Mobile phases in HPLC

Preparation of mobile phases

Careful preparation of the solvents is very important. This can save a considerable amount of time for troubleshooting of malfunctions, ground noise, and so on.

Quality of solvents

All reagents and solvents should be of the highest quality (here you will find an overview of the grades of purity and qualities). HPLC reagents generally cost slightly more than lower quality reagents, but the difference in purity is striking. HPLC reagents do not contain impurities which could lead to undesired peaks in a chromatogram baseline, while lower quality reagents contain traces of impurities that can produce undesirable baseline peaks.

It must be ensured that the water used in the buffer production is of the highest purity. Deionized water often contains traces of organic compounds and is therefore not recommended for HPLC applications. Ultra-pure HPLC water (18mΩ resistance) is produced by passing deionized water through an ion exchange bed. Modern water purification instruments use this mechanism to produce high quality water in high volumes. For preference, water with HPLC quality can be obtained from solvent suppliers.

Important: Do not store HPLC water in plastic containers. Additives of the plastic can get into the water and contaminate it. Always store HPLC water in glass containers.

Buffer solutions

All buffers should be freshly prepared on the day of use. This ensures that the pH of the buffers is not affected by prolonged storage and that no microbial growth occurs. Changes in the pH value, as well as microbial growth, influence the chromatography.

Note that when buffer solutions are stored, they have a limited lifetime. For further guidance on the shelf life of the buffer, reference is made to Pharmacopoeia monographs or the like.

Buffering reagents may contain a stabilizing agent, for example sodium metabisulfite. These stabilizing agents often affect the optical and chromatographic behaviour of buffer solutions, so it is usually advisable to buy reagents which do not contain a stabilizer. The containers of solid reagents are easily contaminated by repeated use. For this reason, we recommend to buy reagents in low container weights.

Filtration

Ideally, all HPLC solvents should be filtered with a 0.45μm filter before use. By using a filter all particles are removed, which could lead to clogging. After filtration, the solvents should be stored in a covered container to prevent re-contamination with dust, etc.

Filtering the HPLC solvents improves both, your chromatography and the wear of the HPLC system. The performance of pump pistons, seals and non-return valves is increased and the lifetime is maximized.

Degassing

Before the freshly prepared mobile phase is pumped into the HPLC system, it should be thoroughly degassed to remove all dissolved gases. Dissolved gas can be removed from the solution by:

• Beading with helium
• Sonication
• Vacuum filtration

If the mobile phase is not degassed, air bubbles can form in the low pressure of the detector cell, which in turn leads to problems such as system instability, false base line peaks,

The most efficient form of degassing is bubbling helium (or another gas with low solubility) through the solvent. When this process is available, we recommend that the mobile phase be continuously degassed at a very low level throughout the analysis. This will inhibit the re-absorption of gases during the analysis time.

Note: Ensure that the solvent container has an air hole to the atmosphere to prevent pressure build-up in the container.