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diatomite composite and pure TiO2 for the purification of phosvitin phosphopeptides
Journal of Chromatography B, 960 (2014) 52–58
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Journal of Chromatography B journal homepage: www.elsevier.com/locate/chromb
Comparison of a novel TiO2 /diatomite composite and pure TiO2 for the purification of phosvitin phosphopeptides Yang Zhang a,b,1 , Junhua Li a,b,1 , Fuge Niu a,b , Jun Sun c , Yuan Dou d , Yuntao Liu a,b , Yujie Su a,b , Bei Zhou e , Qinqin Xu e , Yanjun Yang a,b,∗ a
State Key Laboratory of Food Science and Technology, Jiangnan University, Wuxi, Jiangsu 214122, PR China School of Food Science and Technology, Jiangnan University, Wuxi, Jiangsu 214122, PR China c School of Food and Biological Engineering, Jiangsu University, Zhenjiang, Jiangsu 212013, PR China d Shenzhen Entry-Exit Inspection and Quarantine Bureau, Shenzhen, Guangdong 518045, PR China e Kang de Biologicals Co., Ltd. Nantong b
a r t i c l e
i n f o
Article history: Received 28 December 2013 Accepted 31 March 2014 Available online 5 April 2014 Keywords: TiO2 TiO2 /diatomite composite Phosvitin phosphopeptides Purification Yolk
a b s t r a c t A novel TiO2 /diatomite composite (TD) was prepared and then characterized by scanning electron microscope (SEM) and Fourier Transform Infrared (FTIR). The results of SEM showed that after modification, the porous surface of diatomite was covered with TiO2 . Both diatomite and TD had clear disc-shaped structures with average grain diameters of around 25 m. Then TD and pure TiO2 were applied in the purification of phosvitin phosphopeptides (PPPs) from the digest of egg yolk protein, and a comparative study of adsorption properties of PPPs on TD and TiO2 was performed. In the study of adsorption kinetics, the adsorption equilibrium of PPPs on TD and TiO2 fitted well with the Langmuir model, and the time needed to reach adsorption equilibrium were both around 10 min. The maximum dynamic adsorption capacity of TD (8.15 mg/g) was higher than that of TiO2 (4.96 mg/g). The results of repeated use showed that TD and TiO2 were very stable after being subjected to ten repeated adsorption–elution cycles, and TD could easily be separated from aqueous solution by filtration. On the other hand, the present synthetic technology of TD was very simple, cost-effective, organic solvent-free and available for large-scale preparation. Thus, this separation method not only brings great advantages in the purification of PPPs from egg yolk protein but also provides a promising purification material for the enrichment of phosphopeptides in proteomic researches. © 2014 Elsevier B.V. All rights reserved.
1. Introduction Recently, many researchers have identified that PPPs had a great potential to improve the bioavailability of Ca2+ and thus increased the incorporation of Ca2+ into bones [1,2]. In traditional ways, PPPs are purified from phosvitin, which is the main phosphoprotein in egg yolk containing 10% phosphorus [3]. Although many studies have been carried out, the present purification methods still have some problems.
∗ Corresponding author at: State Key Laboratory of Food Science and Technology, Jiangnan University, Wuxi 214122, Jiangsu, PR China. Tel.: +86 0510 85329080; fax: +86 0510 85329080. E-mail addresses: [email protected], [email protected], [email protected], [email protected] (Y. Yang). 1 These authors contributed equally to this work. http://dx.doi.org/10.1016/j.jchromb.2014.03.038 1570-0232/© 2014 Elsevier B.V. All rights reserved.
As the three main traditional separation methods of PPPs, organic solvent precipitation, ion exchange chromatography and membrane separation, all have their obvious disadvantages [4]. Our research team studied the application of immobilized metal affinity nanoparticles in PPPs purification. These methods were fast, high-loading and efficient, but the stability of these nanoparticles was not so satisfying. And the elution medium used was imidazole, which was toxic and not allowed to be used in food industry [4,5]. In addition, these methods are only used in research laboratories, the magnetic separation technology and equipment for large-scale industrial application are still immature. It is necessary to develop new separation technology for large-scale production of PPPs, which is more stable, specific and easier for expanded industrial application. In recent years, TiO2 is widely used as an affinity support for the enrichment of phosphopeptides, based on the strong selective bidentate binding of phosphorylated peptides onto the TiO2 surface [6–10]. TiO2 is an amphoteric oxide. In acid solutions,
Y. Zhang et al. / J. Chromatogr. B 960 (2014) 52–58
it acts as a Lewis acid with positively charged titanium atoms, displaying anion-exchange properties. While in base solutions, it displays cation-exchange properties. Meanwhile, TiO2 has better corrosion resistance and biocompatibility than other organic or inorganic materials [11–15]. As TiO2 is introduced for phosphopeptides enrichment only at the beginning of this decade, the current researches mainly concentrate in phosphoproteomic. Applying TiO2 in the purification of PPPs has not yet been reported. In our early studies, we discovered that PPPs could be specifically adsorbed onto the surface of TiO2 . However, due to the uneven distribution of grain diameters and surface hydrophilicity of TiO2 powders, they could only be separated from aqueous solutions by high-speed centrifugal, which not only increased the cost of PPPs production but also limited the possibilities of large-scale application of TiO2 . Some researchers have reported several kinds of TiO2 modification, composite materials such as TiO2 microcolumns, Fe3 O4 @TiO2 core–shell microspheres, and SiO2 /TiO2 composite monolithic capillary column have been successfully synthesized and used for the selective enrichment of phosphopeptides [16–19]. But these methods are all laboratory studies, large-scale separation method for phosphopeptides still need further research. In the modification researches of materials chemistry, TiO2 has been loaded on various supporting materials such as glass plates, silica beads, zeolites, activated carbons, and diatomites [20–22]. As diatomite is cheap and widely used as a filter aid in food and pharmaceutical industries, and can easily be separated from aqueous solutions by filtration. What’s more, its natural porous structure and stable physical and chemical properties (under acid and alkaline conditions) makes it a perfect support for materials modification. In this work, a novel TiO2 /diatomite composite was prepared by chemical coprecipitation method and used for the purification of PPPs. The adsorption properties of PPPs on TD were studied and compared with that of pure TiO2 . The successful application of TD in PPPs purification may not only find a new purification method for PPPs production, but also provide a promising separation material for the enrichment of phosphopeptides from other complex biological samples in proteomic researches. 