The common problems encountered in pumping mobile phases are solvent degassing, corrosion, and compressibility. Compressibility has already been discussed and since its influence is minor, we shall not deal with it further. The two other problems, however, need further analysis.
As previously noted, when the mobile phase contains excessive gas which remains dissolved at the pressure produced by the column, the gas may come out of the solution at the column exit or in the detector, resulting in sharp spikes. Spikes are created by microscopic bubbles which change the nature of the flowing stream making it heterogeneous while drift may occur as these microscopic bubbles gradually collect and combine in the detector cell. The main problem is oxygen (from the air) dissolving in polar solvents, particularly water
If needed, degassing may be accomplished by one of the following methods or their combination:
|Subjecting the liquid to vacuum|
|CAUTION: even a simple aspirator can cause the collapse of an ordinary flask; use only heavy-walled flasks.|
|Heating the liquid until boiling occurs.|
|Placing the container of liquid in an ultrasonic bath, or inserting an ultrasonic probe in it.|
|Bubbling a fine stream of helium through the liquid; helium has the unique ability to sparge other gases out of solutions.|
The best results were found by applying vacuum to each solvent for about 5 min, with subsequent helium purging and storing under a helium atmosphere.
Most commercially available instruments are constructed of Type 316 stainless steel.
This material represents the best compromise of corrosion resistance, workability,
strength, and cost. It is inert to all bases, all organic liquids and most nonhalogenated
acids at pH values above 2.0. Type 316 stainless steel is, however, extremely vulnerable
to contact with halogenated acids, like HCl, even at concentrations as low as 0.01 normal.
When attempting to convert a thin-layer or (glass) column chromatographic method to HPLC,
substitute another acid such as nitric, boric, or acetic for halogens. Ion-exchange
systems should be monitored carefully for discolored eluent especially when small particle
packings that exhibit high back pressure (2500+ psi) are used, since several researchers
have observed highly accelerated corrosive reactions at these pressures with some buffer