Getting Started in HPLC

Section 2A. HPLC Solvent Reservoirs

   
The reservoir that holds the mobile phase is often no more than a glass bottle. Often, the reagent bottle that holds our HPLC solvent can be used as a reservoir. Solvent is delivered from the reservoir to the pump by means of Teflon tubing -- called the "inlet line" to the pump. Some HPLC systems like the Agilent 1100 shown at the right have special compartments to hold one or more mobile phase reservoirs. The reservoirs in these systems may have additional features that allow the mobile phase to be degassed and isolated from contact with air.

 

This Agilent 1100 HPLC system has a mobile phase compartment visible at the top of the instrument.


   
The requirements for a solvent reservoir are simple:

  • The reservoir and its attachment to the pump should be made of materials that will not contaminate the mobile phase: Teflon, glass, or stainless steel.

  • The vessel should have some sort of cap to prevent particulate matter from contaminating the mobile phase. Some LC systems provide for this as part of the reservoir design. If you are using a solvent bottle as a reservoir, the top of the bottle can be wrapped in aluminum foil to keep dust out or the bottle cap can be drilled to allow inserting the inlet line through the cap.

  • Don't close the bottle too tightly or removal of mobile phase by the pump will create a vacuum. This prevents mobile phase from flowing the pump, creating a "vapor lock" within the pump.

This "generic" mobile phase reservoir shows the principle components of the reservoir module.


 
Examples of mobile phase reservoirs range from standard laboratory glassware such as beakers or flasks covered with aluminum foil through larger vessels such as media bottles, solvent jugs, or carboys, to purpose-made glassware that includes built-in provision for stirring and degassing.

Stirring capability is not absolutely required, but can help to keep the mobile phase homogeneous as solvent is lost to evaporation. Degassing -- removing dissolved air from the mobile phase -- is not always required, but it is generally recommended for reliable operation. We will have more to say about degassing when we cover pumping systems.


Mobile phase reservoirs range from purpose-made containers through standard solvent jugs.


It is important that particulate matter be kept out of the mobile phase since particulates can damage the pump and injector as well as plug the column. Mobile phases are often filtered before adding them to the reservoir. In addition, a 10-micron frit or inlet filter should be connected to the end of the inlet line that dips into the reservoir. This inlet frit serves more than one purpose. Besides providing extra protection against particulates entering the pump, the inlet filter serves to hold the inlet line at the bottom of the reservoir. The stiffness of the inlet line tends to allow this Teflon tubing to creep out of the reservoir, preventing use of all the mobile phase unless an inlet filter is used to weigh down the end of the tubing. For this reason, inlet filters are often called "sinkers".


"Sinker" frits help keep the inlet line submerged and provide a final line of defense against particulate contamination.


   
The sinker frit is not a substitute for filtering the mobile phase. The typical pore size used in a sinker frit is on the order of 5 - 10 microns; frits of smaller pore size are too likely to plug. In general, the mobile phase should be filtered through a 0.3 to 0.5-micron frit after the solvents and buffers are mixed in order to remove particulate matter. The sinker frit protects against the occasional larger dust particle encountered after the mobile phase has been filtered.

Despite the relatively large pore size, sinker frits can and do get plugged over time. This is especially likely to happen with aqueous buffers as a result of bacterial growth. It is a good idea to periodically check the inlet frit to be certain that mobile phase flows freely. The "siphon test" is a good way to accomplish this:

  1. Make sure the pumping system is primed (i.e., that there is no air in the inlet line)
  2. Disconnect the inlet line where it enters the pump.
  3. Place the disconnected inlet line over a beaker or other collection vessel.
  4. Raise the solvent reservoir about 50 cm and watch what happens. If the mobile phase siphons freely, then the inlet frit is probably OK. If the mobile phase drips out slowly, then either the line is kinked or the inlet frit is partially plugged.


 
   
It is often necessary to remove dissolved air from the mobile phase before the mobile phase is fed to the pump. This procedure is called mobile-phase degassing. The reason for degassing the mobile phase is that dissolved air tends to be released inside the HPLC system. This makes the operation of many HPLC pumps unreliable, leading to fluctuations in flow rate. Bubbles can also get trapped in the detector flow cell, causing problems with this module as well.


 
   
 

 


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Last revised: April 06, 2001.