2. Materials and methods 2.1. Materials Titanium dioxide (TiO2 , chemically pure), Hydrochloric acid (HCl), titanium tetrachloride (TiCl4 ), 95% ethanol, acetone, trypsin (E.C.3.4.21.4.3 × 106 IU/g), sodium hydroxide (NaOH), ammonium molybdate, sodium sulfite, hydroquinone, sulfuric acid (H2 SO4 ), perchloric acid (HClO4 ), nitric acid (HNO3 ), potassium sodium tartrate, copper sulfate pentahydrate (CuSO4 ·5H2 O) were purchased from Sinopharm Chemical Reagent Co., Ltd. Diatomite filter aid STD was purchased from Celite China (Beijing, PR China). The Fresh Egg Yolk powder was from Kang de Biologicals Co., Ltd. Nantong. All the other chemicals were of analytical reagent grade used without further purification and the water used in all experiments was prepared in a three-stage purification system and had an electrical resistivity of 18.2 M cm−1 (highly pure water). 2.2. Preparation of crude yolk polypeptides from egg yolk powder The fresh egg yolk powder was defatted with 95% (v/v) ethanol using Soxhlet’s extraction method and the ethanol was removed by extraction filtration. The residue was dried at room temperature to volatilize the residual ethanol. Firstly, the dried de-fat yolk powder was suspended in 0.1 M NaOH solution and shaken in a homothermal shaker for 3 h (37 ◦ C,
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200 rpm) to reach reaction equilibrium. After that, the mixture was adjusted to pH 8.0 with 0.1 M hydrochloric acid solution and centrifuged for 20 min (8000 × g, 4 ◦ C). Then the suspension was ultra-filtered and the precipitate was washed with highly pure water for five times to remove the free phosphate anion therein. Secondly, the intercept fluid and washed precipitate were dried and transferred to a homothermal enzymatic reactor, and the trypsin was added to the solution above at an enzyme-to-substrate ratio of 1:20 (w/w). The mixture was incubated at 50 ◦ C for 4 h for enzymatic hydrolysis, during which the pH value was maintained at 8.0 with 0.1 M NaOH. The enzymolysis was terminated by incubating the mixture at 95 ◦ C for 15 min and cooling it down to room temperature before adjusting the pH value to 7.0. Then the tryptic digestion solution was centrifuged at 8000 × g (4 ◦ C, 20 min). Finally, the resulting supernatant solution was lyophilized and used as crude egg yolk polypeptides. 2.3. Preparation and characterization of TD For the preparation of TD, aqueous titanium tetrachloride (TiCl4 ) and diatomite were used as the TiO2 resource and support, respectively. Before synthesis, the diatomite was pretreated with 0.1 M HCl solution at a diatomite-to-liquid ratio of 1:20, followed by incubation for 30 min in a shaker at room temperature. The mixture was filtered after adjusting the pH to 7.0, and the precipitation was washed ten times by highly pure water and dried in a thermostatic drier. Through the pretreating process, the impurities in diatomite were removed and the refined diatomite was used for the preparation of TD. Firstly, samples of dried diatomite (2.4 g) were immersed in 50 mL HCl (0.1 M) solution and stirred for 20 min to get a homogeneous suspension. Then 2.24 mL of aqueous TiCl4 solutions were added dropwise to the above suspension under continuous stirring, and the mixture was incubated at 200 rpm for 30 min. As the hydrolytic action of TiCl4 was very fast and the acid environment was helpful to slow down the process, the formation of TiO2 was slow, which contributed to the well precipitation on the surface of diatomite. Secondly, the pH value of the mixture was adjusted to 7.0 gradually and continued stirring for 30 min for the reaction to reach equilibrium. Then mixture was the filtered, and the precipitation was rinsed with deionized water for six times to remove the unreacted chemicals. The initial composite was obtained by drying the precipitation above. Finally, the composite was calcined in a muffle furnace (600 ◦ C, 3 h) for the combination of TiO2 and diatomite to be more stable. After cooling down in a dryer, the TD was obtained. The surface morphology of pure diatomite and prepared TD samples were examined by scanning electron microscope (SEM, S4800, Hitachi Company). Fourier Transform Infrared (FTIR, Nicolte Nexus, Thermo Electrin Corporation) uses infrared radiation to determine the chemical functionalities present in the sample. The samples of diatomite, TiO2 and TD were mixed with potassium bromide (KBr) powder (1:50) and the mixtures were made into pellets under high pressure. The infrared spectra of prepared samples between 400 and 4000 cm−1 were recorded. 2.4. Adsorption of PPPs from aqueous solution Different initial concentrations of crude polypeptides solutions were prepared by dissolving freeze-dried crude egg yolk polypeptides in deionized water, stirring at 200 rpm for 30 min to get a homogeneous peptides solution. 2 g of TiO2 , 2 g of TD and 15 mL of crude polypeptide solutions (10–80 mg/mL, pH 1.0–6.0) were added into 150 mL triangular flasks, the mixed suspensions were shaken in a thermostated shaker (25 ◦ C, 200 rpm) for a certain time, respectively. And during the incubation, the supernatants were withdrawn at suitable time
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Fig. 1. SEM micrographs of pure diatomite (a), TD (b).
intervals. Effects of the pH of crude polypeptide solutions, stirring time and initial concentration on PPPs adsorption were studied. Then PPPs were eluted from the adsorbents by 0.01 M NaOH solutions. Meanwhile, the adsorbents were regenerated using 0.1 M NaOH. The characteristic index used to evaluate the purity of PPPs is the nitrogen/phosphorus molar ratios (N/P), which may reflect the peptide length and density of the phosphoric group. As many studied have indicated: the lower the N/P, the higher the purity of PPPs and the stronger the calcium-binging property [4]. The N/P of the crude egg yolk polypeptides was near 40 as we measured. The eluted PPPs and the supernatants obtained were used to determine the nitrogen (N) and phosphorus (P) contents. The content of N (%) was determined by colorimetric Biuret Method with 6.25 as the N-to-protein conversion factor, and the content of P (%) by the Molybdenum blue colorimetric method (GB/T 5009.872003). Every experiment was carried out in triplicate for the purpose of quality control and statistics. The N/P was calculated by the following equation: CpN ×31 q= CpP ×14
(1)
where q is the N/P of the purified PPPs; CpN and CpP are the quality content (mg/g) of the N and the P of PPPs, respectively; 31 and 14 are the relative molecular weight of P and N, respectively. The purified PPPs was used to measure the zeta potential at different pH (1.0–7.0) by a laser particle size and zeta potential analyzer.
