These web pages are devoted to the Unity through Knowledge Fund (UKF) project approved within the call for 2A proposals “Gaining Experience Grant” under the Conectivity Program in 2010.

TITLE OF THE PROJECT:

“Nanosilver particles versus ionic silver – development of a method for differentiation and quantification“

PROJECT DURATION:

5 months

From 19th April 2010 until 20th September 2010

PROJECT LEADER (MAIN APPLICANT):

Ivana Vinković Vrček, Ph.D., Research Associate

Institute for Medical Research and Occupational Health, Zagreb, Croatia

PROJECT CO-APPLICANT:

Walter Gössler, Ao.Univ.-Prof. Mag. Dr.

Institute of Chemistry – Analytical Chemistry, Karl-Franzens University, Graz, Austria

FUNDING:
Unity Through Knowledge Fund

ADMINISTERING INSTITUTION:
Institute for Medical Research and Occupational Health, Zagreb, Croatia

SUMMARY OF THE PROJECT:

Research and development in the area of new nanomaterials has been given high priority as having an enormous economic potential. The metallic silver nanoparticles are most promising and can be exploited in medicine for burn treatment, dental materials, coating stainless steel materials, textile fabrics, water treatment, sunscreen lotions, etc. Experience from other contaminants and their release into the environment leads to the need for more information and a discussion of technology development from a preventative point of view. The size of a nanoparticle and its differentiation from ionic metal form are key parameters to be determined when attempting to predict the fate, and potential toxicological impact, of engineered nanoparticles released into the environment, which in turn requires robust and sensitive analytical methods. Therefore, the aim of this project is focused on development of spectrometry based method for separation of silver nanoparticles from ionic silver in different biological matrices. In addition, the methods developed within this project will be validated and analytical figures of merit will be determined to evaluate the suitability for routine applications.

OBJECTIVES, SIGNIFICANCE AND CONTRIBUTED ADDED VALUE OF THE PROJECT:

Metallic silver has been in use since ancient times. Upon reaching nanoscale, like other nanomaterials and primarily by virtue of extremely small size, AgNPs exhibit remarkably unusual physicochemical properties and biological activities. The size of a particle and its differentiation from ionic metal form are key parameters to be determined when attempting to predict the fate, and potential toxicological impact, of silver NPs released into the environment. It is important to measure the NPs in the relevant matrix, as properties of NPs may depend on the surrounding matrix and be affected by processing. Analytical techniques based on mass spectrometry (MS) can provide both elemental and molecular information. Their exceptional analytical features could be of great advantage for the analytical characterization at the nanoscale. Recently, ICP-MS has been demonstrated to be a highly valuable tool for ultrasensitive detection and characterization of metalloid-containing NPs. Size distribution analysis of NPs in polydispersed samples could be achieved with different separation techniques. Liquid chromatography can yield quantitative information about shape and nanocrystal size distributions. Such information, added to its low cost, fast operation and reliability, make this separation strategy an attractive option for standardizing nanomaterials directly in the solution state. Coupling of liquid chromatography and ICP-MS for NP analysis has been proposed in innovative bioanalytical applications. Therefore, the aim of this project is focused on development of chromatographic technique interfaced to a multi-element detector (ICPMS) to enable investigations into the behavior and fate of a range of inorganic silver NP's in ‘real-world’ situations. In addition, the methods developed within this work will be validated and analytical figures of merit will be determined to evaluate the suitability for routine applications.

RESULTS OF THE PROJECT:

Development of a chromatographic separation of ionic silver and silver nanoparticles (AgNP) with different particle sizes with direct inductively coupled plasma mass spectroscopy (ICPMS) detection was our main objective.. We started experiments with ionic silver standards and home-made AgNPs. AgNPs were prepared from a standard reduction of the silver salt in sodium citrate. First, we tested the possibility of direct determination of AgNPs in colloid solutions by ICP-MS, without previous digestion/dissolution, avoiding the use of hazardous reagents. The home-made AgNP samples were diluted in three different ways before ICP-MS measurements: (a) in deionised water, (b) in 1% (v/v) trisodium citrate and (c) in 2% (v/v) HNO₃. We have demonstrated that ICP-MS is a sensitive and accurate technique for the direct determination of silver nanoparticles in aqueous colloid solutions. However, our results showed that the sample matrix is the main factor influencing the accuracy and precision of AgNPs determination.

