Solid phase extractive preconcentration of silver from aqueous samples and antimicrobial properties of the clinoptilolite–Ag composite

Abstract

Sorptive properties of the Transcarpathian clinoptilolite compared to Ag(I) were studied in a dynamic way. The sorptive capacity value of clinoptilolite under the optimal conditions is 11.7 mg of Ag per 1 g of zeolite. The solid-phase extraction procedure with natural clinoptilolite thermally activated at 550℃ was used to preconcentrate trace amounts of silver ions in aqueous solutions to be finally determined by the atomic absorption method. It was established that the Ag–clinoptilolite composite has a powerful antimicrobial effect against gram-negative bacteria and yeasts as well as affects significantly vital activity of Staphylococcus aureus.

Introduction

Silver is present in natural waters in trace quantities. It belongs to the physiologically active components of mineral waters. Even at its low concentrations it exhibits therapeutic activity. In most cases application of modern physicochemical methods and newest instrumentation during the microelements determination in natural waters and technological solutions requires the pretreatment of samples which, in particular, includes preconcentration, withdrawal, and/or separation of elements. One of the most effective techniques is the solid-phase extraction (SPE) method with the application of various sorbents—polymeric resins (Ayata et al., 2009; Fisher and Kara, 2016; Tan et al., 2014; Tuzen et al., 2005), modified high dispersed silica (Fisher and Kara, 2016; Tan et al., 2014, Thabano et al., 2009; Zaporozhets et al., 2012; Zougagh et al., 2005), activated carbon (Daorattanachai et al., 2005; Yusof et al., 2007), synthetic zeolites (De Pena et al., 2000). Recently, the popularity of natural zeolites has increased (for SPE applications) (Al-Degs et al., 2008; Chen et al., 2009; Faghihian et al., 2009; Faghihian and Kabiri-Tadi, 2010; Fisher and Kara, 2016; Vasylechko et al, 2015, 1999b, 2001) because of a number of advantages over other sorbents. For example, these natural aluminosilicate minerals contain pores and cavities with strictly defined size and shape which results in a very effective preconcentration and separation of organic and inorganic compounds. In addition, zeolites are characterized by mechanical strength, good stability in the aggressive medium and under thermal treatment, ability to sorb the trace amounts of analytes, high sorption capacity and selectivity, easy modification and regeneration of the sorbent, low cost, and accessibility.

Several composites based on natural clinoptilolite from the deposits from different countries exhibit strong antimicrobial and antiviral activities which particularly refer to surfactant-modified clinoptilolite (Hrenovic et al., 2013; Jevtić et al., 2012), Cu–clinoptilolite (Hrenovic et al., 2013), as well as Ag–clinoptilolite (Concepción-Rosabal et al., 2005; Hrenovic et al., 2013; Lihareva et al., 2010; Top and Ülkii, 2004).

The aim of this paper was to study sorptive properties of Transcarpathian clinoptilolite with respect to trace amounts of Ag(I) in aqueous solutions to develop a method of Ag(I) preconcentration in the SPE mode for further determination using the atomic absorption method and also investigation of the antimicrobial activity of Ag–clinoptilolite composite samples.

Experimental

Clinoptilolite used in these investigations was obtained from the deposit near the village of Sokirnytsia in the Ukrainian Transcarpathian region. The previous analysis showed that the amount of the component was 85–90% and the specific surface area determined by water sorption was 59 m²g⁻¹ (Vasylechko et al., 1999a). The chemical composite of Transcarpathian clinoptilolite is (in %): SiO₂, 67.29; TiO₂, 0.26; Al₂O₃, 12.32; Fe₂O₃, 1.26; FeO, 0.25; MgO, 0.99; CaO, 3.01; Na₂O, 0.66; K₂O, 2.76; H₂O, 10.90 (Tarasevich et al., 1991).

The zeolite samples were ground in a ball mill, the grain fraction of 0.20–0.31 mm size was selected, washed with distilled water and dried at room temperature. The thermal treatment of clinoptilolite was conducted at a required temperature in the oven for 2.5 h.