2.5. Stability of TD and pure TiO2 in repeated use The adsorption and desorption cycles were repeated ten times using the same batch of TiO2 and TD to determine the reusability of the materials. After adsorption, TD was recovered from the reaction mixture by filtration, while TiO2 was by high speed centrifugation. The adsorbents were rinsed with deionized water (at a solid-to-water ratio 1:10) for five times, then they were mixed with the eluent (0.01 M NaOH) and incubated at a thermostated shaker (25 ◦ C, 200 rpm, 30 min) for PPPs to be desorbed completely. After desorption equilibrium, the adsorbents were recovered from the mixture. The N and P contents of the eluted PPPs were determined, and the calculated N/P ratios of the PPPs were used to evaluate the stabilities of the adsorbents.
3. Results and discussion 3.1. The characteristics of TD Typical SEM micrographs of pure diatomite and TD were shown in Fig. 1. It was found that the pure diatomite had a clear discshaped structure with numerous ordered micropores on its surface, which made it an ideal carrier for the synthesis of many composites. Fig. 1b showed that after modification, the surface of diatomite was coated with TiO2 tightly. As can be seen, the grain diameters of diatomite and TD were around 20–30 m. With this size, they could easily be intercepted by a 500-mesh sieve filter cloth.
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Fig. 2. FTIR spectra of pure diatomite (a), TiO2 (b), TD (c). Fig. 3. Effects of pH on the N/P of PPPs adsorbed on TiO2 and TD.
While many commercial TiO2 beads used in the studies of phosphopeptides enrichments were with particle size of 5 m [23–25] or nanoscale TiO2 particles [26,27]. While in this study, the mean particle size of the pure TiO2 powders used was 9.68 m as we measured, which made it difficult to be separated from aqueous solutions except by high-speed centrifugal and limited the possibilities of large-scale application of TiO2 . The specific porous structure of diatomite ensured TD a larger surface area and larger adsorption capacity than pure TiO2 powders. To confirm the binding of TiO2 and diatomite, FTIR technique was used and the FTIR spectra of the samples were shown in Fig. 2. As seen in the spectra, the characteristic peaks of OH group at 3400 cm−1 (stretching vibration) and 1635 cm−1 (bending vibration) were all observed clearly in the spectrograms of diatomite, TiO2 and TD, respectively. These may represented the surface OH group of these materials. As we know, the main component of diatomite was silicon dioxide and the surface OH groups were detected on silicon dioxide due to the terminal Si OH group at the mineral surface [28]. Meanwhile, TiO2 was an amphoteric hydroxide with an amount of OH groups on its surface [11,29]. Thus, the peaks of OH groups were observed on TD, which was the composite of diatomite and TiO2 . The characteristic peaks at around 1078 cm−1 , 782 cm−1 and 455 cm−1 , which were assigned to the asymmetric, symmetric stretching vibrations and bending vibration of Si O Si linkage, were observed in the spectrum of diatomite and TD (line a, c). The strong absorption band between 800 cm−1 and 480 cm−1 was the characteristic absorption of TiO2 (line b). These characteristic absorption band which was also observed in the spectrum of TD was weakened after TiO2 was loaded on the diatomite (line c), demonstrating that Ti O group was involved in the combination. The characteristic peak at 960 cm−1 of TD spectrum could be assigned to asymmetric Si O Ti stretching vibration [10]. According to the related reports, the reaction between TiO2 and the surface Si OH groups of silica support could occur during the calcination steps and form Si O Ti bonds [30]. From the above results, it can be concluded that TiO2 may be bound to the surface of diatomite by covalent bonds.
The optimal pH values for the adsorption of PPPs on TiO2 and TD were investigated in the pH range 1.0–6.0 (Fig. 3). It can be seen from the results that the preferential medium pH for PPPs purification were both at pH 4.0, the N/P molar ratios of the PPPs adsorbed on TD (5.6) and pure TiO2 (5.3) were the lowest. While the pH values of the solution were higher or lower than 4.0, the N/P ratios of PPPs increased. According to the studies of Zhang and Sun [4,5], PPPs with N/P of between 5.0 and 6.0 were with relatively high purity. As is shown in Fig. 4, the zeta potential of PPPs was 0 at pH 2.7, indicated that the isoelectric point (pI) of PPPs was around 2.7. When the pH was below 2.7, an electrostatic repulsion might occur between PPPs and TiO2 , and made it difficult for PPPs to be adsorbed on TiO2 . Thus the N/P ratios of PPPs were higher at pH 1.0 and 2.0. In theory, the negatively charged PPPs could be adsorbed onto the surface of positively charged TiO2 in the range of pH 2.7 to 6.0. At pH 4.0, the electrostatic attraction between TiO2 and PPPs might be much stronger than other pH values, and lead to the lowest N/P and highest purity of PPPs. Another reason maybe that at pH above 4.0, some nonphosphorylated peptides, which was also negatively charged with one or more aspartic acid residues and/or glutamic acid residues, might be adsorbed on adsorbents (non-specific adsorption) [31]. As the pKa values of the side chain carboxylic of aspartic acid and glutamic acid were 3.55 and 4.25, respectively [32].
3.2. The effect of pH TiO2 is an amphoteric hydroxide that can adsorb H2 O onto its surface and then dissociate by the strong electrostatic forces between Ti4+ and neighboring O2− , the “zero point of charge” of TiO2 is around pH 6.0 [11]. The pH value of the aqueous solution has a great influence on the surface electric potential of TiO2 and then influence the adsorption of PPPs.
Fig. 4. Effects of pH on the zeta potential of PPPs.
