HPLC analysis uses liquids as carriers to transport the injected sample to the column and then to the detector. Such carrier can be an aqueous – organic mixture, organic – organic mixture of a buffer in combination with an organic solvent.
It is important to check for complete miscibility of mobile phase solvents before selecting them for any analysis and it is equally important to check that they do not react with each other on mixing. In case the method of analysis is not documented a simple mixing of solvents in a test tube can be helpful in confirmation of both the conditions.
HPLC grade solvents are certified for use in HPLC separations. Even if a small portion of solvent is examined in a beaker it will appear to be crystal clear and suitable for direct use. However, it should be kept in mind that suspended particles or impurities in micron size range are barely seen by the naked eye. Such particles if not removed can in the long run affect the performance of the HPLC system.
Essential precautions in handling and use of HPLC mobile phase will ensure a high level of repeatability and accuracy of analytical results. Bear in mind that water is also an essential component of aqueous mobile phase mixtures. The quality of water used should also conform to the prescribed standards. The earlier article on What is the right quality of water for HPLC analysis is a useful reference.
It is essential to filter the mobile phase components even if you have sourced solvents from reputed suppliers and taken precautions to ensure that buffer salts have been fully dissolved. Suspended particulate matter can result in build up inside columns and result in development of back pressure thereby affecting flow rate and damage to pump components. In the worst scenario there can be mobile phase leakages at joint fittings accompanied with stoppage of the analytical run. Now the question arises on how to minimize such occurrences. Some steps are suggested which will help overcome such situations
- After preparing mobile phase mixture filter through a 0.45 µ size filter using a vacuum
- Prepare mobile phase fresh before use. In case this is not possible every time at least look for any visible suspended impurities or bacterial growth. In such cases it is suggested that the mobile phase should be discarded and another lot freshly prepared
- Use online filters in HPLC solvent bottles to remove any sub- 10 µ size suspended particles. Clean the filters by sonication from time to time
- Filter sample and standard solutions before collection in vials before injection
- Make use of guard columns
The suggested precautions will contribute to the useful life of your HPLC system and at same time improve the reproducibility of your chromatographic separations.