INTRODUCTION

Recent studies have shown a rise in the level of heavy metal in the human body and the everyday environment. These finding have increased the importance of heavy metal analysis which have become a major task in areas such as toxicological, epidemiological and environmental research. Trace metal analysis has a wide range of applications these include medical diagnostics, medical device implantation, and pharmaceutical and food analysis etc. In terms of environmental research, pollutants or just the general level of trace heavy metal may be quantified. In a society in which the effects climate change becomes more apparent it is important to be able to monitor and thereby control environmental pollution so as to find way to improve it as well as maintain of ecological balance.

There are many well-established analytical methods that are available for detecting, measuring, and monitoring heavy metals. Many of the analytical methods are used for detection in environmental samples .Detection in environmental samples is different to that of laboratory samples in that concentration of element in environmental sample may fluctuate with certain samples having very lower concentrations. Subsequently the need for improvement of analysis technique that are able to detect at lower detection limits is continually sort after so as to obtain more precise results.

1. Trace metal analysis techniques

Types of detection method

Atomic absorption spectrometry (AAS): This method of analysis determines the elemental composition of a sample via the absorption of light to measure the concentration of gas-phase atoms. It uses lamps whose core is made of the element. The light source is a lamp with a core made of the analyte of interest, this gives off light that is associated with the element and the sample absorbs this light. The analyte atoms in the sample are vaporized in a furnace and transition to higher electronic energy levels when light is absorbed. The concentration of the specific analyte is dependent on the amount of light absorption. Concentration measurements are determined by a working curve after calibrating the instrument with standards of known concentration. AAS is highly specific therefore each element has to be tested separately, this can be a disadvantage when trying to analyse a mixture, as different lamps must be used when measuring different elements. Another disadvantage to this technique is that the samples must be in solution, or at least volatile in order to be measured.

There are a large number of factors that may interfere with measurement these include formation of non-volatile compounds and smoke formation which will absorb light, giving this method a relatively low level precision in comparison to other methods.

Voltammetry: This studies the behaviour of analytes via the measurement of the potential and current of a sample or cell containing the analyte. There are different categories of voltammetry these include Polarography voltammetry – This process uses DME to produce reproducible effective electrode as a function of time. Linear sweep voltammetry- This process enables pre and post electron transfer reactions to be observed. The potential is linearly increased and the potential range is scanned between the initial potential and final potential Cyclic voltammetry- This is an extension of the linear sweep analysis where by the voltage scan is reversed once maximum current is reach as the as reduction of the analyte would have completed. Anodic stripping voltammetry- determines the specific ionic species by deposition of the analyte and stripping resulting in a redox reaction. The oxidised species registers as a peak Differential pulse voltammetry- Potentiometry: based on measurement of potential of an electrode system consisting of two electrodes, potentiometer and sample containing analyte of interest. This method is advantageous as it can detect ions in presence of whole host of other Substances.

The desire for more accurate result had major challenges as developers had to development new analytical methods and improvement of existing methods. In particular, the introduction of powerful modes of atomic absorption spectrometry such as the graphite furnace other newly established analysis techniques include neutron activation

Graphite furnace atomic absorption spectrometry (GFAAS): Atoms absorb light at particular frequencies depending on the characteristic of the element. The amount of light absorbed may be linearly correlated to the concentration of the analyte and can thereby be quantified. Samples are deposited into small graphite tube which is subsequently heated to vaporize and atomize the analyte. Neutron Activation: A sample is subjected to a neutron flux and radioactive nuclides are produced. As these radioactive nuclides decay, they emit gamma rays whose energies are characteristic for each nuclide. Comparison of the intensity of these gamma rays with those emitted by a standard permit a quantitative measure of the concentrations of the various nuclides.

1.1 Back ground and history of mercury

The dropping mercury electrode was invented in 1922 by Jaroslav Heyrovsky and was the foundation of electroanalytical voltammetry techniques via the development of the first linear sweep voltammetry method of polarography. In the year following between 1947 and 1959 voltammetry at stationary were developed.