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To describe the adsorption kinetics of TiO2 and TD, the pseudosecond-order model was used. The pseudo-second-order model is expressed as follows: 1 t = + qt k2 q2e
1 qe
t
(2)
where k2 is the pseudo-second-order rate constant (min−1 ) of adsorption; qe is the maximum adsorption capacity for pseudosecond-order (mg/g); qt is the amounts of PPPs adsorbed on adsorbents at any time (min) (mg/g). Fig. 5b was obtained by plotting t/qt versus t according to pseudo-second-order model. The adsorption kinetics were expressed as follows: y = 0.18463x + 0.53482, qe = 5.42 mg/g TiO2 , k2 = 0.0637, R2 = 0.99854 (for TiO2 ); y = 0.11923x + 0.1725, qe = 8.39 mg/g TD, k2 = 0.0824, R2 = 0.9981 (for TD). The above results indicated that the theoretical qe value was close to the experimental qe value and the correlation coefficients for the linear plots of t/qt against t were more than 0.99 for both of the two adsorbents. The pseudo-second-order equation fitted well with the experimental data and could be used to describe the adsorption kinetics of PPPs on pure TiO2 and TD by electrostatic interaction. From the results described above, we could conclude that the loading capacity of TiO2 was lower than TD both in practice and theory. 3.4. Adsorption isotherms
Fig. 5. Kinetic curves of the adsorption of PPPs on pure TiO2 and TD (a). Pseudosecond-order kinetic plots for the adsorption of PPPs on TiO2 and TD (b) (initial concentration of polypeptides: 40 mg/mL; pH: 4.0; temperature: 25 ◦ C).
These results showed that TiO2 and TD had a strong selective adsorption of PPPs at pH 4.0, and were efficient to purify PPPs with high purity from crude egg yolk polypeptides solutions.
The adsorption model was studied by using 2 g of pure TiO2 powders or 2 g of TD and 15 mL of polypeptides solution with different initial concentration. The relationship between the adsorption capacity and the initial concentration of polypeptides was shown in Fig. 6a. When the initial concentration of polypeptides increased from 0 to 30 mg/mL, the amount of adsorbed PPPs on TiO2 was increasing fast, then this increasing trends was slowed down at polypeptides concentration above 30 mg/mL and remained relatively unchanged. While for TD, when the initial concentration of polypeptides was above 40 mg/mL, the increasing trend was slowed down. The reason may be that the PPPs adsorption sites of TiO2 and TD were saturated at concentration of 30 mg/mL and 40 mg/mL, respectively. The calculating results showed that at adsorption equilibrium, the N/P of PPPs adsorbed on TiO2 and TD were around 5.17 and 5.47, respectively. These results indicated that the purified PPPs were with relatively high purity. The adsorption behaviors of PPPs on TiO2 and TD were described using Langmuir model for which equation could be expressed as follows:
3.3. Adsorption kinetics
ce 1 Ce = + qe qm k + qm
To ascertain the time required to reach adsorption equilibrium of PPPs, binding experiments were performed using 2 g of pure TiO2 powders or 2 g of TD and 15 mL of crude yolk polypeptides solutions with initial concentration of 40 mg/mL (pH 4.0) at room temperature. Fig. 5a showed that the adsorption rates of PPPs on TiO2 and TD were very fast within 10 min. After 10 min, the rates of adsorption slowed down and then reached reaction equilibrium. At equilibrium, the adsorption capacities of TiO2 and TD were about 4.96 and 8.15 mg/g adsorbent, respectively. As TiO2 was coated on the surface of diatomite, the contact area of TD was larger than that of pure TiO2 , there were greater chances for PPPs to contact with the adsorbent, and thus the maximum adsorption capacity of TD was 38% higher than that of TiO2 . The calculating results showed that the adsorbed PPPs were all with high purity, the N/P of PPPs were about 5.26 (TiO2 ) and 5.51 (TD) at reaction equilibrium.
where Ce (mg/mL) and qe (mg/g adsorbent) are the polypeptides concentration in the aqueous solution and the amount of PPPs adsorbed on the adsorbent at equilibrium, respectively; qm is the maximum adsorption capacity (mg/g adsorbent) and k is the adsorption constant (mL/mg). The experimental data was analyzed and the related results were shown in Fig. 6b. In this work the Langmuir equations for two adsorbents had the expressions as follows: Ce /qe = 0.19955 Ce + 0.07362, qm = 5.01 mg/g pure TiO2 , R2 = 0.9998, k = 2.71; Ce /qe = 0.11591 Ce + 0.04657, qm = 8.63 mg/g TD, R2 = 0.99405, k = 2.49. The high R2 values in the equations indicated that the Langmuir model was very fit to describe the adsorption behavior of PPPs on TiO2 and TD. The Langmuir constant k value from TiO2 was higher than that from TD, which indicated that TiO2 had a higher affinity force for PPPs than TD. This phenomenon might be due to some nonspecific adsorptions occurred on the porous surface of diatomite [33]. The maximum
(3)
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Fig. 7. Effects of repeated uses on the N/P of PPPs purified by TiO2 and TD (pH: 4.0; temperature: 25 ◦ C).
4. Conclusions
Fig. 6. Adsorption isotherms for PPPs adsorbed on TiO2 and TD (a). linear fit (b) (pH: 4.0; temperature: 25 ◦ C).
A TiO2 /diatomite composite was successfully prepared and applied in the enrichment of PPPs from egg yolk protein hydrolysis polypeptide for the first time. The optimized adsorption condition of PPPs was at pH 4.0. The N/P molar ratio of the purified PPPs was around 5.5. The comparison study of the adsorption properties of PPPs onto the pure TiO2 and TD showed that the adsorption models fitted well with the Langmuir model. And after modification, the adsorption capacity was improved. The preparation method of the composite was simple, cost-effective, and organic solvent-free. Meanwhile, with uniform and relatively large diameter, the composite could be separated and recovered from aqueous solutions easily by filtration. The stable N/P ratios of purified PPPs after ten repeated uses indicated the high stability of TD. It is anticipated that TD may have great potential to be used in large-scale purification of PPPs and enrichment of phosphopeptides from other complex biological samples. Acknowledgements
equilibrium adsorption capacities for both adsorbents were close to maximum adsorption capacities calculated from the previous pseudo-second-order kinetic models. 3.5. Stability of TD and TiO2 in repeated use In alkaline environment, TiO2 , TD and PPPs are both negatively charged, the electronic interactions between them are electrostatic repulsion. The PPPs can be efficiently eluted from TiO2 . The adsorbed PPPs were eluted by 0.01 M NaOH solution. Then the adsorbents were regenerated by 0.1 M NaOH. To evaluate the stability of TD and TiO2 , repeated adsorption–desorption experiments were conducted for ten times using the same batch of TD and TiO2 for PPPs purification (Fig. 7). After ten times uses, the N/P of eluted PPPs were still under 6.0, which indicated that TiO2 and TD were very stable, their affinity for PPPs did not decrease after repeated uses. And they both had strong resistance to acid and alkali solutions. This may because that TiO2 and diatomite both had acid and alkali resistance and stable physical and chemical properties. After modification and calcination, TiO2 and diatomite had been conjugated together to form a new stable composite materials which possess the advantages of these two materials, thus the prepared TD was very stable and could be used as affinity materials in the purification of PPPs.
