Nano Sized Particles

Journal of The Institution of Engineers, Singapore Vol. 45 Issue 6 2005

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SEPARATION OF AMINO ACIDS USING NANO-SIZED MAGNETIC PARTICLES

Lim Zhi Min Cerine1, K. Hidajat1 and M. S. Uddin1*

ABSTRACT Nano-sized magnetic particles Fe3O4 with its distinctive traits have created interest in investigating its adsorptive behavior with amino acids. This work investigated the adsorptive behavior of three amino acids, namely glutamic acid, arginine and phenylalanine, on these magnetic particles under varying pH conditions at constant temperature. Several characteristic properties of these particles were evaluated where the specific surface area was found to be 102 m2/g by a nitrogen adsorption method and its isoelectric point was established as 6.9 by evaluating the zeta potentials. From the equilibrium isotherms, it was observed that the adsorption quantities (0.1-1.0 µmol/m2), was comparable to that reported in a study on the adsorption of amino acids on stainless steel [1]. Glutamic acid and arginine were 2-3 folds more adsorbed in the neutral whereas phenylalanine was poorly adsorbed in the acidic and neutral pH regions but showed great improvements in adsorptivity (5-9 folds) in the alkaline range. The exact mechanism for adsorption is still not fully understood but there was no evidence to suggest that adsorption was controlled by the electrostatic forces of attraction, which was thought initially.

INTRODUCTION With the advent of improved technology in the recent years, more emphasis has been placed in the research field towards the invention and contraption of micro and nanotechnology. For example, recent drug delivery techniques for chemotherapy have engaged the use of nano-sized particles as a delivery tool. Lab-on-a-chip has been made possible by the application of nanotechnology. Nanotechnology is also seen in the removal of toxic metal pollutants from waste water. Industrialization has brought about an ever-increasing exposure to toxic heavy metals such as lead and mercury or arsenic where they are irresponsibly dumped into rivers and oceans. As the authorities exert more pressure on these industries, many are inevitably finding more efficient and cost effective ways to improve waste treatment before discharging them to the environment. A process using iron oxide nano particles as a magnet to attract these heavy metals was developed. Because of its high efficiency, the quality of wastewater after treatment improved tremendously. Compared to other modes of removal of such heavy metals, this process has proven to be much more cost effective.

1 Department of Chemical and Biomolecular Engineering, National University of Singapore, 10 Kent Ridge Crescent, Singapore 119260 * Corresponding author. Tel: 65-68742886; Fax: 65-67791936; Email: [email protected]


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The above is a typical example of an adsorption process. Many studies have been made on the adsorptive behavior of molecules on different surfaces because of their large potential for isolating components of interest. There are generally two classes of adsorption namely, physisorption and chemisorption. In physisorption, the forces of attraction are usually weak van der Waals forces with corresponding adsorption energy in the typical range of (5-10) kJ/mol. This magnitude of attraction is very much lower compared to a typical chemical bond and therefore, the structures of adsorbing molecules remain intact. In chemisorption, the adsorption energy is comparable to the energy of a chemical bond and thus the adsorbed molecule may remain intact or dissociate with energy that varies from (30-70) kJ/mol for molecules and (100-400) kJ/mol for atoms. In many instances, however, due to the complex nature of the adsorption process, their behavior may not be easily described and explained even though the mode of adsorption might be known. A common mode of adsorption between amino acids and metal oxide surfaces is by electrostatic forces of attraction since both amino acids and metal oxides are amphoteric in nature i.e. they can exist in different charged conditions, positive or negative, under different environmental conditions. The charges in amino acids are contributed by several functional groups namely the amino group, carboxylic group and side chains (R groups). In metal oxides, it has been reported that their charged properties are contributed by -OH groups present [2]. The charged states of the amino acids and metal oxides are significantly influenced by the pH of the environment. In many metal oxides, when the pH of the environment is lower than their isoelectric point (pI), the -OH group exists as -OH2