Electrolysis is the process by which ionic substances are decomposed (broken down) into simpler substances when an electric current is passed through them. In order for electrolysis to occur mass transfer is required between the electrode .Michael Faraday’s Second Law of Electrolysis was established in 1934 and states that “If the same quantity of electricity (electric charge) is passed through different electrolytes, the mass of a substance librated or deposited altered at an electrode is directly proportional to their chemical equivalents”. This law recognized the quantitative relationshipbetween current and equivalents of elements. The ficks law of diffusion formulae verified this quantitative relationship which enabled

In recent years, mercury electrodes were still widely used in the detection of heavy metals by anodic stripping analysis due to its unique features. Heavy metal ions are reversibly reduced to form amalgams with the thin film of mercury on the electrode surface. The mercury film electrode is formed by electrodeposition onto an electrode which is subsequent stripping of these metals allows the quantification of these metal ions.

Mercury is an important electrode in terms of cathodic process study. Due to its low boiling point mercury as an electrode can be used in many forms these include dropping, streaming and pool configuration.

1.2.1 Types of mercury electrodes

There are different types of mercury electrode these are as follows –

Dropping mercury electrode: This working electrode is usually associated with the voltammetric technique polarography used for environmental analysis, especially for marine study.DME is made from 10-20 cm of glassy capillary tubes with internal diameter of 0.05mm. These dimensions and specification is chose to increase efficiency of the electrode. For example the length of the capillary tubes provides enough static head space to provide a drop time of 3-10 s. There are a number of variations of DME. The vertical orifice capillary has a capillary bent at 90 degrees and removes effect of maxim and depletion where as the Teflon DME is made specifically for use in solution. The reproducibility and vast knowledge about literature make this one of the best electrode for use in electrochemical analysis. Mercury film electrode: a mercury film is formed on an electrode substrate for example glassy carbon, carbon paste and pencil-lead etc. During voltammetric analysis mercury film forms an amalgam with the analyte of interest, which upon oxidation results in a sharp peak, improving resolution between analytes. Hanging mercury drop electrode (HMDE): this is very similar to the DME however HDME produces partial mercury drop of controlled geometry and surface area at the end of a capillary where as DME steadily releases drops of mercury during an experiments. HDME is used for voltammetric techniques requiring stationary electrodes. Mercury-plated electrodes: mercury is electroplated onto a solid electrode. Mercury forms a film or an assembly of micro droplets depending on the substrate on which it is plated.

Electrode substrates

Types of electrode substrate

Glassy carbon electrodes: Produced by placing a thick sheet of glassy carbon into a glassy tube with epoxy cement. The surface of the electrode is then polished until it becomes smooth. Carbon paste electrodes: made by the mixture of graphite and nujol until it develops the consistency of a paste. Carbon cloth electrode: carbon cloth have voltage ranges similar to that of carbon electrode The process of formation is by heating woven hydrocarbon polymer fabric to high temperatures. Platinum electrodes: This involves the use of a fine powder known as platinum black placed on solid platinum. Gold electrode: With the ability to be made into different forms i.e. rods, disc etc it is one of the most commonly used electrodes. Indium tin oxide electrode: transparent electrode generally used for Spectra electrochemical measurement

In this case glassy carbon will be used as our electrode substrate, this is because of the many advantage glassy carbon has as an electrode substrate. Glassy carbon has good conductivity, low electrical resistance, thermal expansion coefficient is small, hard texture, good gas tightness, a wide scope of application of electric potential (from about -1 ~ 1V), chemical stability, can be made of cylindrical, disc, etc. These properties make it ideal for use in voltammetric analysis as it allow the flow of current in a controlled way and facilitate the transfer of charge to and from the analyte electrode shape, use it as a matrix can also be made of mercury film glassy carbon electrode and chemically modified electrodes. In the electrochemical experiments or electroanalytical chemistry has been increasing wide range of applications.

Advantages and disadvantages of mercury electrodes

The mercury film forms Mercury as an electrode is advantageous for many reasons these include its ability to release the contaminated drop and grow a clean drop between each experiment unlike solid electrodes which are required to cleaned and undergo a polishing step in order to prevent contamination of the electrode. This also means the electrode is independent of its past history. Metal ions can be deposited from acidic solutions thought thermodynamic state this is impossible without the formation of hydrogen which causes overvoltage which is associated with the reduction of hydrogen ions. Mercury however has a few limitations, one of which is its ease in oxidation, this causes a limitation in the range of anodic potentials used. When dealing with potential greater than + 0.4 V mercury (I) is formed causing a wave that masks the curve of other oxidized species. This occurs at lower potentials in the presence of precipitating ion or mercury (I) complexes. However the single greatest danger mercury poses as an electrode is its high level of toxicity. Mercury as a compound must be handled carefully, since its toxicity has very serious implications for the health and the environment.