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Comparison of a novel TiO2 /diatomite composite and pure TiO2 for the purification of phosvitin phosphopeptides Yang Zhang a,b,1 , Junhua Li a,b,1 , Fuge Niu a,b , Jun Sun c , Yuan Dou d , Yuntao Liu a,b , Yujie Su a,b , Bei Zhou e , Qinqin Xu e , Yanjun Yang a,b,∗ a
State Key Laboratory of Food Science and Technology, Jiangnan University, Wuxi, Jiangsu 214122, PR China School of Food Science and Technology, Jiangnan University, Wuxi, Jiangsu 214122, PR China c School of Food and Biological Engineering, Jiangsu University, Zhenjiang, Jiangsu 212013, PR China d Shenzhen Entry-Exit Inspection and Quarantine Bureau, Shenzhen, Guangdong 518045, PR China e Kang de Biologicals Co., Ltd. Nantong b
a r t i c l e
i n f o
Article history: Received 28 December 2013 Accepted 31 March 2014 Available online 5 April 2014 Keywords: TiO2 TiO2 /diatomite composite Phosvitin phosphopeptides Purification Yolk
a b s t r a c t A novel TiO2 /diatomite composite (TD) was prepared and then characterized by scanning electron microscope (SEM) and Fourier Transform Infrared (FTIR). The results of SEM showed that after modification, the porous surface of diatomite was covered with TiO2 . Both diatomite and TD had clear disc-shaped structures with average grain diameters of around 25 m. Then TD and pure TiO2 were applied in the purification of phosvitin phosphopeptides (PPPs) from the digest of egg yolk protein, and a comparative study of adsorption properties of PPPs on TD and TiO2 was performed. In the study of adsorption kinetics, the adsorption equilibrium of PPPs on TD and TiO2 fitted well with the Langmuir model, and the time needed to reach adsorption equilibrium were both around 10 min. The maximum dynamic adsorption capacity of TD (8.15 mg/g) was higher than that of TiO2 (4.96 mg/g). The results of repeated use showed that TD and TiO2 were very stable after being subjected to ten repeated adsorption–elution cycles, and TD could easily be separated from aqueous solution by filtration. On the other hand, the present synthetic technology of TD was very simple, cost-effective, organic solvent-free and available for large-scale preparation. Thus, this separation method not only brings great advantages in the purification of PPPs from egg yolk protein but also provides a promising purification material for the enrichment of phosphopeptides in proteomic researches. © 2014 Elsevier B.V. All rights reserved.
1. Introduction Recently, many researchers have identified that PPPs had a great potential to improve the bioavailability of Ca2+ and thus increased the incorporation of Ca2+ into bones [1,2]. In traditional ways, PPPs are purified from phosvitin, which is the main phosphoprotein in egg yolk containing 10% phosphorus [3]. Although many studies have been carried out, the present purification methods still have some problems.
∗ Corresponding author at: State Key Laboratory of Food Science and Technology, Jiangnan University, Wuxi 214122, Jiangsu, PR China. Tel.: +86 0510 85329080; fax: +86 0510 85329080. E-mail addresses: [email protected], [email protected], [email protected], [email protected] (Y. Yang). 1 These authors contributed equally to this work. http://dx.doi.org/10.1016/j.jchromb.2014.03.038 1570-0232/© 2014 Elsevier B.V. All rights reserved.
As the three main traditional separation methods of PPPs, organic solvent precipitation, ion exchange chromatography and membrane separation, all have their obvious disadvantages [4]. Our research team studied the application of immobilized metal affinity nanoparticles in PPPs purification. These methods were fast, high-loading and efficient, but the stability of these nanoparticles was not so satisfying. And the elution medium used was imidazole, which was toxic and not allowed to be used in food industry [4,5]. In addition, these methods are only used in research laboratories, the magnetic separation technology and equipment for large-scale industrial application are still immature. It is necessary to develop new separation technology for large-scale production of PPPs, which is more stable, specific and easier for expanded industrial application. In recent years, TiO2 is widely used as an affinity support for the enrichment of phosphopeptides, based on the strong selective bidentate binding of phosphorylated peptides onto the TiO2 surface [6–10]. TiO2 is an amphoteric oxide. In acid solutions,
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it acts as a Lewis acid with positively charged titanium atoms, displaying anion-exchange properties. While in base solutions, it displays cation-exchange properties. Meanwhile, TiO2 has better corrosion resistance and biocompatibility than other organic or inorganic materials [11–15]. As TiO2 is introduced for phosphopeptides enrichment only at the beginning of this decade, the current researches mainly concentrate in phosphoproteomic. Applying TiO2 in the purification of PPPs has not yet been reported. In our early studies, we discovered that PPPs could be specifically adsorbed onto the surface of TiO2 . However, due to the uneven distribution of grain diameters and surface hydrophilicity of TiO2 powders, they could only be separated from aqueous solutions by high-speed centrifugal, which not only increased the cost of PPPs production but also limited the possibilities of large-scale application of TiO2 . Some researchers have reported several kinds of TiO2 modification, composite materials such as TiO2 microcolumns, Fe3 O4 @TiO2 core–shell microspheres, and SiO2 /TiO2 composite monolithic capillary column have been successfully synthesized and used for the selective enrichment of phosphopeptides [16–19]. But these methods are all laboratory studies, large-scale separation method for phosphopeptides still need further research. In the modification researches of materials chemistry, TiO2 has been loaded on various supporting materials such as glass plates, silica beads, zeolites, activated carbons, and diatomites [20–22]. As diatomite is cheap and widely used as a filter aid in food and pharmaceutical industries, and can easily be separated from aqueous solutions by filtration. What’s more, its natural porous structure and stable physical and chemical properties (under acid and alkaline conditions) makes it a perfect support for materials modification. In this work, a novel TiO2 /diatomite composite was prepared by chemical coprecipitation method and used for the purification of PPPs. The adsorption properties of PPPs on TD were studied and compared with that of pure TiO2 . The successful application of TD in PPPs purification may not only find a new purification method for PPPs production, but also provide a promising separation material for the enrichment of phosphopeptides from other complex biological samples in proteomic researches. 