This section details specific liquid chromatographic procedures for reductive mode LCEC. The section is divided into five parts:
The preparation of both glassy carbon and mercury/gold electrodes is described in detail in the section on WORKING ELECTRODES. Users with questions concerning the cell itself should read this material first.
By far the majority of the LCEC literature deals with oxidative mode electrochemical detection, but considering strictly the electrochemical literature of organic compounds, reduction processes have been examined in much greater detail. Table 5.1 describes some functional group candidates capable of being analyzed by reductive LCEC. Note that within a given functional group, a broad range of reduction potentials may exist due to the effects of substituent groups. Generally, the more delocalized the electrons become, the more easily reducible the substance. In addition, electron-withdrawing groups on an aromatic ring will enhance the reduction reaction.
Table 5.1. Functional Groups Suitable for Reductive Electrochemical Detection.
Reductive mode LCEC requires some mechanical modifications to remove dissolved oxygen from both the mobile phase and sample; it is just as necessary to make sure oxygen does not reenter the system. Oxygen is removed from the mobile phase and sample by bubbling an inert gas (e.g., helium) through the solutions. Refluxing the mobile phase at the same time will thoroughly deoxygenate the system. A mechanical arrangement such as that shown in Figure 5.1 can be set up and dedicated to a specific LC system for this purpose. Table 5.2 details specific parts needed for this modification to modular BASi LCEC systems.
Figure 5.1. Deoxygenation apparatus for LCEC; both mobile phase and sample are deoxygenated.
Table 5.2. Parts list for reductive LCEC.
|1||Connecting plastic tube||Common Lab Supplies|
|2||Gas-trap bubbler||Reliance Glass|
|3||Condenser adapter||Reliance Glass|
|5||3-neck, 1 liter, round-bottom flask||Kimble|
Stainless steel inlet tube for pump
|7||Heating mantle||Scientific Products|
|8||Rubber septum||Common Lab Supplies|
1/16" o.d. stainless steel tubing (when
attaching to valve you may need extra fittings)
|11||Tee to He line||Swagelok|
|12||1/8" o.d. copper tubing||Common Lab Supplies|
|13||Sample vial with rubber septum||BAS|
|14||Vent needle||Scientific Products|
|15||Gas humidifying chamber||Common Lab Supplies|
All plastic tubing in the system must be replaced with stainless steel, because most plastic tubing is permeable to oxygen (PEEK tubing is less permeable than most, and may be an acceptable substitute for stainless steel). A one- or two-liter round-bottom flask with reflux condenser and heating mantle serves as the mobile phase reservoir. One neck is used for the condenser, and the other two necks for the helium inlet and pump outlet. Ordinary laboratory grade helium supplied at ca. 20 psi is tapped at two separate needle valves for independent control of both solvent and sample degassing. A 1/8"-o.d. stainless steel outlet line runs to the inlet check valve of the pump. Large rubber septa seal off the mobile phase from the outside environment. Temperature of the heating mantle can be controlled electronically.
Samples are degassed with the second needle valve. An optional helium saturation chamber filled with water wets the gas thoroughly before it enters the sample container.
The outlet tube from the column to the detector must be replaced with a special steel connector (P/N MF-1029). No plastic tubing may be used! This connector uses special plastic end fittings to electrically isolate the cell from the rest of the LC system (which is grounded). The steel tubing prevents gas intrusion. If you are using a preheater module, the steel tubing in this is appropriate.
Modifications are now completed for reductive LCEC.
If you think this seems like a lot of preparation, you are correct. After the initial setup, a system like this can be fairly reliable for reductive LCEC work, but it has limitations. For example, no gradient elutions can be run under these conditions. For customers requiring a full-featured liquid chromatograph with solvent deoxygenation capabilities, the BASi 200e Analyzer is the instrument of choice.
The following chromatographic startup procedure is recommended for reductive LCEC applications. Before initiating flow through the LC system, heat the mobile phase to 30-35 °C and bubble the inert gas rather vigorously through it for 30-60 minutes. Start pumping the mobile phase through the LCEC system during the last 15 minutes of vigorous bubbling. Even though the mobile phase is degassed, the entire system is not. Oxygen penetrates the stationary phase pores, and it must be flushed out of these pores by initiating the flow of degassed mobile phase through the LC system. This may take several hours.
Degassing of the system requires at least 100-200 mL of preheated mobile phase. Be liberal and flush thoroughly. Turn on the working electrode after flushing and keep the detector at the lowest gain (highest RANGE setting) until the background current has stabilized. Reduce the flow of helium through the mobile phase. Some flow must be maintained to keep the oxygen out, but this need not be as vigorous as during the initial deoxygenating. If the baseline response (background current) begins to gradually increase, the rate of bubbling inert gas through the mobile phase is insufficient to keep the oxygen out of the system and must be increased until the background stabilizes. NOTE: Valuable time can be saved if degassing of the system is performed overnight at a minimal flow rate (0.2-0.3 mL/min). You can turn on the working electrode before leaving the lab in the evening to provide you with a stable system in the morning.
Sample degassing is necessary when working at potentials more negative than 0 to -0.1 V for mercury/gold and -0.3 to -0.4 V for glassy carbon electrodes, as illustrated by hydrodynamic voltammograms in Figure 2.2. The potentials at which the electrodes will be insensitive to dissolved oxygen may vary, depending on the pH, concentration and type of nonaqueous solvent, and the previous history of the electrode surface.
Care must be taken while degassing a sample in order to preserve its original composition. This is extremely important when handling volumes smaller than 500 mL. Presaturating helium gas with mobile phase and maintaining a gentle flow of helium gas through a sample will minimize evaporation of a sample. A presaturation device is pictured in Figure 5.1.
To degas, pass helium into the sample for about 3-5 minutes. This should be regulated at a flow rate as vigorous as allowed by the sample volume (smaller volumes will have to be degassed more gently than larger ones).
To inject, it is best to draw the sample slowly into the injection loop by gentle suction. Exposure to the atmosphere is avoided, and the integrity of the closed system, particularly at the needle seal, is preserved. On BASi chromatographs, you would immerse the waste port of the injection valve into the deoxygenated sample, and fill the loop by aspiration with a syringe mounted in the front (sample injection) port.
Both glassy carbon and mercury/gold electrode surfaces are useful in reductive LCEC. Although mercury is usually the surface of choice with electrochemists, there are fewer reasons for using it in LCEC. In polarography, mercury provides a repeatable, fresh electrode surface, a high hydrogen overvoltage (overpotential), and fast setting times. In liquid chromatography, however, dead volume must be minimized, thereby obviating the use of the usual dropping mercury electrode. For reductive LCEC, the optimal approach is to employ a thin-layer cell. The dropping mercury electrode is replaced by a glassy carbon surface or a mercury film.
To select the proper electrode, follow these guidelines:
Hg + RSH â†’ HgSR + e- + H+ Ep = +0.2 to +0.4 V
It is the complex of mercury and thiol that is actually undergoing the oxidation.
On carbon, it has been shown that disulfides are the usual product, with the reaction taking place at a much higher applied potential: