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What is ASV?

Anodic Stripping Voltammetry (ASV) is an analytical technique that specifically detects heavy metals such as Arsenic, Cadmium, Lead, Mercury and others. Heavy metals are usually toxic to humans and animals (and plants in some cases). ASV does not detect the light metals such as Sodium, Potassium, Aluminium, Calcium and Magnesium. It cannot detect inorganic elements such as Sulphur, Iodine, Bromine and Phosphorus or anions such as Sulphate, Chloride, Nitrate etc.

ASV (Anodic Stripping Voltammetry) essentially works by electroplating certain metals in solution onto an electrode. This concentrates the metal. The metals on the electrode are then sequentially stripped off, which generates a current that can be measured. The current (milliamps) is proportional to the amount of metal being stripped off. The potential (voltage in millivolts) at which the metal is stripped off is characteristic for each metal. This means the metal can be identified as well as quantified.

This method was first described in the 1920s. JAROSLAV HEYROVSKY won the Nobel Prize for chemistry in 1959 for developing it. It is therefore older than Atomic Absorbtion (AAS) or ICP (Induced Coupled Plasma) and is essentially a European invention. The method has found widespread acceptance, but only in specialist laboratories that need to analyse metals in the low part per trillion (nanogram per litre) range, without needing to concentrate the sample.

The original methods used liquid Mercury as the electrode. These devices are called Hanging Mercury Drop devices (HMD). Some devices allow the Mercury to form drops continuously; a mini analysis is carried out as each drop forms. These are called Dropping Mercury Electrodes (DME). DME electrodes consume significant amounts of liquid mercury.

Another way around the problem was to use 3 electrodes. The working electrode is where the metal is plated and stripped off, the counter electrode measures the current flow and the important reference electrode that is used to ensure the potential of the working electrode is maintained correctly. This helps to virtually eliminate the effects of the electrical field building up on the working electrode. This system is now used on HMD systems as well because it allows much lower detection limits to be achieved.

The Cogent Environmental system is a new type of 3 electrode device. Instead of liquid mercury as the electrode, this device uses a glassy carbon electrode that is plated with a very thin film of Mercury. (Mercury Thin Film Electrode, MTFE) This is carried out at the beginning of an analytical run and lasts for between 10 and 30 subsequent analyses. The Mercury is contained as a salt in the supporting buffer used. This means only a very small amount of Mercury is used and ensures the operator never comes into contact with liquid Mercury. The amount of Mercury used per analysis is measured in parts per trillion. If however the analysis is for Arsenic, Selenium or Mercury, a Gold electrode can be used, removing the need for any Mercury.

What is CSV and how does it differ from ASV?

Cathodic Stripping Voltammetry (CSV) is essentially the opposite of ASV. In ASV the potential is changed in the negative to positive direction. In CSV the potential is changed in a positive to negative direction. The following graphic displays this concept.

What is the difference between Linear, Square Wave, and Differential Pulse sweep types?

The preceding graphical display shows the difference between the ASV and CSV methods, but it is also in essence a linear sweep. The appearance of the sweep is that of a staircase, but if the steps are made infinitesimally small, the appearance is that of a line, thus the term Linear Sweep meaning that the potential is changed in a linear fashion over time.

The following graphic displays the parameters of a Differential Pulse Sweep. In a differential pulse sweep, the step size, pulse amplitude or height, pulse width, and rest width are all variable factors that can be used to increase sensitivity of a particular application. The VAS software provided with the instrument has a mathematics function that will calculate recommended values, or they can be modified independently.

The differential pulse potential wave form consists of small pulses (of constant amplitude) superimposed upon a staircase wave form. In the example waveform shown, the current would be sampled at two points during the pulse. One measurement is taken at the beginning of the pulse, and one at the end of the pulse; the difference between these two values being the result that is displayed (or recorded as data).

Differential Pulse Waveform

In Square Wave Voltammetry, the potential wave form consists of a square wave of constant amplitude superimposed on a staircase wave form. The current is measured at the end of each half-cycle, and the current measured on the reverse half-cycle (ir) is subtracted from the current measured on the forward half-cycle (if). This difference current (if – ir) is displayed as a function of the applied potential (again recorded as data). The parameters of this waveform are displayed in the following graphic.

Square Wave Waveform

Analysis

To analyse a sample, the metals must be in solution. For effluent and water the metal is already in solution (though some sample types may still require a digestion), but soil samples need to have the metal extracted.