2. Materials and methods 2.1. Materials Titanium dioxide (TiO2 , chemically pure), Hydrochloric acid (HCl), titanium tetrachloride (TiCl4 ), 95% ethanol, acetone, trypsin (E.C.3.4.21.4.3 × 106 IU/g), sodium hydroxide (NaOH), ammonium molybdate, sodium sulfite, hydroquinone, sulfuric acid (H2 SO4 ), perchloric acid (HClO4 ), nitric acid (HNO3 ), potassium sodium tartrate, copper sulfate pentahydrate (CuSO4 ·5H2 O) were purchased from Sinopharm Chemical Reagent Co., Ltd. Diatomite filter aid STD was purchased from Celite China (Beijing, PR China). The Fresh Egg Yolk powder was from Kang de Biologicals Co., Ltd. Nantong. All the other chemicals were of analytical reagent grade used without further purification and the water used in all experiments was prepared in a three-stage purification system and had an electrical resistivity of 18.2 M cm−1 (highly pure water). 2.2. Preparation of crude yolk polypeptides from egg yolk powder The fresh egg yolk powder was defatted with 95% (v/v) ethanol using Soxhlet’s extraction method and the ethanol was removed by extraction filtration. The residue was dried at room temperature to volatilize the residual ethanol. Firstly, the dried de-fat yolk powder was suspended in 0.1 M NaOH solution and shaken in a homothermal shaker for 3 h (37 ◦ C,
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200 rpm) to reach reaction equilibrium. After that, the mixture was adjusted to pH 8.0 with 0.1 M hydrochloric acid solution and centrifuged for 20 min (8000 × g, 4 ◦ C). Then the suspension was ultra-filtered and the precipitate was washed with highly pure water for five times to remove the free phosphate anion therein. Secondly, the intercept fluid and washed precipitate were dried and transferred to a homothermal enzymatic reactor, and the trypsin was added to the solution above at an enzyme-to-substrate ratio of 1:20 (w/w). The mixture was incubated at 50 ◦ C for 4 h for enzymatic hydrolysis, during which the pH value was maintained at 8.0 with 0.1 M NaOH. The enzymolysis was terminated by incubating the mixture at 95 ◦ C for 15 min and cooling it down to room temperature before adjusting the pH value to 7.0. Then the tryptic digestion solution was centrifuged at 8000 × g (4 ◦ C, 20 min). Finally, the resulting supernatant solution was lyophilized and used as crude egg yolk polypeptides. 2.3. Preparation and characterization of TD For the preparation of TD, aqueous titanium tetrachloride (TiCl4 ) and diatomite were used as the TiO2 resource and support, respectively. Before synthesis, the diatomite was pretreated with 0.1 M HCl solution at a diatomite-to-liquid ratio of 1:20, followed by incubation for 30 min in a shaker at room temperature. The mixture was filtered after adjusting the pH to 7.0, and the precipitation was washed ten times by highly pure water and dried in a thermostatic drier. Through the pretreating process, the impurities in diatomite were removed and the refined diatomite was used for the preparation of TD. Firstly, samples of dried diatomite (2.4 g) were immersed in 50 mL HCl (0.1 M) solution and stirred for 20 min to get a homogeneous suspension. Then 2.24 mL of aqueous TiCl4 solutions were added dropwise to the above suspension under continuous stirring, and the mixture was incubated at 200 rpm for 30 min. As the hydrolytic action of TiCl4 was very fast and the acid environment was helpful to slow down the process, the formation of TiO2 was slow, which contributed to the well precipitation on the surface of diatomite. Secondly, the pH value of the mixture was adjusted to 7.0 gradually and continued stirring for 30 min for the reaction to reach equilibrium. Then mixture was the filtered, and the precipitation was rinsed with deionized water for six times to remove the unreacted chemicals. The initial composite was obtained by drying the precipitation above. Finally, the composite was calcined in a muffle furnace (600 ◦ C, 3 h) for the combination of TiO2 and diatomite to be more stable. After cooling down in a dryer, the TD was obtained. The surface morphology of pure diatomite and prepared TD samples were examined by scanning electron microscope (SEM, S4800, Hitachi Company). Fourier Transform Infrared (FTIR, Nicolte Nexus, Thermo Electrin Corporation) uses infrared radiation to determine the chemical functionalities present in the sample. The samples of diatomite, TiO2 and TD were mixed with potassium bromide (KBr) powder (1:50) and the mixtures were made into pellets under high pressure. The infrared spectra of prepared samples between 400 and 4000 cm−1 were recorded. 2.4. Adsorption of PPPs from aqueous solution Different initial concentrations of crude polypeptides solutions were prepared by dissolving freeze-dried crude egg yolk polypeptides in deionized water, stirring at 200 rpm for 30 min to get a homogeneous peptides solution. 2 g of TiO2 , 2 g of TD and 15 mL of crude polypeptide solutions (10–80 mg/mL, pH 1.0–6.0) were added into 150 mL triangular flasks, the mixed suspensions were shaken in a thermostated shaker (25 ◦ C, 200 rpm) for a certain time, respectively. And during the incubation, the supernatants were withdrawn at suitable time
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Fig. 1. SEM micrographs of pure diatomite (a), TD (b).
intervals. Effects of the pH of crude polypeptide solutions, stirring time and initial concentration on PPPs adsorption were studied. Then PPPs were eluted from the adsorbents by 0.01 M NaOH solutions. Meanwhile, the adsorbents were regenerated using 0.1 M NaOH. The characteristic index used to evaluate the purity of PPPs is the nitrogen/phosphorus molar ratios (N/P), which may reflect the peptide length and density of the phosphoric group. As many studied have indicated: the lower the N/P, the higher the purity of PPPs and the stronger the calcium-binging property [4]. The N/P of the crude egg yolk polypeptides was near 40 as we measured. The eluted PPPs and the supernatants obtained were used to determine the nitrogen (N) and phosphorus (P) contents. The content of N (%) was determined by colorimetric Biuret Method with 6.25 as the N-to-protein conversion factor, and the content of P (%) by the Molybdenum blue colorimetric method (GB/T 5009.872003). Every experiment was carried out in triplicate for the purpose of quality control and statistics. The N/P was calculated by the following equation: CpN ×31 q= CpP ×14
(1)
where q is the N/P of the purified PPPs; CpN and CpP are the quality content (mg/g) of the N and the P of PPPs, respectively; 31 and 14 are the relative molecular weight of P and N, respectively. The purified PPPs was used to measure the zeta potential at different pH (1.0–7.0) by a laser particle size and zeta potential analyzer.
