OH)2 SiO, Si(OH)2 SiO, Si(O)2

13.67 86.27 0.06

14.90 85.05 0.05

16.57 83.39 0.04

Lysine (pH = 8.9) LysH2+ 3 LysH+2 LysH± Lys

0.00 60.47 39.04 0.49

0.00 60.34 39.23 0.41

0.00 58.84 40.80 0.36

Silica (pH = 8.9) SiOH, Si(OH)2 SiO, Si(OH)2 SiO, Si(O)2

0.00 28.47 71.53

0.00 30.56 69.44

0.00 33.30 66.70

Glutamic acid (pH = 3.9) GluH+3 1.45 GluH±2 74.46 GluH 24.09 Glu2 0.00

1.51 77.78 20.71 0.00

1.57 81.24 17.19 0.00

Silica (pH = 3.9) SiOH, Si(OH)2 SiO, Si(OH)2 SiO, Si(O)2

81.49 18.51 0.00

83.31 16.69 0.00

79.92 20.08 0.00

a Each species is represented as AHnx where A = Gly, Lys, Glu; x = # ionisable protons; n = amount of charge on species; ± = zwitterion. The silica species are not adjusted to account for the Q2:Q3 ratio. Adjustments are made in the text when considered.

3.2.1. Adsorption from pure water solutions Two isotherm models stand out in their application to solution adsorption analyses: the Langmuir and the Freundlich isotherm models [27]. Pedagogically, the Langmuir isotherm model should be fitted initially to solution adsorption data when a plateau is observed in the amount adsorbed with increasing equilibrium solution concentration. The plateau would be equivalent to a monolayer amount adsorbed. The Freundlich isotherm model is also often fitted to solution adsorption data because no apparent plateau is observed in the relatively higher equilibrium solution concentrations. Its application is often favoured over the Langmuir model because of the apparent continuing increase in amount adsorbed with increasing equilibrium solution concentration, and (sometimes) a slightly higher-valued regression coefficient for the data fit. If the isotherm data were to show a (relatively) sharp increase in the initial amount adsorbed at relatively low solution concentrations followed by a steady increase in amount adsorbed, alternative isotherm models would be expected to be more applicable. For our adsorption data, the Sips or Langmuir–Freundlich (L–F) model, Eq. (1), was applicable to each set of data and gave a superior fit compared with the aforementioned models. An additional advantage of the L–F model (over the Freundlich) is the direct calculation of the apparent equilibrium constant, KLF, which also relates to the heat of adsorption. The curve fitting for each set of data was a minimisation of the average of the sum of residuals squared, and a maximisation of the Pearson correlation coefficient.

qads ¼

qm K LF C eq


1=n

1 þ K LF C eq


1=n

ð1Þ

At low Ceq, when KLFCeq  1, the equation reduces to the Freundlich isotherm. When the surface becomes homogeneous, n ? 1, the equation reduces to the Langmuir isotherm model. The exponent 1/n has the same implication towards surface heterogeneity as in the Freundlich isotherm; relatively high values imply localised adsorption by a strongly heterogeneous surface, as a narrow distribution of adsorption energy. Each of the isotherm data points was the result of several repeated measurements; we suggest the isotherm shapes are reproducible and correct. Fig. 5 shows the adsorption isotherms of (a) glycine, (b) lysine, and (c) glutamic acid by Aerosil 200 from pure water solution. The L–F equation of best fit applied to the adsorption data and their uncertainties is plotted alongside the data. The overall isotherm fit for glutamic acid depicts a convex shape, whereas glycine and lysine have concave shape, relative to the equilibrium concentration axis, suggesting glutamic acid has initially weaker interaction with the silica surface than those of glycine and lysine.Amounts adsorbed are expressed as mmol/g, allowing for direct comparisons to be made. For adsorption mechanism discussion purposes, a value of 1.0 mmol/L was chosen as it is the highest common value of Ceq, the data in each isotherm are well represented, and the value is considerably lower than most other published results for similar systems. When Ceq = 1.0 mmol/L, the amount adsorbed, q, for glycine is 0.21 mmol/g, for lysine q = 0.10 mmol/g, and for glutamic acid q = 0.30 mmol/g. These q values equate to 0.65, 0.31 and 0.93 molecules/nm2, respectively. Previous studies have proposed that glycine may interact with silica surfaces through a series of different interactions, such as hydrogen bonding [38–40] and electrostatic interactions [1]. The protonation state of the surface silanol groups is important in determining which mechanism is favourable. The surface comprises of 19% Q3 silanols, and 81% Q2 silanols [34,36]. This would suggest that at pH 5.3 the silica surface has approximately 16.4% of the total silanol groups in a deprotonated state, equivalent to 0.23 deprotonated silanol groups (SiO)/nm2. Since the amount of