Mercury has a major impact on the environment due to its ability to progressively build up in successive trophic levels as well as along the food chain by a process of biomagnification. Over time this build up migrate across both biotic (other organisms) and abiotic (soil, air, and water) sources.

At the top levels of the aquatic food web are fish-eating species, such as humans, seabirds, seals etc .In a study performed by the environmental protection agency US of fur-bearing animals in Wisconsin, the species with the highest tissue levels of mercury were otter and mink, which are top mammalian predators in the aquatic food chain. Top avian predators of aquatic food chains include osprey and bald eagle. Thus, mercury is shown transferred and accumulated through several food web levels. Aquatic food webs tend to have more levels than terrestrial webs, where wildlife predators rarely feed on each other, and therefore the aquatic biomagnification typically reaches higher values .In terms of health hazard mercury exposure may result in severe neurological effects, symptoms include convulsions, fits, and highly erratic movements. This was seen in Minamata, Japan, from about 1950-1952 where birds experienced severe difficulties in flying as well as domestic animals, especially cats whose diets were high in seafood exhibiting abnormal behaviour. This exposure was caused by the release of methyl mercury in the industrial wastewater from the Chisso Corporation’s chemical factory, which continued from 1932 to 1968.

Chemically modified electrodes

The ability of an electrode can be increased by means of chemical modification through the addition of atoms, molecules or nano particles to the surface of the bare electrode increasing its functionality over a wide range. Due to the many disadvantages mercury poses as an electrode, potential replacements for mercury are continually sought. The introduction of bismuth as an alternative has many significant advantages. It has been demonstrated that Bi is a good alternative since it is less toxic and easy to handle. The abilities of bismuth as an electrode are comparable to that of mercury and are attributed Thin Bi film is thus a good substitute candidate for the detection of heavy metals. Bi will be deposited onto the surface of a glassy carbon electrode (GCE) using electrodeposition thereby causing heavy metal ions to form an amalgam with Bi. ASV will be used to determine the concentration of heavy metals.

The method by which bismuth is coated on to a electrode plays a huge part in its potential as an electrode. There three main methods by which bismuth may be coated onto an electrode.

Ex-situ plating: This process involves the bismuth ions in a solution being moved toward the working electrode which in this case is glassy carbon, by an electric field form by passing a current through the electrode there by forming a bismuth film on the electrode. The coated electrode can then be transferred into a sample containing the analyte to be quantified. For the best formation of the film bismuth must be pre-plated in a low ph condition this is because bismuth has a tendency to hydrolyse when in alkaline conditions dues to the presence of water molecule splitting. Acidic conditions catalyse this polymer degradation.

Figure The molecular structure of bismuth nitrate pent hydrate

In-situ plating:

In this process the bismuth film is coats the electrode while the analysis process take s place. It should be noted however in order to do this the concentration of bismuth must exceed that of the analyte of interest. This is because of the possibility of interference due to saturation effects. The method of coating the electrode is superior in that it reduces experimental time as coating and analysis is done simultaneously however this method can only be performed by anodic stripping as it requires a preconcentration step. In this sense this method is not as versatile as ex-situ plating which can use any analysis method to analyse sample.

Bismuth pre-cursor:

A bismuth precursor can be used to modify an electrode if potential of about -0.1 V is applied onto an electrode resulting in the formation of metallic bismuth.

Bi2O3(s) +3H2O+6e–2Bi(s) + 6OH–

Fig The reduction reaction that takes place at the electrode between the electrode and bismuth precursor

This method is usually prolific with carbon paste electrodes as it eliminates the need to use anodic stripping to form the film as well as a preplating step before for analysis of sample can take place.

Bismuth modified electrode are physically and chemically stable therefore it retains its useful properties in the presence of air, moisture or heat, and under the expected conditions of application.Thus facilitate numerous measurement in different solutions without destroying the film once the electrode is coated with bismuth. This stability can be achieved by the addition of bromide ions (via sodium bromide) to the bismuth solution during the preplating stage of modification.