2.5. Stability of TD and pure TiO2 in repeated use The adsorption and desorption cycles were repeated ten times using the same batch of TiO2 and TD to determine the reusability of the materials. After adsorption, TD was recovered from the reaction mixture by filtration, while TiO2 was by high speed centrifugation. The adsorbents were rinsed with deionized water (at a solid-to-water ratio 1:10) for five times, then they were mixed with the eluent (0.01 M NaOH) and incubated at a thermostated shaker (25 ◦ C, 200 rpm, 30 min) for PPPs to be desorbed completely. After desorption equilibrium, the adsorbents were recovered from the mixture. The N and P contents of the eluted PPPs were determined, and the calculated N/P ratios of the PPPs were used to evaluate the stabilities of the adsorbents.
3. Results and discussion 3.1. The characteristics of TD Typical SEM micrographs of pure diatomite and TD were shown in Fig. 1. It was found that the pure diatomite had a clear discshaped structure with numerous ordered micropores on its surface, which made it an ideal carrier for the synthesis of many composites. Fig. 1b showed that after modification, the surface of diatomite was coated with TiO2 tightly. As can be seen, the grain diameters of diatomite and TD were around 20–30 m. With this size, they could easily be intercepted by a 500-mesh sieve filter cloth.
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Fig. 2. FTIR spectra of pure diatomite (a), TiO2 (b), TD (c). Fig. 3. Effects of pH on the N/P of PPPs adsorbed on TiO2 and TD.
While many commercial TiO2 beads used in the studies of phosphopeptides enrichments were with particle size of 5 m [23–25] or nanoscale TiO2 particles [26,27]. While in this study, the mean particle size of the pure TiO2 powders used was 9.68 m as we measured, which made it difficult to be separated from aqueous solutions except by high-speed centrifugal and limited the possibilities of large-scale application of TiO2 . The specific porous structure of diatomite ensured TD a larger surface area and larger adsorption capacity than pure TiO2 powders. To confirm the binding of TiO2 and diatomite, FTIR technique was used and the FTIR spectra of the samples were shown in Fig. 2. As seen in the spectra, the characteristic peaks of OH group at 3400 cm−1 (stretching vibration) and 1635 cm−1 (bending vibration) were all observed clearly in the spectrograms of diatomite, TiO2 and TD, respectively. These may represented the surface OH group of these materials. As we know, the main component of diatomite was silicon dioxide and the surface OH groups were detected on silicon dioxide due to the terminal Si OH group at the mineral surface [28]. Meanwhile, TiO2 was an amphoteric hydroxide with an amount of OH groups on its surface [11,29]. Thus, the peaks of OH groups were observed on TD, which was the composite of diatomite and TiO2 . The characteristic peaks at around 1078 cm−1 , 782 cm−1 and 455 cm−1 , which were assigned to the asymmetric, symmetric stretching vibrations and bending vibration of Si O Si linkage, were observed in the spectrum of diatomite and TD (line a, c). The strong absorption band between 800 cm−1 and 480 cm−1 was the characteristic absorption of TiO2 (line b). These characteristic absorption band which was also observed in the spectrum of TD was weakened after TiO2 was loaded on the diatomite (line c), demonstrating that Ti O group was involved in the combination. The characteristic peak at 960 cm−1 of TD spectrum could be assigned to asymmetric Si O Ti stretching vibration [10]. According to the related reports, the reaction between TiO2 and the surface Si OH groups of silica support could occur during the calcination steps and form Si O Ti bonds [30]. From the above results, it can be concluded that TiO2 may be bound to the surface of diatomite by covalent bonds.
The optimal pH values for the adsorption of PPPs on TiO2 and TD were investigated in the pH range 1.0–6.0 (Fig. 3). It can be seen from the results that the preferential medium pH for PPPs purification were both at pH 4.0, the N/P molar ratios of the PPPs adsorbed on TD (5.6) and pure TiO2 (5.3) were the lowest. While the pH values of the solution were higher or lower than 4.0, the N/P ratios of PPPs increased. According to the studies of Zhang and Sun [4,5], PPPs with N/P of between 5.0 and 6.0 were with relatively high purity. As is shown in Fig. 4, the zeta potential of PPPs was 0 at pH 2.7, indicated that the isoelectric point (pI) of PPPs was around 2.7. When the pH was below 2.7, an electrostatic repulsion might occur between PPPs and TiO2 , and made it difficult for PPPs to be adsorbed on TiO2 . Thus the N/P ratios of PPPs were higher at pH 1.0 and 2.0. In theory, the negatively charged PPPs could be adsorbed onto the surface of positively charged TiO2 in the range of pH 2.7 to 6.0. At pH 4.0, the electrostatic attraction between TiO2 and PPPs might be much stronger than other pH values, and lead to the lowest N/P and highest purity of PPPs. Another reason maybe that at pH above 4.0, some nonphosphorylated peptides, which was also negatively charged with one or more aspartic acid residues and/or glutamic acid residues, might be adsorbed on adsorbents (non-specific adsorption) [31]. As the pKa values of the side chain carboxylic of aspartic acid and glutamic acid were 3.55 and 4.25, respectively [32].
3.2. The effect of pH TiO2 is an amphoteric hydroxide that can adsorb H2 O onto its surface and then dissociate by the strong electrostatic forces between Ti4+ and neighboring O2− , the “zero point of charge” of TiO2 is around pH 6.0 [11]. The pH value of the aqueous solution has a great influence on the surface electric potential of TiO2 and then influence the adsorption of PPPs.
Fig. 4. Effects of pH on the zeta potential of PPPs.
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To describe the adsorption kinetics of TiO2 and TD, the pseudosecond-order model was used. The pseudo-second-order model is expressed as follows: 1 t = + qt k2 q2e
1 qe
t
(2)
where k2 is the pseudo-second-order rate constant (min−1 ) of adsorption; qe is the maximum adsorption capacity for pseudosecond-order (mg/g); qt is the amounts of PPPs adsorbed on adsorbents at any time (min) (mg/g). Fig. 5b was obtained by plotting t/qt versus t according to pseudo-second-order model. The adsorption kinetics were expressed as follows: y = 0.18463x + 0.53482, qe = 5.42 mg/g TiO2 , k2 = 0.0637, R2 = 0.99854 (for TiO2 ); y = 0.11923x + 0.1725, qe = 8.39 mg/g TD, k2 = 0.0824, R2 = 0.9981 (for TD). The above results indicated that the theoretical qe value was close to the experimental qe value and the correlation coefficients for the linear plots of t/qt against t were more than 0.99 for both of the two adsorbents. The pseudo-second-order equation fitted well with the experimental data and could be used to describe the adsorption kinetics of PPPs on pure TiO2 and TD by electrostatic interaction. From the results described above, we could conclude that the loading capacity of TiO2 was lower than TD both in practice and theory. 3.4. Adsorption isotherms
Fig. 5. Kinetic curves of the adsorption of PPPs on pure TiO2 and TD (a). Pseudosecond-order kinetic plots for the adsorption of PPPs on TiO2 and TD (b) (initial concentration of polypeptides: 40 mg/mL; pH: 4.0; temperature: 25 ◦ C).