The liquid sample is added to supporting electrolyte (buffer) to ensure the oxidation states of the metal ions are optimised for electrochemistry. This also dilutes the sample, which removes many of the potentially interfering compounds (cf immunoassay methods). Another component of the buffer removes any dissolved oxygen in the sample that would interfere with the analysis.

The analysis proceeds by initially plating the working electrode with Mercury or Gold (some methods are done on solid electrodes and this step may not be required). Several quick runs with a standard are performed to stabilise the Mercury or Gold film and to confirm the analyser is working correctly. The diluted sample is then added to the cell and the working electrode is given a negative potential relative to the reference electrode. The value can be varied depending on which metals are to be analysed. The negative potential attracts the positive metal ions to it, where electrons combine with the metal ions to produce the metal. The use of the Mercury film enhances the process as when the metal ion is reduced to the metallic state, it forms an amalgam with the Mercury, which stabilises it during the stripping phase. Mercury on glassy carbon also has a high over-potential relative to Hydrogen. This means the potential can be set that allows metals such as Zinc to be plated onto the electrode, without producing hydrogen gas. Hydrogen is very reducing and will interfere with the subsequent stripping. The potential is then held for around 60 seconds (up to 300 in some applications) while the metal accumulates on the electrode, effectively concentrating the metal in the sample onto a small area. Not all of the metal in solution is reduced onto the electrode, but the plating time selected is long enough to reduce sufficient metal onto the electrode to give a good signal.

During the plating process, the sample is mixed at high speed. This ensures that the metal ion concentration at the electrode/sample interface is the same as the concentration in the bulk sample. By mixing the sample, the major factor that pulls the ions to the working electrode is the negative potential and not diffusion, convection or other random movement in the sample. This also helps prevent a capacitive build up on the electrode where a layer of positive ions shield the negative electrode from other ions in the sample. By ensuring the negative potential is the dominant factor during the analysis, the reproducibility of the analysis is dramatically improved. An added bonus is the complex mathematical formula used to calculate the amount of metal deposited for a given time at a given potential is simplified.

The potential is then allowed to become less negative and the metals re-oxidise (or are stripped from the electrode), which generates electrons (2 for each Cu atom, 3 for As etc). Each metal will strip from the electrode at a specific potential, which allows for identification of a metal. The data can be plotted to give a graph of current against potential. This graph is called a voltammogram.

The rate at which the potential is changed is called the sweep rate and is another variable that can be altered to optimise an analysis. The faster the sweep rate (mV/sec) the better the resolution and better detection limits. This is because at high sweep rates, the metals on the electrode quickly strip off from the electrode, giving a narrow peak on the voltammogram. A slow sweep rate allows the metal to strip off slowly, giving a broader peak, which is more variable in size. However, this slow sweep can separate two metals that have similar stripping potentials. By applying different waveforms to the sweep, stripping potentials can be shifted, which is useful when 2 metals of interest strip at a similar potential.

The generation of electrons is measured by the counter electrode as a current produced in the cell. The current in micro or nano amps is proportional to the metal concentration on the electrode. As each metal strips from the electrode, a graph (voltammogram) is produced showing a series of peaks corresponding to current (metal concentration) at specific potentials. By selecting a potential “window” where a specific metal is expected to appear, ASV can be used to identify and quantify the metal concentration in the sample.

Quantification

The calibration curves for individual metals can be linear over 2 orders of magnitude. Most ASV instruments can therefore use a single concentration of standard to analyses samples between 10ppb and 1,000ppb. The calibration curve also has a characteristic gradient which is useful for initial QC of the instrument performance. This method is called calibration curve comparison.

As with all analytical methods there can be interferences. The matrix and presence of other metals or substances can change the potential at which a metal strips from the electrode. Certain metals have similar stripping potentials so a slight shift in stripping potential can cause peak overlap. (similar to using GC or HPLC.) For this reason the analysis is always run with a standard of the metal of interest to identify the exact stripping potential.

To minimise the effects of interference, methods have been developed that use specific buffers that are best suited to various matrixes and metals together with a procedure known as standard additions. The instrument is first calibrated using a known concentration of the metal of interest in specific buffer. The concentration selected should be in the same order of magnitude as the expected concentration of metal in the sample. The current produced should match the expected value for that instrument. If it does the system passes the initial QC check.