The functionalization of conducting substrates is widely used in electroanalysis in order to confer both selectivity and sensitivity. Ion-exchange membranes are interesting materials for this purpose. Nafion will be drop casted on GCE and then positively charged ions will be incorporated within the negatively charged Nafion film. The application of suitable reduction potential allows the ions to be reduced to their metallic states. ASV will then be employed for the stripping and quantification of these ions. Cu, Zn, and Pb will be the heavy metals ions that will be examined.

– chemically modified electrodes (plenty of paper in literature), then Nafion (its uses, applications and so on…)

Electrochemistry

HERE YOU CAM IMPROVE. PLEASE DESCRIBE CYCLIC VOLTAMMETRY AND WHY WE USED differential pulse voltammetry for the quantification of heavy metals

Electrochemistry is associated with the analysis of chemical reactions in a solution at the boundary of an electron conductor and ionic conductors where transfer of electrons between the electrode and electrolyte take place. This electron transfer is a mechanistic description of the thermodynamic concept of redox, in which the oxidation states of both reaction partners.

In general, electrochemistry deals with situations where cations transfer across from a solution –electrode interface via reaction with electrons within the interface. Anions are also transformed within this interface to produce electron, this is all through a series of oxidation and reduction reactions resulting in electrons moving from the anode connected by an external electric circuit to the cathode as a current which is carried by the ions of the supporting electrolyte. The solubility and stability of sample determines which solvent system should be used. Once this is determined compatible supporting electrolyte can be used without causing interference with the oxidation-reduction reaction.

Cyclic voltammetry

It enables the electrode potential to be rapidly scanned in search of redox couples. Once located, a couple can then be characterized from the potentials of peaks on the cyclic voltammogram and from changes caused by variation of the scan rate.

Differential pulse voltammetry

Electrochemistry has found extensive applications for the study of chemical reactions.

Anodic stripping voltammetry

This method of analysis which quantitatively determines the specific ionic species; the analyte of interest is electroplated on the working electrode through a deposition step. During a stripping stage the analyte is oxidised and the current is measured. The oxidation of elemental species is registered as peaks in the current signal at the potential at which the species is being oxidised. The stripping may either be linear, square, wave etc in the case of this experiment it is pulse and cyclic.

Four steps of anodic stripping

Cleaning: the potential is held at a oxidize state greater than that of the analyte for a period of time to remove it from the electrode. Potential held at a lower potential: The potential of the system is held sufficiently low enough to reduce the analyte and deposit it on the electrode. Deposited material spread evenly on electrode: If solid inert electrode is used this step is not needed. Working electrode is raised to a higher potential and stripping (oxidization) of the analyte as analyte is oxidised it emits electrons which are measured as current.

– Reference electrode is an electrode with potential which is a) independentof analyte (or other) ions in solution; b)Independent of temperature.In case of figure 2, the electrode sensitive to hydrogen ions is an indicator electrode. Potentialof an indicator electrode depends mainly on the concentration of the analyte ions (in this case hydrogen ions).

2. Experimental determination of trace metals in the Samples

2.1. Apparatus

In these experiments, a glassy carbon electrode (GCE) of 3-mm diameter was used as the working electrode with an Ag/AgCl reference electrode and a platinum counter electrode. The glassy carbon electrode was polished with alumina powder (0.3 and 05 alpha) and polishing pad. These three electrodes are used in conjunction with an ivium biopotentiostat which was interfaced with a personal computer. A conventional three-electrode cell arrangement was used for voltammetric measurements. Differential Pulse Stripping Voltammetry was performed anodically to detect trace metals. Experiment was performed at room temperature (22? C±1)

2.2. Reagents

All chemicals used in this study were of analytical reagent grade and used without further purification. Bismuth (II) nitrate pentahydry, sodium bromide, potassium hexcyanoferrate(II), copper(II)nitrate hydrate, lead(II)nitrate, hexaammineruthenium(III)chloride, iron(II)nitrate Methanol and zinc nitrate hexahydrate standard stock solutions (1000 mg/L, atomic absorption standard solution) were obtained from sigma-Aldrich. Potassium hexcyanoferrate (II) and hexaammineruthenium (III) chloride are redox mediators.0.1 M acetate buffer (pH 4.65) also obtained from sigma-Aldrich was used as supporting electrolyte. Glassy carbon was supplied by sigma-Aldrich and deionised water (18 M? cm) was used throughout. Nafion®117 polymer from sigma-Aldrich and Bismuth (II) nitrate pentahydry was used to produce electrode films. All chemicals used in the experiment are for research and development purposes.

