BAS offers several models of Ag/AgCl reference electrodes. Each reference electrode is individually inspected and tested for adherence to an acceptable range relative to a standard calomel electrode. The model RE-6 is used by the epsilon system when configured with any of the standard flowcells. It features glass-body construction with a porous ceramic frit at the solution interface. The filling solution is 3 M NaCl gel that has been saturated with AgCl; the gel is semi-solid and will appear cloudy with occasional particles.
The purpose of the reference electrode is to provide a stable, reproducible voltage to which the working (detector) electrode potential may be referenced (see PRINCIPLES). A reference electrode may be considered a small battery whose voltage (potential) is determined by the chemistry taking place between a solid conductor (usually a metal salt) and the electrolytic solution around it. Ideally, if a small current is passed through the electrode, the potential change is negligible, and in any case, returns to the initial value when the current ceases. In addition, the potential value should not vary with time and should be reproducible from electrode to electrode. The most common reference electrodes meeting these criteria are the mercury/mercurous chloride (calomel) and the silver/silver chloride (Ag/AgCl) electrodes.
Every Ag/AgCl reference electrode is shipped with a colored plastic sheath that covers the porous tip and retards drying. Immediately upon receipt, remove this sheath by rolling it down from the glass body to the tip. The plastic will roll down and slide off the end of the electrode. Do not tug at the sheath or hold the electrode by its lead while you are doing this. If you have trouble removing the sheath, make a small cut at the upper edge of the sheath using small scissors, and try again.
BE CAREFUL NOT TO BREAK OFF THE END OF THE ELECTRODE!
ONCE THE SHEATH IS REMOVED, STORE THE ELECTRODE TIP IN 3 M NaCl AS SHOWN BELOW.
The Ag/AgCl reference electrodes are easily ruined by drying. Keep the tips wetted at all times and store in 3 M NaCl when not in use.
The Ag/AgCl reference electrodes are easily ruined by drying. Keep the tips wetted at all times and store in 3 M NaCl when not in use. A reference electrode storage vial is available from BAS (MR-5275. Be sure that you check the electrodes periodically and replace the solution in the storage vessel with fresh 3 M NaCl to keep the tips wet. DO NOT ENTIRELY IMMERSE REFERENCE ELECTRODES. Keep the connecting pins dry, or they will corrode and contaminate the reference electrode.
Reference electrodes will naturally change with use due to the transport of ions and solvent across the junction. The rate of change is a function of the difference in composition between the sample solution (i.e., mobile phase in liquid chromatography) and the filling solution (3 M NaCl gel). In LCEC usage, it is advisable to change to a new reference electrode frequently, at least once per month. Storing the reference electrode in 3 M NaCl between experiments will extend its lifetime. In spite of all attempts to extend their lifetimes, reference electrodes are still expendable items, so be certain to have spares on hand as needed.
We recommend that three reference electrodes be rotated in your LCEC system. Keep one electrode in your system for about two weeks. Turn the detector to STANDBY before removing and replacing the electrode. (Failure to do so can ruin a glassy carbon working electrode.) Rinse excess 3 M NaCl storage solution off the replacement reference electrode before inserting it into the cell. Replace the bushing and O-ring if they show signs of wear. Turn the cell on. Place the first electrode into the storage container. In another two weeks, replace the reference electrode with the third reference electrode provided in the kit. By rotating the three reference electrodes provided with your detector on a continuous basis, you can maximize their lifetimes. Depending on the mobile phase conditions and detector use, the reference electrodes can last from 3-6 months. When you replace reference electrodes, replace all three of them at the same time.
If you are concerned about the viability of a particular Ag/AgCl reference electrode, you can test it using a simple voltmeter, additional reference electrodes of the same type (or a calomel reference electrode), and a small beaker of 3 M NaCl.
Read the potential difference between the electrodes on the voltmeter. Ideally the difference between two electrodes of the same type would be zero. However, in actual practice there is commonly some variation. If the two electrodes are of the same type (e.g., Ag/AgCl vs. Ag/AgCl, or calomel vs. calomel) the meter should read 0 ± 20 mV. If your reading for any pair of electrodes is significantly different, you should have another electrode of the same type handy to help distinguish which of the two is bad. When comparing an Ag/AgCl reference electrode to a calomel electrode, make the calomel the black (negative) input on the voltmeter. The meter should then read -35 ± 20 mV. A single-probe combination pH electrode is not suitable for this test.
The UniJet detector is shipped with an embedded silver wire, which gets coated with AgCl to form the Ag/AgCl reference electrode. It should be regenerated each time the mobile phase is changed or the electrode is polished . After polishing and rinsing with water and methanol, apply a drop of the reference electrode coating solution (CF-2200) to the electrode surface:
Do not allow the solution to get on the working electrode or on your skin. Leave the solution on the electrode for 5 minutes. Rinse the electrode with water. The reference electrode should be a dull bronze color and uniform in appearance.
The UniJet reference electrode is a pseudo-Ag/AgCl reference electrode. The reference electrode potential is determined by the quantity of Cl- in the mobile phase. The higher the Cl- concentration, the closer the reference will become to a standard Ag/AgCl. We recommend using 10 mM NaCl in any mobile phase to increase the stability of the reference electrode and prevent alterations in the potential. The UniJet reference electrode is about 100 mV more positive than the standard RE-6 Ag/AgCl reference electrode. Thus, the potential set on the detector should be 100 mV less than was used with an RE-6.