The sorption properties of clinoptilolite were studied under dynamic conditions in a SPE mode. Metal solutions were passed through a sorption cartridge filled with 0.6 g of the sorbent at 5 mlmin⁻¹ velocity using the peristaltic pump. The investigation techniques under dynamic conditions are described in detail in the paper (Vasylechko et al., 1996). The passage moment of Ag(I) ions was determined by spectrophotometry with Sulfarsazene. The absorbance of the complex solution was measured at 540 nm (DR/400 V spectrophotometer HACH) against the solutions which contained all components, except Ag(I). This method is highly sensitive method for Ag(I) determination (LDL = 100 ng ml⁻¹). For Ag(I) desorption from the zeolite bed, 25 ml of the eluent was passed through the sorption cartridge at a flow rate of 0.5 ml min⁻¹. The Ag(I) content in the eluates was determined by the flame version (propane–butane–air) of atomic absorption spectrophotometry using an AAS-1N atomic absorption spectrophotometer (Carl Zeiss, Jena) at λ = 328.1 nm. The adsorption and desorption studies were carried out at a temperature of 20 ± 1℃.

The strains of gram-negative bacteria Escherichia coli ATCC 25922, gram-positive bacteria Staphylococcus aureus ATCC 25923, Bacillus subtilis ATCC 31324, yeasts Saccharomyces cerevisiae D 67.5, and filamentous fungus Aspergillus niger IMB16706, obtained from the Microbial culture collection of Ivan Franko National University of Lviv were used as the reference cultures to determine the antimicrobial properties of the fine dispersed Ag–clinoptilolite. The express test for determination of antimicrobial properties of Ag–zeolite was carried out according to the below technique: 100 µl of cells suspension of each test culture at the concentration of 106 cells ml⁻¹ were sown on a L-agar (Kieser et al., 2000) in the case of bacteria or Sabouraud medium (g l⁻¹: dextrose—40, peptone—10, agar—20; pH 5.6) in the case of A. niger. The weighed mass of the investigated samples (0.01 g) was put on the agar plates sown with the test cultures. The cultures were grown at 37℃ for 24 h (bacteria) or at 25℃ for 48 h (fungi). The detection was made visually by indicating the growth inhibition zones of the test cultures.

The quantitative determination of the antimicrobial activity of Ag–clinoptilolite was carried out according to the below method. The weighted amounts of fine dispersed zeolite (0.1, 0.5, and 1.0 mg), Ag–clinoptilolite (0.1, 0.5, and 1.0 mg), or metallic silver (0.21 and 1.0 µg) were put into the microtubes with 0.9 ml of sterile distilled water. The silver mass of 0.21 µg corresponded to its quantity in 1 mg of clinoptilolite–Ag composite. After that 0.1 ml of E. coli (titer 1.5 · 10¹² cells ml⁻¹), S. aureus (titer 1.1 · 10¹⁰ cells ml⁻¹), or S. cerevisiae (titer 2.0 · 10¹⁰ cells ml⁻¹) cells suspension was put into the tubes. The aqueous cell suspensions of test cultures without Ag–clinoptilolite were used as the reference. The mixtures were shaken for 2 min and seeded with dilution (10⁻⁵–10⁻⁹) in the respective media mentioned above. The number of colonies that survived was counted after 24 h. Fine dispersive samples (particles size < 1 µm) were used for the investigations.

All used reagents were of analytical grade. The 0.05 % solution of Sulfarsazene was prepared using 0.05 М borax and other reagent solutions using bidistilled water. The standard solution of Ag(I) (1.0 mg ml⁻¹) was obtained via dissolving of AgNO₃ and the content of Ag(I) was determined titrimetrically using the Moore’s technique.

Results and discussion

Sorption of Ag(I) on clinoptilolite was studied depending on the medium acidity. Ag(I) is the most effectively sorbed from the slightly alkaline solutions. The distinct maximum at pH 8.0 can be observed on the curve showing the dependence of sorption efficiency on the acidity, although the satisfactory sorption is obtained in the weakly acidic and neutral solutions in a pH range of 4–7 (Figure 1). Such way of the sorption process is caused by the chemical features of the zeolite surface, as well as by the forms of Ag(I) existence in aqueous solutions at different pH values. At low pH values the dissociation of OH groups, that are sorption active on the surface, is almost completely suppressed which is the reason for a small value of sorption capacity of clinoptilolite with respect to Ag(I). On changing the pH value one form of Ag(I) is transformed into another. According to Baes and Mesmer (1976) at low concentrations (∼10⁻⁵ M) in the weakly alkaline solutions Ag(I) mainly exists in the Ag(Н₂О)₂⁺ form and partly as the soluble form [Ag(OH)(H₂O)]⁰. This indicates that Ag(I) is sorbed on clinoptilolite mainly according to the ion-exchange mechanism. In strongly alkaline solutions, the anionic hydroxocomplex [Ag(OH)₂)]⁻ is partly formed and does not sorb on the zeolite.

Figure 1.