2.3. Electrode preparation

2.3.1 Cleaning and Setting up the GCE

This involves the glassy carbon surface being polished with micro-sized abrasives in order to expose a new surface. A plastic Petri dish is prepared and a polishing pad placed into the dish. A few grams of 0.3 alpha alumina powder was placed onto the polishing pad and a solution was made by the addition of deionised water. The GCE was held at right angle to the pad, and polished in a circular motion until the GCE looked like a mirror surface. The electrode was rinsed with distilled water and air dried.

Ex-situ preparation of bismuth film electrode

The ex-situ bismuth film electrode was prepared by deposition of metallic bismuth onto a glassy carbon substrate electrode from 0.1M acetate buffer solution (ph 4.65) containing 100mg/L bismuth (II) nitrate pentahydry by applying a potential of -1.0V for 5mins with the biopotentiostat while the solution was stirred. Bismuth film electrode in the presence of bromide ions (sodium bromide) was prepared similarly from 0.1 M acetate buffer solution (ph 4.5) containing 50mg/L bismuth (II) and 50mg/L sodium bromide by applying a potential of 0.3V for 60s. This electrode was then transfer into a sample to analyse the analyte of interest.

2.4. Procedure

Setting up the ivium biopotentiostat

The ivium biopotentiostat, the computer CPU and the computer monitor are turn on at the mains. The computer is manually turned by pressing the on button on the CPU, after which the monitor will display a login screen. Once logged in the ivium soft icon on the desktop was double clicked to open the program. The parameters for the ivium biopotentiostat must be set. These settings change dependent on which method is being used.

For example Differential pulse stripping voltammetry was used to quantify amount of copper in a 1 mM solution of potassium nitrate in deionised water by the following method. Click on Method and under the list of electroanalysis select Differential Pulse Stripping Voltammetry. In the Parameter Box, set the E start (V) = -0.35 and the E end (V) = 0.6 (the potential range within which the analyte is determined), Current Range =100mA, Pulse time (ms) =10, Pulse amplitude (mV) =10, E step (mV) =25,

Scan rate (V/s) =0.05 and the pre-treatment Time (sec) = 300.

Cyclic voltammetry was used to analyse the affect of scan rate by increasing the scan rate on Nafion modified GCE in a1mM solution of potassium nitrate in 0.1 M acetate buffer at ph 4.65 at different scan -rates :

Click on Method and under the list of cyclic voltammetry select standard. In the Parameter Box, set the E start (V) = -0.4, Vertex 1(V) =0.7, Vertex 2(V) =-0.4, Current Range =1mA, E step (mV) =10 and Scan rate (V/s) =0.1.

Once the settings were selected and the electrodes were connected the system was run by selecting start. The run is completed when the graph appears on the screen and the computer makes a beeping sound. The graph of results was then analysed.

Preparation of a 1.0 mM Potassium chloride solution

To prepare a solution of 1.0 mM potassium nitrate approximately 0.0046 g of potassium nitrate was weigh out in a clean dry weighing boat using a balance and placed into a 50 ml beaker. Solution is made up by dilution to a volume of 15ml with deionised water and mix well until the potassium chloride dissolves.

Preparation of a 1.0 mM Potassium chloride and hexaamineruthenine solution

To prepare a solution of 1.0 mM potassium nitrate and hexaamineruthenine approximately 0.0046 g of potassium nitrate and 0.111g of hexaamineruthenine was weigh out in a clean dry weighing boat using a balance and placed into a 50 ml beaker. Solution is made up by dilution to a volume of 15ml with deionised water and mix well until the potassium

Cyclic voltammetry calculations for the effect of scan rate

The cyclic voltammogram is characterized by the peak potential Ep, at which the current reaches a maximum value and by the value of the current ip. When the reduction process is reversible, the peak current is again given by the relation –

Ip = 0.4463 nFA (Da) 1/2Cb – (1)

With

a= = at 25?C – (2)

This relation results from the set of differential equations for fick’s law of diffusion. In term of adjustable parameters the peak current is given by the Randle-Sevcik equation. This equation describes the effect of scan rate on the peak current ip. The equation is given by-

Ip= 2.69 ?105n3/2AD1/2Cbv1/2at 25?C– (3)

Figure Cyclic voltammogram where ipc and ipa show the peak cathodic and anodic current respectively for a reversible reaction.