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0.50

(a)

Amount ads., q, mmole/g

0.45 0.40 0.35 0.30 0.25 0.20 0.15 0.10 0.05 0.00 0.0

0.5

1.0

1.5

2.0

2.5

3.0

3.5

Equil. Conc, C eq, mmol/L 0.14

(b) Amount ads., q, mmole/g

0.12 0.10 0.08 0.06 0.04 0.02 0.00 0.0

0.5

1.0

1.5

2.0

Equil. Conc, C eq, mmol/L 0.35 0.30

Amount ads., q, mmole/g

glycine adsorbed at Ceq = 1.0 mmol/L is 0.65 molecules/nm2, not all glycine can interact via electrostatic interactions with the available deprotonated silanol groups. At higher Ceq and q this is even more evident. In contrast, at lower Ceq, the principal interaction is most likely the stronger electrostatic interaction, possibly up to Ceq  0.25 mmol/L (0.23 molecules/nm2 of adsorbed glycine). Over the experimental concentration range the shape of the isotherm can be described as an L3a isotherm, following the classifications of Giles et al. [41]. The general L3 isotherm classification shows a ‘step’ at higher Ceq, whereas the L3a continues with an upward trend. A further increase in Ceq for our experimental conditions may show a shape similar to the typical L3 isotherm, but for the purposes of our discussion, the L3a classification best describes the isotherm in Fig. 2a. The L3a isotherm is typical of adsorbate–adsorbent interactions, with only minor contributions from adsorbate–adsorbate interactions, similar to a classical Langmuir interpretation for solution adsorption. This interpretation suggests there is little interaction between glycine molecules in the adsorbed phase, and thus minimal clustering of glycine molecules around a particular adsorbed molecule. Meng et al. suggested glycine crystallites form at amounts adsorbed >0.4 molecules/nm2 at natural pH [1], with their corresponding Ceq  100 mmol/L; this concentration is a 100-fold increase of our value of Ceq which gave 0.65 molecules/nm2 adsorbed. Clearly our results contrast sharply with theirs, suggesting a more amenable surface presented by Aerosil 200 than theirs, Aerosil 380. No clear indication was given regarding surface hydroxyl concentration of their silica samples. The structure of zwitterionic glycine renders it favourable for other types of intermolecular bonding, such as hydrogen bonding with protonated silanol groups through the carbonyl oxygen, and ion–dipole interactions between the charged amino group and the oxygen constituent of the protonated silanol. Alongside electrostatic interactions, it seems likely that all three of these interactions are possible, in order to account for the amount of glycine adsorbed. This interpretation compares favourably with the DFT modelling for (gas phase) glycine adsorption [39]. The solution conditions in the lysine adsorption system give 76.94% surface silanol de-protonation, equivalent to a total of 1.08 SiO/nm2, in contrast to the 0.23 SiO/nm2 at the pH conditions for glycine adsorption. At Ceq = 1.0 mmol/L, only 0.31 molecules of lysine adsorb per nm2, significantly less than the total number of available de-protonated silanol sites. In these solution conditions lysine exists as 69.5% LysH+2 and 39% LysH±, and a negligible quantity of Lys. Thus, the possible interactions would be similar to that of glycine, with hydrogen bonding and ion–dipole bonds having less probability than electrostatic interactions. The shape of the adsorption isotherm in Fig. 2b is also L3a. As well as limited adsorbate–adsorbate interactions, the L-shaped isotherm model proposes that when an adsorbed molecule is linear or has a planar surface, the major axis is parallel to the surface [27]. Mudunkotuwa and Grassian performed similar adsorption analyses for histidine adsorption by TiO2 nanoparticles and proposed that the adsorption process is reversible, and occurs through a variety of different configurations and modes of interaction [22]. Their results gave a similarly shaped L3a isotherm, and they interpret the adsorption of histidine as parallel to the surface, with interaction occurring via a combination of electrostatic, hydrogen bonding, and ion–dipole interactions. A parallel interaction involving multiple binding sites may explain the decreased amount adsorbed compared with glycine. Kitadai et al. suggested that lysine is adsorbed predominately in the cationic state through electrostatic interactions [15], however their interpretation did not propose a structural conformation of adsorbed lysine and appears to have overlooked silica surface contributions. It is likely that electrostatic interactions are the dominant interactions, although there are other interactions involved when lysine adsorbs onto silica. A