The clinoptilolite sorption capacity of silver (I) as function of pH solutions (□) and thermal pretreatment of sorbents (•) (concentration of Ag(I) = 1.0 µg ml⁻¹; pH 8.0; flow rate—5 ml min⁻¹; time of heat treatment—2.5 h).

Sorptive properties of clinoptilolite regarding Ag(I) depend on the temperature of its thermal pretreatment in a complicated way (Figure 1). The zeolite samples calcined at 550℃ exhibit the maximal sorption efficiency. Withdrawal of ligand (cluster) water and isolation of the water molecules from the surface of Transcarpathian clinoptilolite is likely to enhance Ag(I) sorption that takes place at the temperatures >180℃ (Vasylechko et al., 1999a). Some decrease in sorption capacity of clinoptilolite calcined at the temperatures >550℃ could be caused by the surface dehydroxylation. As a result, the sorption efficiency of Ag(I) with the zeolite samples treated at 700℃ increases. This could be facilitated by the formation of siloxane bonds found in the place of tetrahedral vacancies in the clinoptilolite structure at such high temperatures (Tomazović et al., 1996). It is known (Vasylechko et al., 2003) that both Si–O–Si group and the surface ОН groups are the functional ones interacting with heavy metal ions. At 800℃ Transcarpathian clinoptilolite is almost completely amorphized (Vasylechko et al., 2003), so it does not sorb Ag(I) (Figure 1). The sorption capacity of clinoptilolite under the optimal conditions reaches the value of 11.7 mg Ag g⁻¹.

The quantitative (100%) elution of Ag from the zeolite surface was reached using the solutions of 3.5 M HNO₃, 0.5 M NaNO₃, and 0.5 M KNO₃. The fact that the neutral solutions of NaNO₃ and KNO₃ provide the full desorption of silver additionally proves the ion-exchange mechanism of Ag(I) sorption on the clinoptilolite.

It was established that the (10–100)-fold excess of common cations (Na⁺, K⁺, Ca²⁺, NH₄⁺, Zn²⁺, Mg²⁺) of water does not affect the adsorption capacity of clinoptilolite with respect to Ag(I).

The SPE technique for Ag(I) preconcentration before its FAAS determination in water was proposed.

Sample preconcentration procedure

The sorbent was ground in a ball mill to a 0.20–0.31 mm size and washed with distilled water. After drying at room temperature, the clinoptilolite sample was heated in the oven at 550℃ for 2.5 h and then stored in a desiccator. The diluted solutions of NaOH or HNO₃ were added to the analyzed water up to the рН value of 8.0. Then 0.5–2.5 l of the obtained solution was passed through the cartridge containing 0.6 g of the sorbent using the peristaltic pump with a flow rate of 5 ml min⁻¹. After the sample loading, the cartridge was washed with 50 ml of bidistilled water at the same flow rate. Whereas Ag(I) ions were desorbed with 25 ml of a 3.5 M nitric acid solution at a flow rate 0.5mlmin⁻¹. Silver content in the solution was determined by the atomic absorption method as described in the experimental part. In general, the proposed method for determination of Ag(I) ions had a linearity range from 1.5 to 200 ng ml⁻¹. The detection limit was found to be 0.45 ng ml⁻¹. The method was tested using tap water. As can be seen from Table 1, the components of water do not have a considerable effect on the determination of trace amounts of silver (I) ions. The Ag recovery from spiked tap water was above 96%.

Table 1.

The results of FAAS determination of silver (I) in the tap water after the solid phase preconcentration using clinoptilolite (n = 3; P = 0.95).

Volume of water sample (ml)	Enrichment factor a	Concentration of Ag(I) (ng ml−1)		Recovery (%)	RSD (%)
		Added	Found
500	20	100	97 ± 1.8	97	0.75
2000	80	10	9.7 ± 0.25	97	1.0
2500	100	2.5	2.4 ± 0.17	96	2.9
2500	100	0	ND b

RSD: relative standard deviation.

a

Enrichment factor = volume of sample/volume of eluent.

b

ND < detection limit.

Antimicrobial properties of the clinoptilolite composites with silver

During the express test, the growth inhibition zones have been found around the clinoptilolite crystals with the adsorbed Ag(I) ions for most of the test cultures except S. cerevisiae. The largest growth inhibition zone was observed in the case of B. subtilis (Figure 2(c)), on the other hand, the Ag–clinoptilolite inhibited the growth of A. niger with the lowest efficiency (Figure 2(e)). At the same time neither the fine dispersed clinoptilolite nor silver created growth inhibition zones of the test cultures.