Starting with chromatographic separation experiments we faced with two major problems: severe adsorption of ionic silver or AgNPs on stationary phases and chromatographic system limitations according to the acidity of mobile phase. After initial experiments it became immediately apparent that the silver in both form, ionic and nano, has strong interaction with stationary phase matrix. We conducted experiments with three different types of chromatography systems: (a) ion exchange chromatography (IEC), (b) reversed phase chromatography (RPC) and (c) size exclusion chromatography (SEC). In each experiment, we observed severe adsorption of ionic silver or silver particles on stationary phase. However, we have faced with adsorption of ionic silver on chromatographic devices in flow-injection experiments as well. The loss of trace amounts of silver ions on different container walls has been recognized for some time. Such adsorption appears to be associated with many factors. During the first two project months, we greatly enhanced our understanding of ionic silver nature, what features need to be taken into the account when choosing the appropriate mobile phase, and which can be left out. Thus, our main observation was that the chromatography of silver requires aggressive elution condition. The main point of our interest was whether the use of concentrated acids or strong complexing agents would lead to quantitative elution along with reasonable separation of ionic silver from the AgNPs. Eluents developed for classical ion exchange chromatography were not really effective in elution of silver ions. In contrast, the few successful high performance elutions of silver ions by using strong acids like HCl or HNO₃ did not separate Ag ions from Ag nanoparticles. We compared the effects of several different mobile-phase additives, sodium citrate, sodium chloride, pyridine, sodium dodecyl sulfate (SDS), thiocynate, thiourea… The effects of mixed surfactant on the elution and separation of AgNPs from ionic silver were investigated. Different compositions of mobile phase were employed to investigate the adsorption problems. After initial studies it became immediately apparent that the chromatography of silver was dramatically improved by addition of SDS which was absolutely necessary; otherwise the analytes could not be eluted from the column due to their irreversible adsorption on the packing material. The peak shapes were nearly symmetrical and increasing the retention time produced the normal increase in peak broadening. It is possible that the interaction between nanoparticles and surfactant induces the alignment of surfactant around silver particles. Although SDS can solve the adsorption problem of packing material, the separation resolution was still not high enough to resolve ionic silver from AgNPs.

In contrast, the few successful HPLC separations of silver have been based on the ion-interaction chromatography of very stable complexes such as the Ag(CN)*- species

using silica phases. However, we did not want to administer such hazardous material. We tried to solve our problems by complexing silver with thiocynate, thiourea and cysteine. In each cases, the elution of silver ions was improved.

With the addition of pyridine and isopropanol in acidic mobile phase containing SDS, we obtained the reasonable separation of ionic Ag from AgNPs. However, after one week of employing such mobile phase in our LC-ICPMS system, we observed severe dissolving of our Ni skimmer cone, the interface through which the ions pass and which maintains the high vacuum in the mass spectrometer region. Its small orifice seems to be very sensitive to such combination of mobile phase additives. This raises costs of such analysis.

We are preparing scientific publication based on our results and findings. Owing to these preliminary results a further development and rigorous study of the LC-ICPMS procedure are undergoing in our laboratories.

FUTURE RESEARCH TASKS AND ACTIVITIES:

Further studies are required for determining the exact interaction mechanism between surfactant and silver particles. Although, our results are less than ideal, it has to be stressed that this particular area of inorganic speciation analysis is at very formative stage. The quantitative aspect of our methodology is still to be fully addressed.

Important data gaps include knowledge of stability of different types of nanosilver on long timescales, effects of water chemistry on reactivity and bioavailability, as well as the likelihood and nature of associations with natural particulates. Our further experiments are directed toward understanding the size, dispersion and aggregation of AgNPs as well as sophisticated equilibria between silver ion and silver nanoparticles under analytical conditions of pH, major ion concentration and surfactant concentration.

Understanding their size, dispersion and aggregation under environmentally conditions of pH, major ion concentration and HS concentration is essential in trying to determine their fate and behaviour using laboratory experiments. In addition, are likely to affect the behaviour and effects of AgNPs on the separation process.

Further studies are underway involving study of properties such as the dynamics of dispersion, rate of dissolution, characteristics of the silver aggregates, surface area and surface characteristics. Ongoing work focuses also on the improvement of the column in terms of peak resolution, quantitative and particle species analysis (ionic vs. particle form). The problem of applying LC methodology is the irreversible adsorption of the particles by column packing material, due to the high surface area of stationary phase and high surface activity of nanoparticles. This problem limits the types of columns that can be used for the separation of nanoparticles.

We plan to introduce complementary methods to confirm and/or explain the results obtained so far. So further investigations are necessary to prove reliability of the HPLC method for seperation of AgNPs from ionic silver.