+ and the metal oxides become positively charged. When the pH is higher than their pI, the -OH group deprotonates to -O- and the metal oxides become negatively charged. When the right pH conditions prevail, amino acid molecules and the adsorptive surfaces are oppositely charged and the molecules are attracted to the metal oxide surfaces. A change in pH conditions can cause both to be either positively or negatively charged and the molecules are released from the surfaces if adsorption is reversible. In this manner, the amino acids can be adsorbed and desorbed by a simple change in pH conditions. Ionic strength of the environment can also affect the adsorption process, especially when molecules are adsorbed by means of electrostatic interactions. As the ionic strength of the environment increases, the adsorbed molecules are displaced by these ions and are released back into the solution if the adsorption is reversible. Ionic strength of a solution can also be varied to selectively adsorb targeted molecules while allowing other impurities to be separated. This is conceptually similar to the ionic exchange columns that are used predominantly in the biomedical and pharmaceutical industries for purification of fine chemical and drugs and as an analytical tool to study the properties of many biological macromolecules. The study of adsorption of amino acids on metal oxide particles has received increasing attention because of its practical importance and potential in the biomedical industry. Notably, amino acids are the basic constituents of proteins, biological macromolecules of critical importance to the human body. Inspired by nano-technology and the possibility of adsorption of amino acids on magnetic surfaces, the present work studied the adsorptive behavior of three amino acids namely, glutamic acid (acidic), arginine (basic)


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and phenylalanine (aromatic) on nano-sized magnetic particles under varying pH conditions.

EXPERIMENTAL

Materials For the synthesis of the nano-sized magnetic particles: iron (II) cholride tetrahydrate (99%), iron (III) chloride hexahydrate (97%), ammonium hydroxide (25% NH3 in water) and Mili-Q water deionized and deoxygenated by sparging N2 gas. Amino acids used: L-glutamic acid monosodium salt (min 99% purity), L-arginine hydrochloride (min 98% purity), L-phenylalanine (sigma grade purity) were purchased from Sigma®.

Calibration and Quantification Techniques In order to quantify the amounts of amino acid at equilibrium, appropriate assays and calibration curves were established. Aromatic amino acids have been found to absorb light strongly at wavelengths between 250 to 280 nm. Phenylalanine, in particular, absorbs significantly at 258 nm and was directly analyzed using the UV/Vis spectrophotometer (Shimadzu UV-1601). Ninhydrin was used to quantify the other two amino acids, glutamic acid and arginine [4]. Assaying with ninhydrin is a common method used in the quantization of amino acids in the biotechnology industry because of its high reproducibility and sensitivity [12]. In recent years, chromatography techniques, e.g. high performance liquid chromatography (HPLC), have been more extensively used for such purposes because of their high speed, resolution and reproducibility. Nevertheless, both are established methods for amino acid analysis. The ninhydrin assay is a colorimetric method. Ninhydrin reacts with the amino group present in amino acids and is reduced to hydrindantin. In the process, ammonia and carbon dioxide are released. The ammonia, in turn, reacts with ninhydrin and hydrindantin to give a deep purple colored complex, also known as Ruhemann’s purple [7]. It is this colored complex that allows spectrophoretic methods such as UV/Vis to be employed. Its absorbance was found to be strongest at 570 nm. Technically, the ninhydrin assay was carried out as such. To 5 ml of the supernatant in a 30 ml sample vial, 1 ml of ethanolic ninhydrin was added. The sample vial was subsequently clamped in a heated water bath at 80oC for 7 min. The solution which had turned purplish was allowed to cool at 25oC for 7 min and absorbance measured with UV/Vis spectrophotometer at the wavelength of 570 nm. A wavelength scan for maximum peak absorbance for both spectrophoretic methods was found to correspond well to the optimum absorption wavelength cited in literatures [12]. Precaution was taken to ensure that the selected buffers' components do not interfere at these wavelengths. A negative control was used to eliminate background contribution from other factors. As the assays were found to be dependent on pH, a calibration curve was constructed for each amino acid at each pH evaluated.


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Synthesis of Nano-Sized Magnetic Particles The magnetic particles, Fe3O4, were synthesized in accordance to the protocol by a chemical co-precipitation method [10-11] where the molar ratio was maintained at Fe2+ : Fe3+ = 1: 2. Prior to the precipitation process, 50 ml of Mili-Q water was equilibrated to 80oC and deaerated by sparging nitrogen gas for 5 min to achieve inert condition. Following that, 0.86 gm of FeCl2.4H2O and 2.35 gm of FeCl3.6H2O were dissolved under vigorous stirring at 1015 rpm to attain approximately 1 g of dried Fe3O4 precipitate. It was allowed to run for 4 minutes before the addition of 5 ml of NH4OH for precipitation and stirred for another 10 min. The solution was subsequently cooled to room temperature and rinsed with Mili-Q water to remove any unreacted chemicals. Lastly, a magnetic field was used to separate the phases and the wet Fe3O4 precipitate was freeze dried before it was used for experiments.

Adsorption Experiments 100 mg of the magnetic particles was added to 20 ml of 0.25 mM glutamic acid buffered at pH 4.55 contained in a 30 ml sample vial. The contents were vigorously mixed at 180 rpm using a mechanical shaker for 16 hours to allow equilibrium to be established. A magnetic field was used to separate the phases and the supernatant of the solution was withdrawn for analysis. The experiment was repeated with varied amino acid concentrations (0.25 to 4.0 mM) at different pH values (pH 4.55 to pH 9) for each of the three amino acids. The amount of amino acid adsorbed was determined as the difference between the concentrations of the pure amino acid and the supernatant after equilibrium was established. The isotherms were then charted for comparison. To ensure that 16 hours was sufficient for equilibration, a series of simple experiments were carried out to determine the amino acid concentrations at regular time intervals. 2.5 mM of glutamic acid, arginine and phenylalanine were separately buffered at pH 7 and vigorously mixed with 100 mg of magnet at the same experimental condition of 180 rpm. Measurements were made at the 12th, 16th, 20th and 24th hour.

Characterization To assess the effectiveness of the magnetic particles as an adsorbent for the amino acids, some of their relevant properties such as the specific surface area and the isoelectric point were evaluated using BET analyzer (Model: NOVA 1000) and through zeta potential determination (Brookhaven Zeta Plus 90 analyzer) respectively. In determining the specific surface area of the magnetic particles, a nitrogen adsorption method was employed and the BET (Brunauer, Emmett and Teller) equation was applied. The BET method is a universal approach used to determine the specific surface area of adsorbents and to characterize porous materials. In principle, the specific surface area is measured by adsorption of gaseous nitrogen, typically operated at 77K, the boiling point of nitrogen. On the other hand, the pI of the magnetic particles was determined by its electrokinetics or zeta potential values and is shown in Figure 1. The test samples were prepared by adding 20 mg of wet magnetic particles to 10-3 M NaNO3 solution. Prior to


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that, the pH of the solutions were adjusted to a range of initial values from 3 to 11 by titrating with diluted HNO3 or NaOH. As shown in Figure 1, the pI of the magnetic particles is 6.9. The pI values of glutamic acid, arginine and phenylalanine have been well investigated and were reported by Lehninger [3] to be 3.22, 10.76 and 5.48 respectively.

-60

-40

-20

0

20

40

60

0 2 4 6 8 10 12 14

pH

Zeta

Pot

entia

l (m

V)

Figure 1: Zeta potentials of Fe3O4 at different pH values

RESULTS AND DISCUSSION

Adsorption Isotherms Figures 2 display the adsorption isotherms of the various amino acids on the surface of the nano-sized magnetic particles at equilibrium under different pH conditions. Both the acidic (glutamic acid) and basic (arginine) amino acids adsorbed significantly better at pH 7 compared to that at pH 4.55. Additionally, at pH 7, adsorption was most significant at low amino acid concentrations, i.e. < 1.5 mM. Beyond this, the extent of adsorption decreased with increasing amino acid concentrations. At concentrations above 3 mM, adsorption became ‘negative’. This is unlike saturation isotherms that are commonly seen in gaseous adsorptions. ‘Negative’ adsorption could be explained by the preferential adsorption of water molecules to amino acid molecules at high amino acid concentrations resulting in amino acids to be more concentrated at equilibrium. Such adsorption phenomenon can be found in many liquid mixture systems [15]. ‘Negative’ adsorption is also explained by Seader and Henley [9]. For a binary liquid mixture, one component is referred to as the solute and the other the solvent. It is then assumed that at low concentrations, only the solute component will be adsorbed. However, at high concentrations the distinction between solute and solvent becomes arbitrary and this assumption may not hold as the solvent molecules may also be adsorbed in this region. Such adsorption isotherms may exhibit various shapes over a


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large concentration range, unlike the saturation isotherms found commonly in gas adsorptions.

Figure 2: Adsorption isotherms of (a) glutamic acid, (b) arginine, and (c) phenylalanine on nano-sized magnetic particles

On the other hand, the adsorption for phenylalanine was most significant at the alkaline pH range (pH 8.25 and pH 9) and the adsorbed amounts for pH 4.55 and pH 7 remained low over the entire range of equilibrium concentration studied. It was also observed that the adsorption isotherms for phenylalanine at the alkaline pH values had a similar decreasing trend at higher concentrations. Among the employed modeling of adsorption, the Langmuir model and the Freundlich empirical model are the typically used models whereby the equilibrium data were represented adequately (Figures 3 and 4). The general forms of the Langmuir and Freundlich models are given by the following equations (1) and (2) respectively:

mQKC1

KCQ+

= (1)

n1

kCQ = (2)

where Q and C are the specific amount of adsorbate adsorbed (µmol/ m2) and the equilibrium concentration of adsorbate (mM), respectively. Qm is the maximum loading corresponding to complete coverage of adsorbent surface (µmol/ m2), K is the adsorption


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equilibrium constant (1/mM), k is the indicator of the adsorption capacity of the adsorbent and n is the indicator of the adsorption intensity. Conversely, the adsorption isotherm for aromatic phenylalanine at pH 7 gave a linear relationship between Q (µmol/m2) and C (mM), which follows the Henry's Law: bCQ = (3)

where b is the empirical temperature-dependent constant.

Figure 3: Langmuir fit of adsorption data for (a) glutamic acid, and (b) arginine at pH 7

Figure 4: Freundlich fit of adsorption data for (a) glutamic acid, and (b) arginine at pH 7

Consequently, by fitting the experimental data using Origin version 6.0, the correlation coefficient values are summarized in Table 1.


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Table 1: Summary of equilibrium parameters of the adsorption isotherms Amino Acids (pH 7) Glutamic Arginine Phenylalanine Model: Langmuir Correlated Values K = 9.32 K = 10.78 - Qm = 1.10 Qm = 0.63 R2 0.694 0.884 - Model: Freundlich Correlated Values k = 0.99 k = 0.57 - n = 4.67 n = 4.46 R2 0.713 0.798 - Model: Henry's Law Correlated Values - - b = 0.0924 R2 - - 0.891

From the adsorption isotherms observed, different isotherm patterns were established from the experiments performed. In principle, convex isotherms are favorable, because greater amounts of adsorbate can be obtained with low solute concentrations. On the other hand, concave isotherms are unfavorable. For phenylalanine, the amount adsorbed was minimal; hence the isotherm is apparently linear and follows the Henry's Law. From Table 1, arginine displays better adsorption affinity to the binding sites of Fe3O4 as compared to glutamic acid with a slightly higher K value though the Qmax value is smaller. However, no conclusive evidence can be established because of the poor correlations for conditions at pH 4.55 and pH values in the alkaline region. The adsorption quantities of the amino acids were generally comparable to that reported by Imamura et al. [1]. However, for these experiments, it was observed that glutamic acid showed better adsorption in the neutral rather than acidic pH. To note, for both glutamic acid and arginine in the alkaline regions, it was not feasible to assay using ninhydrin which will be elaborated further in the following subsection. From the results above, there was no clear evidence to suggest that the main mode of adsorption between amino acids and magnetic particles was by the electrostatic forces of attraction. Otherwise, glutamic acid would have been adsorbed to a much greater extent in the acidic rather than in the neutral region whereby the glutamic acid (pI = 3.22) being negatively charged should repel or adsorb poorly to the negatively charged magnetic particles (pI = 6.9) at pH 7. In addition, the extent of adsorption of aromatic amino acids such as phenylalanine would have been oblivious to changes in pH. However, the adsorptions of phenylalanine were in fact dependent on the pH conditions.

Calibration Using Ninhydrin In the course of the experiments, it was found that the ninhydrin assay was not suitable for glutamic acid and arginine at pH 9. Many of the calibration solutions turned dark yellow instead of purple. Initially, it was thought that the low ninhydrin concentrations could be the cause. Therefore, ninhydrin concentrations were increased to allow


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formation of the purple complex for some of the amino acid concentrations. However, this did not work for lower amino acid concentrations unless even more intense ninhydrin concentrations were used. Nonetheless, doing so resulted in very dark colored solutions for the higher end amino acid concentrations such that absorbance measurements were inaccurate and inappropriate (absorbance values of 2 and above were observed). Optimizing the ninhydrin concentrations would only allow calibration charts of very tight ranges (less than 1 mM) of amino acid concentrations which would not facilitate the quantitation of amino acids in this study. The limitation of the ninhydrin assay at pH 9 may be due to the possible blockage of the alpha amino group of the amino acids at low ninhydrin concentrations by the constituting components in the alkaline buffer. From the reaction scheme [7], such blockage would render the amino group from the amino acid unavailable for reaction with ninhydrin in the initiating reaction. There could also be some non-specific interactions between ninhydrin and the buffer components at low ninhydrin concentrations such that ninhydrin is no longer effective. Sheng et al. [12] indicated that although it has been generally accepted that the ninhydrin assay can be used for almost all amino acids, each amino acid does have its own unique characteristics and may undergo a much more complex reaction scheme than the generally accepted scheme [7]. The extent of colour formation will also differ with varying ninhydrin concentrations, incubation times, temperatures and pH values. Some amino acids are more affected by these conditions than others. This possibly explains the difficulties faced when trying to obtain the calibration curves for glutamic acid and arginine at pH 9. Due to the shortage of time for a more rigorous approach to obtain calibration curves for glutamic acid and arginine at pH 9, the experiments at this alkaline region were aborted. It was also noted that some of the calibration curves were not straight lines, as would be if depicted by Beer Lambert’s law. However, these curves were satisfactorily reproducible, particularly within the concentration ranges of the calibration curves and were acceptably used to quantify the amounts of amino acids.