RESULTS AND DISCUSSION

Effects of scan rate

The effects of Scan rate was examined by increasing the scan rate from 50mV/s to 1000mV/s for a 1 mM solution of potassium chloride + hexaamineruthenine in deionised water. As the scan rate increased the current increased The 2,4-DNPHMCPE showed increase in the peak current with increase in scan rate

Figure Cyclic voltammetry of GCE in 1 mM solution of potassium chloride + hexaamineruthenine in deionised water at different scan -rates a) 1000 mV/s b) 750mV/s c)400 mV/s d)100mv/s e)50mV/s

Figure Cyclic voltammetry of Nafion modified GCE in a 1 mM solution of potassium chloride + hexaamineruthenine in deionised water at different scan -rates a) 1000 mV/s b) 750mV/s c)400 mV/s d)100mv/s e)50mV/s

Nafion after(04/02/2011)

Figure Cyclic voltammetry of Nafion modified GCE in a 1 mM solution of potassium chloride + hexaamineruthenine in deionised water at different scan -rates a) 750 mV/s b) 400mV/s c)200 mV/s d)100mv/s e)50mV/s f)20mV/s

Bismuth in acetate baseline

Figure Differential pulse voltammetry of bismuth modified GCE in a 0.1 M solution of acetate buffer at ph 4.65 with different concentrations of lead. a) 120µL b) 100µL c) 80µLd) 40µL

Figure Differential pulse voltammetry of bismuth modified GCE in a 0.1 M solution of acetate buffer at ph 4.65 with different concentrations of zinc. a) 400µL b) 300µL c) 200µLd) 150µL e) 100µL

Figure Differential pulse voltammetry of bismuth modified GCE in a 0.1 M solution of acetate buffer at ph 4.65 with different concentrations of zinc. a) 600µL b) 500µL

Figure Differential pulse voltammetry of bismuth modified GCE in a 0.1 M solution of acetate buffer at ph 4.65 with different concentrations of lead. a) 120µL b) 100µL c) 80µLd) 40µL

Figure Differential pulse voltammetry of GCE in a 1mM solution of potassium nitrate and deionised water different concentrations of copper (II) nitrate added. a) 750µL b) 450µL c) 150µL

Figure Differential pulse voltammetry Nafion modified of GCE in a 1mM solution of potassium nitrate and deionised water with different concentrations of copper (II) nitrate additions. a) 650µL b) 450µL c) 150µL

Figure Differential pulse voltammetry of bismuth modified GCE in a 0.1 M solution of acetate buffer at ph 4.65 with different concentrations of lead. a) 120µL b) 100µL c) 80µLd) 40µL

Figure Differential pulse voltammetry of bismuth modified GCE in a 0.1 M solution of acetate buffer at ph 4.65 with different concentrations of lead. a) 120µL b) 100µL c) 80µLd) 40µL

Figure cyclic voltammetry of Nafion modified GCE in a1mM solution of iron (II) in 0.1 acetate buffer at ph 4.65 at different scan -rates. a) 100mV/s b) 50mV/s c) 20mV/s

Figure cyclic voltammetry of Nafion modified GCE in a1mM solution of potassium nitrate in 0.1 M acetate buffer at ph 4.65 at different scan -rates. a) 100mV/s b) 50mV/s c) 20mV/s

Conclusion

ASA = Anodic stripping voltammetry

AAS = Atomic absorption spectrometry

Bi = Bismuth

DME = Drop mercury electrode

Reference

Electrode kinetics: principles and methodology By C. H. Bamford, R. G. Compton

Fundamentals of analytical chemistry, eighth edition

Electrochemistry for chemists

http://en.wikipedia.org/wiki/Hanging_mercury_drop_electrode

http://www.chem.unep.ch/mercury/report/Chapter5.htm

http://www.epa.gov/ttn/oarpg/t3/reports/volume6.pdf

http://electrochem.cwru.edu/encycl/art-p01-plants.htm

http://en.wikipedia.org/wiki/Electric_field

http://en.wikipedia.org/wiki/Molar_concentration