Figure 2.

Results of the express test for the determination of antimicrobial properties of the clinoptilolite (left) and Ag–clinoptilolite (right) connected with the growth of E. coli ATCC 25922 (a), S. aureus ATCC 25923 (b), B. subtilis ATCC 31324 (c), S. cerevisiae D 67.5 (d), and A. niger IMB16706 (e).

The dependence of E. coli, S. aureus, and S. cerevisiae survival on the concentration of the clinoptilolite, silver, and Ag–clinoptilolite composite was studied. It was proved that metallic silver at the concentration corresponding to one particle of silver in a 1 mg of Ag–clinoptilolite composite (0.2 µg ml⁻¹) eradicates 82.0% of S. aureus cells, 96.3% of E. coli, and 99.4 % of S. cerevisiae (Figure 3). The increase of silver concentration 100-fold enhanced the reduction of the bacterial test culture cells negligibly. The silver composite with the clinoptilolite at the concentration of 0.1 mg ml⁻¹ (containing 0.02 µg of silver) eradicated 87.3% of S. aureus cells, 91.3% of E. coli, and 99.9% of S. cerevisiae (Figure 4). Such reduction of the bacterial cells rate of the test cultures is similar to the influence of 0.2 µg ml⁻¹ metallic silver. The increase of Ag–clinoptilolite concentration to a value of 0.5 mgml⁻¹ decreased the survival of bacteria for another several percent and caused significant decline in yeasts survival to a value of 1.5 × 10⁻⁵% of all cells.

Figure 3.

Influence of the fine dispersed silver on the survival of E. coli, S. aureus, and S. cerevisiae. For axis of abscissas—concentration, mg ml⁻¹; for axis of ordinates—lg% of survival.

Figure 4.

Dependence of сell survival of strains E. coli (a), S. aureus 25923 (b), and S. cerevisiae (c) on the concentration of clinoptilolite and clinoptilolite–Ag. For axis of abscissas—concentration, mg ml⁻¹; for the axis of ordinates—lg% of survival.

On applying 1 mgml⁻¹ of Ag–clinoptilolite, the survival of E. coli decreased to 3.5 × 10⁻⁵% of cells, yeasts died completely, and as for S. aureus only 3.6% of cells remained viable. Clinoptilolite itself decreased the survival of bacteria less efficiently (only 10%), whereas 96–98% of yeasts died at the clinoptilolite concentration equal to 0.5 and 1.0 µg ml⁻¹.

The obtained results indicate that the composite of the clinoptilolite with adsorbed Ag(I) has a powerful antimicrobial effect regarding gram-negative bacteria and yeasts. It has also significant influence on the viability of S. aureus. Moreover, metallic silver inhibits the viability of E. coli, S. cerevisiae, and S. aureus less efficiently. Clinoptilolite itself is characterized by a poor antibacterial activity. This is obviously associated with the structural features of the cell wall of investigated test cultures.

Conclusions

The sorption properties of Ukrainian Transcarpathian clinoptilolite regarding the aqueous solution of silver (I) ions using the dynamic technique were investigated. It was established that Ag(I) is recovered with the highest efficiency from the slightly alkaline solutions mainly according to the ion-exchange mechanism. In general, the optimal conditions for silver sorption on Transcarpathian clinoptilolite are as follows: temperature of the preliminary thermal treatment of 550℃, Ag(I) solution flow rate of 5 ml min⁻¹, zeolite grains size of 0.20–0.31 mm, pH 8.0. The maximal adsorption capacity of Transcarpathian clinoptilolite with respect to Ag(I) ions of 11.7 mgg⁻¹ can be achieved in this case.

It was established that the (10–100)-fold excess of common water cations does not affect the adsorption capacity of clinoptilolite with respect to Ag(I).

3.5 M HNO₃ solutions were found to be the best eluents of silver from clinoptilolite. The developed SPE technique offers a possibility of silver(I) trace amounts preconcentration in the presence of matrices components of water before FAAS determination. An enrichment factor of 100 was obtained under the optimal conditions. A wide range of linearity (1.5–200 ng ml⁻¹) with the detection limit of 0.45 ng ml⁻¹ was achieved. The developed procedure was applied for the determination of silver (I) ions in the tap water, whereas the recoveries and RSD values were 96–97 and 0.75–2.9%, respectively.

It was determined that the Ag–clinoptilolite composite has a powerful antimicrobial effect regarding gram-negative bacteria and yeasts as well as great influence on the viability of S. aureus. The clinoptilolite itself is characterized by poor antibacterial activity.