(c)

0.25 0.20 0.15 0.10 0.05 0.00 0.0

0.2

0.4

0.6

0.8

1.0

1.2

Equil. Conc, C eq, mmol/L Fig. 2. Adsorption of (a) glycine, (b) lysine, and (c) glutamic acid by Aerosil 200 silica at 298 K from pure aqueous solution, with the L–F equation of overall best fit plotted as a solid line on each isotherm.

parallel adsorption mechanism involving the LysH+2 species and two SiO sites, or the interaction between the LysH± species and a single SiO site are the most probable mechanisms for lysine adsorption when taking into account the isotherm shape, as well as the work performed with other amino acids with similar functional groups. The solution conditions for glutamic acid adsorption indicate only 3.8% of the surface silanol groups are in a deprotonated state, equivalent to 0.05 SiO/nm2 and 1.35 SiOH/nm2. When Ceq = 1.0 mmol/L, there are 0.93 molecules/nm2 of adsorbed glutamic acid. At pH 3.9 glutamic acid is predominantly found in the zwitterionic

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state, with 25% in the GluH and 1.5% in the GluH+3 state (Table 1). There is limited ability for glutamic acid to adsorb via electrostatic interaction, so other forces such as hydrogen bonding and ion–dipole interactions must become influential. Hydrogen bonding would occur between the protonated carboxylic acid, and the protonated silanol group. Ion–dipole bonds would most likely occur between protonated amino groups of glutamic acid and the surface hydroxyl oxygen. Hydrogen bonding is the most likely interaction due to the higher number of carboxylic acid groups present. The isotherm in Fig. 2c is classified as S1. As with lysine and glycine, the shape of the isotherm may change with increasing Ceq, however for the experimental concentration range the suitable classification is S1. This classification suggests that there is strong adsorbate–adsorbate interaction within the adsorbed phase, particularly if the adsorbed molecules have a major axis perpendicular to the adsorbent surface [27]. Our finding is consistent with that of Bouchoucha et al. who described an S-shaped isotherm for glutamic acid adsorption by silica, and proposed that due to the adsorbent and adsorbate conditions, hydrogen bonding was likely the predominant mode of interaction [12]. In contrast to their findings, which reached a maximum adsorption of 0.3 mmol/g at Ceq = 40 mmol/L, similar adsorption capacity was achieved at Ceq = 1.0 mmol/L in our system for a silica with 30% less available surface area. Bouchoucha et al. also postulated that at higher Ceq, adsorbed glutamic acid could act as a nucleus for crystallite formation, consistent with the accepted interpretation of the isotherm shape in both their system and ours. Crystallite or multiple layer formation may explain the higher amounts of glutamic acid adsorbed compared with glycine and lysine at equivalent solution concentrations. 3.2.2. Adsorption from sodium chloride solution The effect of solvated sodium and chloride ions on the adsorption of (a) glycine, (b) lysine, and (c) glutamic acid at the silica–solution interface is shown in Fig. 3. No data are given for 100 mmol/L solutions as there was no adsorption noted within experimental uncertainty and detector limits for the method employed for amino acid quantification. Similar results were reported by Vlasova and Golovkova for lysine adsorption by silica [14]. The L–F model again gave the best fit to the experimental data for each adsorption system. Table 2 summarises the L–F constants for the model of best fit in both pure water and 10 mmol/L sodium chloride solution. As expected, from the amounts adsorbed for each adsorptive, the binding constant KLF decreases for each amino acid in the presence of sodium chloride, as does the value for 1/n for glycine and lysine. The qm values may not properly reflect the actual monolayer equivalent amount adsorbed because the equilibrium solution concentrations in these experiments are considerably lower than required to support the monolayer amount adsorbed. The effect of 10 and 100 mmol/L sodium chloride on the speciation of glycine, lysine and glutamic acid (Table 1) was considered as a possible reason for the decrease in adsorption. Glycine is unaffected by the addition of electrolyte at pH 5.3, and the speciation remained the same as in pure water. Similarly, the silica surface is mostly unaffected with only 0.5% less charged sites available. Lysine shows a small (<2% in 100 mmol/L sodium chloride) decrease in the amount of available LysH+2 species, and a similarly small increase in the amount of available Lys±, while the silica surface has 0.4% less charged surface sites. In contrast, glutamic acid experiences 7% increase in the amount of GluH±2 present, and a similar decrease in the amount of GluH present, while the silica surface experiences 3% reduction in the amount of available charged sites. Clearly, the effect of sodium chloride on the speciation of glycine and silica at pH 5.3 does not account for the