CONCLUSIONS In summary, the adsorptive behaviors of glutamic acid, arginine and phenylalanine, on the nano-sized magnetic particles were studied under different pH conditions. From the adsorption data gathered and by fitting the experimental results to empirical models, the adsorption quantities between the investigated amino acids and the magnetic particles were comparable to that of the adsorption study between amino acids and stainless steel. In addition, there was no clear evidence in support of electrostatic forces of attraction as the main mode of adsorption since the extent of adsorption for glutamic acid was significantly reduced in the acidic pH region. Furthermore, phenylalanine showed a considerable increase in adsorption at the alkaline regions. Adsorption between aromatic amino acids such as phenylalanine should be oblivious to changes in pH conditions if electrostatic attractions played the major role. The isotherms for glutamic acid and arginine at pH 7 resemble the adsorption isotherms in many liquid mixtures. The ‘negative’ adsorption observed could be explained by the preferential adsorption of water


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molecules over amino acid molecules at high amino acid concentrations. This illustrates the complex nature of adsorption and that it cannot be easily described by a single process. Although the adsorption mechanism may not be understood at this point, there are clearly some trends observed from this study and may be used as a gauge for future projects. This study can be further extended by determining the influence of ionic strength on the adsorption process and exploring other assay techniques such as the HPLC system for the evaluation of glutamic acid and arginine at the alkaline pH range.

REFERENCES [1] Imamura, K., Mimura, T., Okamoto, M., Sakiyama, T. and Nakanishi, K. (2000). Adsorption Behavior of Amino Acids on a Stainless Steel Surface, J. Colloid Interface Sci., 229: pp237, 246. [2] Kittaka, S. (1974). J. Colloid Interface Sci., 48: pp327, 334. [3] Lehninger, A. L. (2000). Lehninger Principles of Biochemistry, 3rd ed: pp118. [4] Nam, S. W. Laboratory protocol www.glue.umd.edu/~nsw/ench485/lab3a.htm [5] Nyquist, R. A. and Kagel, R. O. (1971). Infrared Spectra of Inorganic Compounds: pp495. [6] Popplewell, J. and Sakhnini, L. (1995). The Dependence of the Physical and Magnetic Properties of Magnetic Fluids on Particle Size, 149, (1-2): pp174-180. [7] Ruhemann, S. (1911). J. Chem Soc., 99: pp1486-1510. [8] Schwarz, J. A. and Contescu, C. I. (1999). Surfaces of Nanoparticles and Porous Materials (Surfactant Science Series, Vol. 78): Chapter 3, 4, 8, 9, 17, 26 and 29. [9] Seader, J. D. and Henley, E. J. (1998). Separation Process Principles: Chapter 4 and 15 pp204-207 and pp778-879. [10] Shamim, N., Peng, Z. G., Hong, L., Hidajat, K. and Uddin, M. S. (2003). Nano-sized Magnetic Particles: Synthesis, Characterization and surface modification, 4th BUET conference paper. [11] Shen, L., Laibinis, P. E. and Hatton, T. A. (1999). Langmuir, 15: pp447-453. [12] Sheng, S., Kraft, J. J. and Schuster, S. M. (1993). A Specific Quantitative Colorimetric Assay for L- Asparagine, Analytical Biochemistry, 211: pp242-249. [13] Tentorio, A. and Canova, L. (1989). Adsorption of α-Amino Acids on Spherical TiO2 Particles, Colloids and Surfaces, 39: pp311-319.


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[14] Treybal, R. E. (1981). Mass Transfer Operations, 3rd ed: Chapter 8 and 11 pp275-335 and pp565-649. [15] Valenzuela, D. P. and Myers, A. L. (1989). Adsorption Equilibrium Data Handbook. [16] Voet, D., Voet, J. G. and Pratt, C. W. (1999). Fundamentals of Biochemistry: Chapter 4 pp77-88.

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