The sample is then analysed and an initial metal concentration calculated. A small volume of a known concentration of the standard is then added to the sample and it is re-analysed. A second, small volume of the same standard is then added to this sample and it is analysed again. The three results are compared. As ASV produces a linear calibration curve over 2 orders of magnitude, the results should also produce a linear curve with a gradient similar to that expected for the target metal. For simple shifts in the line such as a slight curve, parallel line or slight divergence, a simple calculation can take the analytical result and convert it to a compensated result. The VAS software supplied with the instruments can compensate for more complex shifts in the calibration curve.

By using this procedure, most of the effects of interference are suppressed, giving a true valid result. It is interesting to note that most routine AAS or ICP analyses do not use the standard additions method to compensate for matrix effects, even though different matrixes can significantly affect this type of analysis.

VAS is a powerful, but easy to use software program that gives the PDV6000plus the functionality of laboratory based instruments. It also allows the PDV6000plus unit to be programmed with specific methods and firmware upgrades.

VAS is fully 32-bit compliant. Written to run on the current Windows platforms. Supports Windows 98, ME, NT, 2000, XP and Vista.

Minimum System Requirements

• IBM compatible computer with 300 MHz Pentium, Celeron, or AMD K7
• Standard 9 pin RS232C serial port and cable
• Colour monitor, 256 colours, 800×600 resolution
• Windows 98, ME, NT, 2000 or XP
• 64 megabytes of RAM. (128 MB recommended)
• 10 megabytes of hard drive space available
• CD drive

Supported Analysers

VAS can be used with the following voltammetric analysers:

• PDV3000*
• TEA3000*
• PDV6000*
• PDV6000plus

(* no longer manufactured – now replaced by the PDV6000plus)

How VAS works

Voltammetry is an electrochemical technique which can be used to determine the concentrations of certain substances in liquids. The method involves placing the liquid into a special cell with three electrodes embedded into it. Firstly a reducing potential is applied to accumulate, or plate, metal ions on the carbon working electrode. The potential is then ramped in a positive direction to a final potential, causing the metal to be stripped off back into solution. During this last phase the current in the cell is measured and analysed to determine the metal ion concentration.

Graphing the cell current versus cell voltage during the sweep portion of the voltammetric cycle gives a curve like:

Figure 1: Data Curve for Cadmium, Lead and Copper

As different metals give a peak at their own characteristic potential, each distinct peak corresponds to a particular element in the solution. In the above example they are cadmium, lead and copper in that order. The size of the peak (uA) is proportional to the concentration of that metal in the cell.

There are two methods of analysis supported by VAS: standard comparison and standard addition. Both of these methods rely upon using a standard solution of known concentration as a reference. For more information see Standard Comparison Analysis and Standard Addition Analysis.

Detection Windows

To differentiate and identify peaks on the voltammetric curve VAS uses detection windows. A detection window is a range of voltages in which VAS will search for a peak. Before finding peaks with VAS it is necessary to set a detection window for each element you wish to measure in the sample. If you know in advance where to place the detection windows you can set them from the Initiate Run dialog or they can be placed visually once the standard or sample curve has been taken.

Peak Measurement

To measure the concentration of an element using voltammetry the size of the peak in the voltammetric curve is measured. The size of the peak is proportional to the concentration of the metal producing it, so that doubling the concentration doubles the size of the peak.

When a voltammetric curve is generated, there is an underlying component due to the electrolyte and the voltammetric cell itself, called the background current. To compensate for this, a peak baseline is drawn. This should simulate the background as closely as possible.

The two main methods of measuring peak size are peak height and peak area. Both of these measurements are taken from the baseline.

Figure 2: Peak Size Calculation By Peak Height or Peak Area

It is important to note that the absolute peak height is not taken into consideration when either the height or area is measured. For this reason a vertical shift of the entire voltammetric curve will not effect the results.

Blanks and Artificial Blanks

As mentioned above, when a voltammetric curve is generated, peaks of interest are superimposed on a background current. Generally the baseline described above is sufficient compensation for this. However, to counteract extreme cases of this background current a blank curve (from a cell containing only electrolyte) can be sampled and subtracted from the standard and sample curves.

Figure 3: Blank Subtraction

Blank subtraction also serves to remove any bias introduced by impurities already present in the cell. For these reasons it is recommended that a blank curve be taken before a sample is analysed. The cell should not be rinsed after the blank is taken but the other solution should be injected immediately. If no blank is available, either because the data has already been taken or it would be too slow to take a blank, it is possible to have VAS calculate an artificial blank. This is used in exactly the same way as a blank except that it clearly cannot counteract any impurities in the cell.

