Preparation of Mobile Phase for HPLC

PEAK SOLUTIONS
A resource for chromatographers

The following is an excerpt from 'An Introduction to HPLC for Pharmaceutical Analysis' by Oona McPolin, available to purchase through the MTS website. 

Solvent grades
There are HPLC grade solvents available for the common solvents used and it is recommended that these are used for the best results. They are typically ≥ 99.9% pure. Within the HPLC grade there are different types available from some suppliers. These include ‘gradient grade’, and ‘LC-MS’. When running gradient elution and also when using a mass spectrometer as a detector the solvent used needs to be very pure to eliminate interferences which might affect the results. Acetonitrile is also available in a ‘far UV’ grade, this relates to the use of a UV detector in the far UV range, i.e. below 200nm. The difference in these grades relates to purity, very high purity solvents are usually obtained by multiple distillations. If a specialised grade is required for an analysis then this information should be included in the details of the HPLC analytical method.

Water for HPLC may be sourced from solvent suppliers in HPLC grade bottles or some type of water purification system can be used[1]. A water purification system for HPLC should generate ultra pure water (18 MΩ resistivity), it is extremely important that the system is well maintained.
Different types of methods have different tolerances to the grade of the solvents used in the mobile phase. This should be assessed thoroughly during the method development process and any requirements included in the analytical method.

Measuring solvents
When measuring out the solvents to be used in a mobile phase each solvent is measured separately using a measuring cylinder of an appropriate size rather than measuring one and making up to volume with the other. The reason for this is that the volume of the mixture is smaller than the individual volumes due to a contraction effect. This is particularly applicable to methanol but is true for other water miscible solvents to some extent.

Mixing the mobile phase
When the mobile phase contains added buffer, ion-pair reagent or other additive, it is customary to weigh out these substances using HPLC grade reagents and add them to the aqueous portion of the mobile phase mixture. The pH is then adjusted on the aqueous portion prior to mixing with the organic portion. There are a number of options about how to perform the mixing of the mobile phase:

Isocratic methods
 The aqueous and the organic portions of the mobile phase may be measured separately and mixed in a container by the analyst (premixing) or each portion may be placed on the HPLC system and mixed in the correct proportions by the HPLC pump (online mixing).

Gradient methods
The aqueous and the organic portions of the mobile phase for both the starting conditions and for the final conditions of the gradient may be measured separately and mixed in a container by the analyst (premixing). This will result in two mobile phases, e.g. for the gradient 20 to 80 %B over 20 minutes, a mobile phase of 20 %B and a mobile phase of 80 %B are prepared by the analyst, these are then placed on the HPLC system and mixed during the analysis by the HPLC pump. The other option, as in the isocratic method, is to place the aqueous and organic portions on the HPLC system and mix in the correct proportions by the HPLC pump (online mixing).

There are advantages and disadvantages to each approach. Premixing means that the pump does not have to perform mixing in the isocratic case and performs the least amount of mixing in the gradient case. If the pump being used is not capable of this type of mixing then the premixing option is preferred. Premixed mobile phases are subject to analyst error and different batches will be slightly different to each other. Online mixing removes the analyst error and is often more convenient to operate. Bottles received from the supplier containing solvent and premade reagent can be placed directly on the system preventing contamination associated with measurement. Plastic coated bottles are available for this purpose. However, if the pump is unable to deliver a well mixed mobile phase then online mixing is not suitable. The final decision depends on the pump capabilities, the analyst preference and the application.

Reference: 
1. S. Mabic, C.Regnault, J. Krol, LC•GC Eur., 18(7), 2005, ‘The Misunderstood Laboratory Solvent: Reagent Water for HPLC’.

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