Fig. 3. Adsorption of (a) glycine, (b) lysine, and (c) glutamic acid by Aerosil 200 silica at 298 K from 10 mmol/L sodium chloride solution, with the L–F equation of best fit plotted as a dashed line on each isotherm. The data for adsorption from pure water solutions (solid line) is plotted alongside for comparison.

Table 2 Langmuir–Freundlich coefficients for glycine, lysine, and glutamic acid adsorption from pure water and from 10 mmol/L sodium chloride solution by Aerosil 200 silica at 25 °C. Pure water

Glycine Lysine Glutamic acid

10 mmol/L NaCl

KLF

qm

1/n

KLF

qm

1/n

0.145 0.130 0.177

1.356 0.497 2.651

0.868 0.651 1.202

0.008 0.001 0.117

1.422 0.434 3.207

0.547 0.390 1.402

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1

Fraction dissociated

0.9 0.8 0.7 0.6 0.5 0.4 0.3 0.2 0.1 0 2.0

3.0

4.0

5.0

6.0

7.0

8.0

9.0

10.0

pH Fig. 4. Speciation plot for glycine dissociation over the pH range 2.0–10.0 (—) showing the effect of 10 mmol/L sodium chloride (- - -).

(a) 0.20 pH 8.0

Natural pH

Absorbance

decrease in adsorption. Lysine and silica at pH 8.9 experience slight changes in the available species upon the addition of sodium chloride, but this does not account for the large decrease in adsorption. The speciation is affected minimally and the species present still have the ability to interact with the surface. Glutamic acid experiences a greater effect on speciation due to the addition of sodium and chloride ions than glycine and lysine, but this still does not account for the complete inhibition of adsorption in 100 mmol/L sodium chloride solution. The isotherms for amino acid adsorption from 10 mmol/L solution (Fig. 3) depict similar shapes to those of adsorption from pure water solutions suggesting a similar interpretation of the interaction modes to those in pure water solution would be correct. In 10 mmol/L sodium chloride solution, the amount of available sodium ions is between 3–6 times that of the amino acids initially available for adsorption. The presence of solvated sodium and chloride ions in the adsorption system dramatically decreases the amount adsorbed for each amino acid, as represented in Fig. 3 and summarised in Table 3. Sodium ions form a positively charged electric double layer around the negatively charged silica surface, altering the apparent surface charge [14,36]. Chloride ions have also been shown to affect the acid–base nature of the surface silanol groups [42]. NMR studies have also shown reversible, nonspecific attraction forces exist between sodium ions and deprotonated surface silanol groups [43]. Irreversible binding of sodium ions would almost completely inhibit amino acid adsorption even at low sodium chloride concentrations. This ability of sodium ions to reversibly interact with the de-protonated silanol groups results in competition with amino acids for the limited binding sites, decreasing the amount of amino acid adsorbed. Sodium ions may form ion–dipole interactions with protonated surface hydroxyl groups, similar to the proposed interactions between protonated amino groups and the surface hydroxyl groups, leading to further competition at binding sites. Ion–dipole interactions can also form between sodium ions and carbonyl oxygen groups of amino acids. The interaction energy of ion–dipole bonds is greater than that of hydrogen bonds, therefore preventing the formation of hydrogen bonds between amino acids and the surface silanol sites.