Note: Wherever a blank or artificial blank can be used, if both are selected then VAS will use only the blank if one is available, or only the artificial blank if no true blank is present. Under no circumstances will both be used together.

The Standard Comparison Method

Standard comparison analysis compares the signal from the sample with the signal from a standard. The ASV technique produces a linear response for most metals across a concentration range of 2 orders of magnitude, which allows a simple comparison to be made. It is assumed that peak size is linear with concentration and the standard is used to find the slope of the relationship. This is then used to calculate the concentration of the sample. With this technique it is best to use a standard concentration that is within the same range as the sample concentration.

Figure 4: Standard Comparison

The Standard Addition Method

Standard addition analysis initially analyses a sample and then one or more standard addition curves. A standard addition curve is taken by adding a small amount of standard solution into the sample already in the cell. This is repeated up to five times, usually adding the same amount of standard each time so that the concentration of standard steadily increases.

The sample and standard additions data is then plotted on a graph and a line of regression is fitted. This is then used to calculate the concentration present in the sample.

Figure 5: Standard Addition

This technique can compensate for matrix effects that enhance or depress the sample result. Because more information is being used the errors should be less for the standard addition technique than for standard comparison. For this reason it is normally a more accurate technique, but is more time consuming. For simple screening of samples where samples need to be classified as above or below a certain concentration, the standard comparison technique is perfectly acceptable.

VAS will display the regression graph in the results window when the standard additions technique is used. A correlation value will also be shown, where 1 = sample and standards are all on the regression line and the result will be reliable. Normally expect a correlation value > 0.999. A correlation value any lower means the sample and standards are scattered about the regression line and the result will be less reliable.

The VAS Data Structure

The data storage system in VAS works on a number of levels. The first file level is called projects and is equivalent to a complete book. A project contains all the data, which was taken for a common purpose. For example, the samples taken from a test site on a river over one week would all go into a single project, as would the data collected while trying to detect lead and zinc in fruit juice samples. The projects file can be given a title to reflect the contents of the project.

The level below projects contains the main data files, with a single project containing many. One file called Run Configurations will contain the configuration settings for the analysis (run time, hardware settings etc). Several configurations can be stored in this file. Other files are created for each sample and contain all of the data needed to determine the concentration of the target analyte. Each file can be given a description to identify the sample. Another file called the Report file contains a summary of all of the results for the project.

In each sample file, sub files are created for each discreet set of analytical data that is produced. For example, if a standard curve analysis is being used then the sample file may contain sub files for a standard voltammogram, a sample voltammogram, a blank voltammogram and the results from processing these voltammograms. Opening these sub files will show the voltammogram graph for that run.

As an example the project called ‘Fruit Juice’ is shown below:

Figure 6: VAS Fruit Juice Project

This project contains three files – a Report, an Orange Juice sample and an Apple Juice sample. The report contains a summary of the data in the project (from the Orange Juice and Apple Juice files). The Orange Juice and Apple Juice files both contain all of the data necessary to calculate the concentrations in a single sample. Each file has four data members in it – a Sample Voltammogram, a Standard Voltammogram, a Blank Voltammogram and Results.

Managing Projects

Projects are used to keep your data organised into logical and consistent units. It is important that all data that belongs together goes in the same project and that sample and run data from different projects is not mixed together in a single project.

Projects are created and manipulated with the commands on the Project menu. Before beginning work you need to make a new project or open an existing one. Because all of the data in a project is visible in a single scrolling window it is a good idea not to make your projects too big.

You can open more than one project at a time in VAS. To change between them you use the commands on the Window menu. To move data between projects or within a project use the Cut, Copy and Paste commands on the Edit menu.

To back up your data or to exchange information with another user of VAS you may want to export and import projects.

Managing Files

There are a number of different types of files in VAS. They are:

• Samples – Store the data associated with a single sample
• Run Configurations – Store run configuration settings
• Reports – Store summaries of data in a project

Using Comments

Where appropriate you should comment the different sorts of data in the following ways:

• Sample Files – Which chemical sample the file is for (e.g. Holding tank C – 10:00am sample) and any special events concerning the taking of the sample
• Curve data members – Any unusual run configuration options used in generating the curve
• Run Configuration data members – What was different about that run configuration (e.g. 1000 mV/s sweep rate with 60 sec plate)
• Report – General notes

As an example, consider the following files:

Figure 7: Use Comments To Identify VAS Data

Here comments have been used to label the run configurations, allowing you or someone later to see at a glance what each one was intended for. The sample file has been named (“Sample A72”) to identify its source and a comment has been attached as a reminder of exactly when it was taken.