These results showed that TiO2 and TD had a strong selective adsorption of PPPs at pH 4.0, and were efficient to purify PPPs with high purity from crude egg yolk polypeptides solutions.
The adsorption model was studied by using 2 g of pure TiO2 powders or 2 g of TD and 15 mL of polypeptides solution with different initial concentration. The relationship between the adsorption capacity and the initial concentration of polypeptides was shown in Fig. 6a. When the initial concentration of polypeptides increased from 0 to 30 mg/mL, the amount of adsorbed PPPs on TiO2 was increasing fast, then this increasing trends was slowed down at polypeptides concentration above 30 mg/mL and remained relatively unchanged. While for TD, when the initial concentration of polypeptides was above 40 mg/mL, the increasing trend was slowed down. The reason may be that the PPPs adsorption sites of TiO2 and TD were saturated at concentration of 30 mg/mL and 40 mg/mL, respectively. The calculating results showed that at adsorption equilibrium, the N/P of PPPs adsorbed on TiO2 and TD were around 5.17 and 5.47, respectively. These results indicated that the purified PPPs were with relatively high purity. The adsorption behaviors of PPPs on TiO2 and TD were described using Langmuir model for which equation could be expressed as follows:
3.3. Adsorption kinetics
ce 1 Ce = + qe qm k + qm
To ascertain the time required to reach adsorption equilibrium of PPPs, binding experiments were performed using 2 g of pure TiO2 powders or 2 g of TD and 15 mL of crude yolk polypeptides solutions with initial concentration of 40 mg/mL (pH 4.0) at room temperature. Fig. 5a showed that the adsorption rates of PPPs on TiO2 and TD were very fast within 10 min. After 10 min, the rates of adsorption slowed down and then reached reaction equilibrium. At equilibrium, the adsorption capacities of TiO2 and TD were about 4.96 and 8.15 mg/g adsorbent, respectively. As TiO2 was coated on the surface of diatomite, the contact area of TD was larger than that of pure TiO2 , there were greater chances for PPPs to contact with the adsorbent, and thus the maximum adsorption capacity of TD was 38% higher than that of TiO2 . The calculating results showed that the adsorbed PPPs were all with high purity, the N/P of PPPs were about 5.26 (TiO2 ) and 5.51 (TD) at reaction equilibrium.
where Ce (mg/mL) and qe (mg/g adsorbent) are the polypeptides concentration in the aqueous solution and the amount of PPPs adsorbed on the adsorbent at equilibrium, respectively; qm is the maximum adsorption capacity (mg/g adsorbent) and k is the adsorption constant (mL/mg). The experimental data was analyzed and the related results were shown in Fig. 6b. In this work the Langmuir equations for two adsorbents had the expressions as follows: Ce /qe = 0.19955 Ce + 0.07362, qm = 5.01 mg/g pure TiO2 , R2 = 0.9998, k = 2.71; Ce /qe = 0.11591 Ce + 0.04657, qm = 8.63 mg/g TD, R2 = 0.99405, k = 2.49. The high R2 values in the equations indicated that the Langmuir model was very fit to describe the adsorption behavior of PPPs on TiO2 and TD. The Langmuir constant k value from TiO2 was higher than that from TD, which indicated that TiO2 had a higher affinity force for PPPs than TD. This phenomenon might be due to some nonspecific adsorptions occurred on the porous surface of diatomite [33]. The maximum
(3)
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Fig. 7. Effects of repeated uses on the N/P of PPPs purified by TiO2 and TD (pH: 4.0; temperature: 25 ◦ C).
4. Conclusions
Fig. 6. Adsorption isotherms for PPPs adsorbed on TiO2 and TD (a). linear fit (b) (pH: 4.0; temperature: 25 ◦ C).
A TiO2 /diatomite composite was successfully prepared and applied in the enrichment of PPPs from egg yolk protein hydrolysis polypeptide for the first time. The optimized adsorption condition of PPPs was at pH 4.0. The N/P molar ratio of the purified PPPs was around 5.5. The comparison study of the adsorption properties of PPPs onto the pure TiO2 and TD showed that the adsorption models fitted well with the Langmuir model. And after modification, the adsorption capacity was improved. The preparation method of the composite was simple, cost-effective, and organic solvent-free. Meanwhile, with uniform and relatively large diameter, the composite could be separated and recovered from aqueous solutions easily by filtration. The stable N/P ratios of purified PPPs after ten repeated uses indicated the high stability of TD. It is anticipated that TD may have great potential to be used in large-scale purification of PPPs and enrichment of phosphopeptides from other complex biological samples. Acknowledgements
equilibrium adsorption capacities for both adsorbents were close to maximum adsorption capacities calculated from the previous pseudo-second-order kinetic models. 3.5. Stability of TD and TiO2 in repeated use In alkaline environment, TiO2 , TD and PPPs are both negatively charged, the electronic interactions between them are electrostatic repulsion. The PPPs can be efficiently eluted from TiO2 . The adsorbed PPPs were eluted by 0.01 M NaOH solution. Then the adsorbents were regenerated by 0.1 M NaOH. To evaluate the stability of TD and TiO2 , repeated adsorption–desorption experiments were conducted for ten times using the same batch of TD and TiO2 for PPPs purification (Fig. 7). After ten times uses, the N/P of eluted PPPs were still under 6.0, which indicated that TiO2 and TD were very stable, their affinity for PPPs did not decrease after repeated uses. And they both had strong resistance to acid and alkali solutions. This may because that TiO2 and diatomite both had acid and alkali resistance and stable physical and chemical properties. After modification and calcination, TiO2 and diatomite had been conjugated together to form a new stable composite materials which possess the advantages of these two materials, thus the prepared TD was very stable and could be used as affinity materials in the purification of PPPs.
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