0.05

pH 4.0

(b)

Filtrate

Adsorbed

3.2.3. Solution infrared analysis Infrared techniques have been employed to develop an understanding of the mechanism of amino acid adsorption by metal oxides in aqueous solution [15,19,20,22]. Infrared spectroscopy enables visualisation of the ionised amino acid species present in solution by revealing functional groups within the molecules [18]. Fig. 4 shows a speciation diagram for glycine in pure water and the effect of 10 mmol/L sodium chloride present in solution, with the latter calculated via Pitzer’s approach [37]. Such diagrams provide an insight into the theoretical abundance of each species in solution at any specified pH. Fig. 5a shows the ATR-IR spectra of 400 mmol/L glycine solutions; unaltered (natural) pH at 5.3, and two pH values either side at pH 4.0 and 8.0. The values of 4.0 and 8.0 were chosen as they are on the cusp of a species change (Fig. 4) and the speciation is not significantly affected by additional dissolved ions. There are four

Table 3 Amounts adsorbed in molecules/nm2 for each amino acid in both pure water and 10 mmol/L sodium chloride solutions at Ceq = 1.0 mmol/L. Amount adsorbed (molecules/nm2)

Glycine Lysine Glutamic acid

Pure water

10 mmol/L NaCl

0.65 0.31 0.93

0.30 0.06 0.47

1775

1675

1575

1475

1375

1275

Wavenumber (cm-1) Fig. 5. Infrared spectra of glycine; (a) solvated glycine at adsorption (natural) pH = 5.3 and at pH = 4.0 and 8.0, (b) spectrum for glycine solvated within the filtrate, equivalent to equilibrium solution and glycine as an adsorbed species. Adsorbed spectrum has water, silica and filtrate contributions subtracted.

peaks present for each spectrum, at 1600, 1512, 1412 and
 1330 cm1, associated with the tas ðCOO Þ; dsd NHþ 3 ; tss ðCOO Þ and dðCH2 Þ, respectively [17,18]. The aforementioned peaks in the infrared spectrum confirm the presence of the zwitterionic species. Fig. 5b shows the IR spectra of the adsorption filtrate and the amino acid adsorbed by the silica surface. The filtrate spectrum consists of the solvated glycine with the water spectrum contributions subtracted, and thus, contains the same peaks as those previously attributed to the zwitterionic glycine molecule, and at the same wavenumber value as the solvated phase. The spectrum for adsorbed amino acid showed a single asymmetric peak centred on 1635 cm1, with the asymmetry towards a lower energy. Studies have shown peak deconvolution to be a useful tool for revealing obscured peaks within spectra of solvated or adsorbed amino acids [18,22]. Fig. 6 shows the result of deconvolution of the adsorbed phase spectrum from Fig. 5b.

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0.05

Absorbance

0.04 0.03 0.02 0.01 0 1750

1700

1650

1600

1550

1500

1450

Wavenumber (cm-1) Fig. 6. The result of peak deconvolution analysis for the adsorbed glycine spectrum.

