Choice of the right buffer and pH for a mobile phase is one thing, but there are a few more things to consider and take care of when working with a buffered mobile phase in your HPLC or UHPLC analysis. To keep it simple I prepared an overview of the (in my opinion) most important points:
- Also in HPLC, the quality of output is determined by the quality of input. Therefore, all solvents used should be of at least HPLC or MS quality so that there are no concerns about contamination that may lead to ghost peaks or buildup of blockages in the system. Ultrapure water should always be used to prepare any aqueous solvents, such as buffer solutions.
- Dedicated safety caps (figure 1) are a good solution to protect the mobile phase from contamination and loss of solvent due to evaporation. They seal the bottles, while ensuring safe outflow of HPLC solvents through fitted capillaries. Foil or parafilm should NOT be used for covering mobile phase bottles, as it doesn’t provide an airtight closure and can result in extraction of leachables.
- Ideally, buffers should be freshly prepared on the day of use. Ensure that the solution is clear, homogeneous and free from non-dissolved particles, filter through a 0.45 μm filter if necessary. In case that buffer solutions are stored, ensure they are still good for use before putting them on your system, as they have limited lifetime.
- Most buffers provide ideal conditions to support microbial growth. A buffer solution that is kept at room temperature for an extended period of time, will become cloudy, indicating growth of microorganisms. These contaminants can cause a range of problems, such as clogging in the system, coated check valves and chromatographic interferences. Therefore, some precautions should be observed in practice:
- It is mandatory to check the adjusted pH BEFORE adding any organic solvent. Ensure the pH meter is accurately calibrated before the measurement. While not recommended, if you choose to measure the pH in the mobile phase bottle, clean the probe beforehand, to avoid contamination.
- Seals and other wetted parts can be damaged by high salt content and/or extreme pH conditions. Use of a rear seal wash (typically 10 - 20 % v/v IPA in water) to remove any potentially detrimental mobile phase residues is useful to enhance the lifetime of piston and seals.
- Buffer solubility is an important point to consider and depends on buffer type, concentration, pH and type of organic solvent employed. Ideally, the salt should be completely water-soluble, and care must be taken, that no precipitation occurs during gradient analysis, when the aqueous solution is mixed with an organic solvent. It's best to test the mixture in advance to ensure that it remains in solution, as insoluble crystals can build up and cause blockages in the instrument. An easy test can be performed prior to the experiment by mixing different portions of buffer and organic solvent in a test tube as shown in picture 3. When the mixture turns cloudy, the salt is not soluble in this mobile phase composition.
- When using a pH buffer, acid or base only in mobile phase A, in gradient analysis, ionic strength of the mobile phase will decrease with the increasing amount of organic solvent (mobile phase B). Addition of the same concentration of buffer, acid or base also to the organic solvent (mobile phase B) can counter this effect and minimises problems with a sloping baseline, pH and ion-strength gradients and improves linearity for charged compounds as shown in figure 4.
A lot of the problems that occur during HPLC or UHPLC analysis can be attributed to the mobile phase quality, or blockages in the tubing, due to particulate introduced via solvent delivery. Therefore, it is worth paying attention to good practice on solvent selection and preparation, to avoid any lasting trouble at a later stage. However – I will come to troubleshooting advice at some point in My Blog, explaining how to deal with those problems should they occur after all. But first – after dealing with the mobile phase for some time, I’ll talk about the stationary phase in my next article.
