Peter Jones, application chemist at Metrohm UK, provides this introduction to amperometric detection - which utilises a flowing current to initiate a chemical conversion of electro-active analytes
Amperometric detection utilises a flowing current to initiate a chemical conversion of electro-active analytes, ie those substances which undergo a reduction or oxidation reaction according to the potential applied.
Due to the nature of the electrode processes there are three key factors which must be controlled in order to obtain stable and reproducible results: temperature, pH, and mass transfer (the eluent flow through the reactor should be continuous and without pulsation).
The reactions at the working electrode are influenced by the temperature, but this will also affect the background reactions producing the baseline current.
Therefore, thermal stability is a necessary precondition of the analysis in order to maintain a stable baseline and reproducible signal response.
Similarly, pH alterations affect the current/potential curves (voltammograms) and the possible results of poor pH control include reduction of signal intensity and lower signal to noise ratios.
Further, redox reactions are dependant on mass transfer at the electrode/electrolyte interface and therefore a constant eluent flow is necessary both for baseline stability and reproducible signals, therefore any pulsation from the pump delivering the eluent to the reactor cell must be eliminated.
The 817 Bioscan from Metrohm is a PC controlled instrument that facilitates the analysis of electro-active components utilising the most advanced three-electrode, flow-through (wall-jet) cell with a unique patented solid state reference electrode.
The solid state reference electrode eliminates standard maintenance issues with normal three electrode systems utilising Ag/AgCl reference electrodes, and delivers significant improvement in signal to noise ratio.
Housed in an electromagnetically shielded cabinet (Faraday cage), the 817 Bioscan also contains an oven - accurate to 0.5C and precise to 0.1C, over a temperature range from 10C above ambient to 60C.
The cabinet also contains a pulsation absorber and a 3m pre-heating capillary.
However, the flow-through cell should not be operated above 45C over long periods of time.
Operating principle.
The 817 Bioscan operates as a fully integrated cell and detector system and the principle of operation is the same as in any other high performance liquid chromatography.
The separating column is placed in the thermostatically controlled cabinet along with the pulsation absorber, flow-through cell and pre-heating capillary, this allows for unparalleled thermal stability.
The 817 Bioscan is a complete detection system combining the cell, oven, pulsation dampener and preheating capillary into a single compact unit.
The only other requirements for operation are a pump to deliver the eluent and a standard six-port injection valve for introducing sample to the system.
These units can be obtained as separate modules from Metrohm or by using the system as a direct detector after the LC separation existing HPLC systems can be used.
Most applications, however, require the use of a sodium hydroxide eluent, it is therefore necessary that the pump and injector should be of the metal free variety - such as the all Peek 812 valve unit or 709/818 pump unit available from Metrohm.
Limits of detection are dependant on the analyte but are typically in the range of 10-100ppb with PAD.
Control and data acquisition.
The design of the 817 Bioscan allows the data to be retrieved direct to PC via RS232 connection, or to a third party data acquisition system using either an analogue or digital signal output.
There are a further two relay outputs and two transistor-transistor-logic (TTL) outputs which could be used to automate any of the Metrohm range of IC sample changers, liquid handling devices, and a large number of third party LC modules and autosamplers.
The only control requirement is that the Metrohm software (IC Net 2.3) operates in the background to turn on the oven and flow-through cell, the cell may alternatively be controlled by a remote TTL input signal.
Modes of operation.
The maintenance-free flow-through (wall jet) cell is available with a range of working electrode materials designed to encompass different operating potentials in both acid and alkaline conditions.
Further, the volume of the flow-through cell can be altered by changing the cell spacer allowing end-users the ability to select the sensitivity of response directly.
The cell body is made from the highly fluorinated KelF (CTFE) polymer material providing the cell with excellent chemical resistance.
The three modes of operation for the 817 Bioscan are DC, scan, and pulse.
DC mode applies a constant potential at the working electrode and the current produced is plotted against time.
DC mode is typically used for inorganic electrochemically active anions such as cyanides and sulphides as well as species such as iodide, sulphite and nitrite.
DC mode can also be used for vitamin complexes and certain pharmaceutical products.
Scan mode is used to examine the voltammograms of analyte dissolved in eluent, by passing a solution of the analyte through the detector cell, a current potential curve is generated which, when compared with a current potential curve for the eluent alone can be used to optimise the parameters for DC or pulse mode.
Scan mode, however, does not involve a chromatographic separation and is used to optimise the conditions for the other analysis modes.
Pulse mode or pulsed amperometric detection (PAD) is the most versatile of the measuring techniques for LC applications, as it employs a measuring potential and two cleaning potentials to ensure the electrochemical regeneration of the electrode surface.
During an electrochemical analysis, reaction products can adsorb onto the surface of the electrode, radically altering the structure, and electrochemical properties of the electrode.
In a PAD system, the analysis is performed by a cyclic series of potentials applied to the working electrode.
Firstly a measuring potential is applied during the last stage of the application of this potential (after a suitable equilibration time) a measurement of the current is made.
Secondly, a large positive potential is applied to the electrode which causes the oxidative removal of any reaction products, and finally a negative potential is applied to reduce the electrode to its base metallic state (eg Au).
The entire three stage process lasts typically for one second and is repeated throughout the chromatographic separation - taking a new measurement, with an essentially brand new electrode surface, every second.
A typical application for the 817 Bioscan is the determination of carbohydrates amino acids, alcohols, glycols and antibiotics in the food, biological, and pharmaceutical industries.
Carbohydrates - or saccharides - are the most abundant class of organic substances found in nature.
Saccharides are created by green plants during photosynthesis and are a necessary dietary supplement even for carnivores.
However, despite being a rich source of energy they contain no nitrogen and no animal could survive on carbohydrate alone, as nitrogen is necessary to form amino-acids and proteins.
The saccharide compounds form polymers of varying sizes, many of which, such as cellulose and starch are important to both plant and industrial processes; as such they are sub divided according to the number of saccharide groups within a molecule.
Monosaccharides contain one saccharide group, disaccharides contain two, while trisaccharides contain three groups.
The series of compounds with between four and ten saccharide groups are known as oligosaccharides, while any compound containing more than ten saccharide groups is classed as a polysaccharide.
Column selection.
Aside from the selection of working electrode material, there are many columns which can be used to optimise the separation of carbohydrates (and other electrochemically active species).
The most versatile column, for PAD applications, in the Metrohm range is the Metrosep Carb-1, which has the highest capacity of any exchange column available and is available in either 250 or 150mm lengths (with a recommended 15mm guard column).
Post column reaction may be required for certain applications, this means the LC separation can be achieved not only using the more traditional anion exchange resins but also by cation exchange, reversed and normal phase resins without compromising the separation column.