Deconvolution analysis was performed using a Lorentzian distribution function resulting in a primary absorbance at 1635 cm1 and a secondary peak at 1600 cm1. The peak at 1600 cm1 corresponds to the tas ðCOO Þ. Kitadai et al. suggested the peak at 1635 cm1 corresponds to the d(OH) of water [15]. Interestingly, their paper also described water (solvent) spectrum subtraction. In our solvated amino acid ATR-IR analyses, solvent contributions were subtracted and only the peaks due to solvated amino acids were observed [18]. For solution adsorption ATR-IR analyses we again subtracted solvent and solvated amino acid contributions and no d(OH) was observed. Sato also observed a peak
shift from 1610 to 1627 cm1, associating it with the dad NHþ of 3 glycine when adsorbed by kaolinite [23]. Furthermore, Mundunkotuwa and Grassian described a peak at 1637 cm1 and associ
ated it in part with the dad NHþ of histidine, and also ring 3 structure contributions. We suggest the peak at 1635 cm1 in
Fig. 6 is associated with the dad NHþ 3 of glycine adsorbed by silica, and that the mode of interaction is more favourable for asymmetric deformation. Since the tas ðCOO Þ peak is stationary, there must only be limited interaction of this moiety with the silica surface (hydrogen bonding) at the experimental pH. A similar spectrum resulted for the adsorbed phase in 10 mmol/L sodium chloride solution. The schematic concluded from these interpretations is NH+3 groups present towards the deprotonated silanol groups with the COO group presenting away from the surface towards the solvent. Such an arrangement is also consistent for lysine, which interacts with the surface via the a and e-amino groups, with the COO directed into the solvent phase. The presence of sodium chloride does not affect the mechanism of adsorption, only the amount adsorbed.

(3) Electrostatic interactions between the protonated amino group and negatively charged surface sites account for the adsorption of glycine up to 0.23 molecules/nm2. At higher amounts adsorbed the adsorption occurs through a combination of electrostatic, hydrogen bonding, and ion–dipole interactions. (4) A parallel adsorption mechanism involving both amino groups is the most probable configuration for lysine adsorbed by the silica surface, dominated by electrostatic interactions at the experimental pH conditions. This showed similarities to a recently reported adsorption mechanism for histidine on TiO2 [22]. (5) Glutamic acid adsorption occurs through hydrogen bonding with protonated surface silanol group, with ion–dipole interactions also being probable. The high amount adsorbed, as well as the S1 isotherm shape, suggests clustering of glutamic acid within the adsorbed phase. (6) The presence of 10 mmol/L sodium chloride dramatically reduces the amount adsorbed for the amino acids studied, while the addition of 100 mmol/L completely suppresses adsorption, consistent with observations made by Vlasova and Golovkova [14]. Ionic strength effects on the dissociation of the amino acids and the acid–base properties of the silica surface have only minimal effect on the amount adsorbed. Shielding of charged surface sites and amino acid functional groups by oppositely charged ions is the primary cause of the decreased amount adsorbed. (7) Deconvolution of the adsorbed phase spectra clearly indicated the presence of a strong peak associated with the
dad NHþ at an increased energy for vibration, and the 3  tas ðCOO Þ was unaffected. There was no difference between the adsorbed phase peaks in pure water and 10 mmol/L sodium chloride solution, indicating the mechanism of adsorption is unaffected by the presence of sodium and chloride ions. Innovations derived from the current work include linking the number of available surface sites with the number of adsorbed amino acid species per unit surface area, as a function of system pH, to formulate an interpretation of the mechanism of adsorption; and, confirming the mechanism via ATR-IR spectroscopy of the adsorbed phase with background spectra removed. To put the above work in the context of a deeper understanding of amino acid interactions with mineral and oxide surfaces in general, several additional directions should be further considered as effects of the following on the current amino acid adsorption processes: (i) temperature; (ii) mixed electrolyte; (iii) other amino acids and mineral surfaces; (iv) amino acid mixtures; and (v) computer simulation including solvent effects.

4. Conclusions The current work presents an in-depth analysis of the effect of ionic strength on the adsorption of glycine, lysine and glutamic acid by non-porous silica at the natural (unaltered) solution pH. A comparison of pure water solution and added 1:1 electrolyte was used to highlight the effects of electrolyte on isothermal amount adsorbed, and ATR-IR spectroscopy demonstrated its effect on the mechanism of adsorption. The findings are as follows: (1) The combination of nitrogen and water adsorption analyses is useful for determining the number of hydrophilic bonding sites/nm2 of adsorbent. In the case of Aerosil 200 silica there were 1.4 hydrophilic sites (silanol groups)/nm2. (2) The Langmuir–Freundlich isotherm model was sufficiently flexible to fit the adsorption isotherms for each amino acid classified as either L3a or S1.

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