Try to use file names to identify what the files are for and comments to store additional information. By using sensible filenames and comments you make the contents of your data much clearer to others and to yourself (especially when you come back in the future).

Exporting and Importing Projects

The data in VAS projects is stored on disk in a format optimised for speed of access. You should never directly edit or copy this data but should only deal with it through VAS.

If you want to exchange data with another VAS user or you want to backup your data onto hard drive, CD-ROM, USB drive or floppy disks you will need to export and import projects. Exporting a project writes an entire VAS project into a single file on disk, while importing allows you to take such a file and expand it into a full VAS project.

If you are backing up your data then you can delete the projects from VAS once you have exported them. If you need them at a future date then you can simply import them again.

Generating Reports

VAS allows you to prepare a summary of all data in the project with the Report... command on the Analysis menu. Generating a report takes each sample file in the project and extracts the results.

For example, for the Fruit Juice project shown in The VAS Data Structure, a report would include the information from the results in the Orange Juice and Apple Juice files. This is the actual concentrations found in the sample as well as data on the peak sizes and positions and the original standard concentrations.

Printing Data

Nearly all of the data that VAS uses or generates can be printed on a standard Windows printer. There are two main methods of printing data. Firstly, you can use the Print... command from the File menu. This allows you to print whatever data is currently being manipulated on the screen, whether it be a project or a graph. The second method is to click on the Print... button in those dialog boxes where it is provided. This will print the current contents of the dialog.

Generally, if you want to print the contents of a project or a graph, use the Print command on the menu. If you want to print other data, open the data (double-click or the Open command) and click the Print button where it is available.

Collecting Data

Before using the data viewing and analysis portions of VAS it is necessary to collect voltammetric data from your instrument.

The following sections contain detailed information on collecting data.

Graphing Data

The graphing of collected data forms an integral component of voltammetric analysis. VAS automatically converts the data received during the analysis into a graphical form. VAS has been designed to identify and select the correct peaks and compensate for changes in curve position during the analysis and to provide accurate results with a minimum of user intervention. To ensure that the best results are obtained with difficult samples the graphical output can be manipulated allowing the analysis process be fine-tuned.

The VAS graphing subsystem allows visual judgement of the quality of the collected curve, manual adjustment of detection windows and peaks, visual confirmation of the effects of blank and artificial blank subtraction and multiple curves to be displayed and overlapped within the same window.

Graphing a Curve

To graph a collected curve simply open the curve member within the file. To open a curve select the curve member within the project window and select the Open... command from the File menu.

Opening a curve displays the graph window (shown below). Within the window you can see the graph itself and the axes (current vertical and voltage horizontal). The detection windows for the curve’s file are displayed immediately above the curve and the peaks on the curve corresponding to each detection window are shaded with diagonal lines (if the curve has defined peaks and peak shading has been selected from the Display menu). If the curve has an artificial blank then it will be appear in green on the graph (if artificial blanks are displayed and in the Setup, Options dialog box, the Coloured By Type graphs are selected).

Figure 8: VAS Graph Window

The graph window displays all the open curves within one window by overlaying them. There is no limit on the number of curves which may be open at once.

• Click on the name of the current curve in the toolbar. A list of all curves displayed drops down and you can select the one you want
• Press the Tab key to cycle through the curves displayed. Keep pressing Tab until the curve you want is selected
• Click on the curve directly

Graphing Options

There are many options for configuring the graph window to display only the information that you require. By default only some information is displayed, additional information can be displayed if desired and information that is ordinarily displayed can be hidden.

There are two main ways to turn the display of curve features on and off:

• Select the feature from the Display menu. If the feature was on (with a tick next to it) it will be turned off, and vice versa
• Click on the tool-bar icon for each feature. If it was on (button pressed in) it will be turned off, just like the menu

For more information on the available graphing options refer to the online help or the VAS Reference Manual.

Graphing Multiple Curves

It is possible to have multiple curves displayed on the graph window at the same time. There are several ways of opening multiple curves:

Multiple curves appear on the graph window as shown below. Notice that the selected curve has its peaks shaded while all other displayed curves do not.

Figure 9: VAS Graph Window Displaying Multiple Curves