The desirable properties of the HPLC mobile phase were covered in an earlier article. Practices adopted for preparation and handling of the mobile phase are equally important. At this stage extreme care is required to ensure repeatability and accuracy of results. Useful tips are provided in this article that will help you achieve the desired objectives of your analysis.
- Use HPLC grade water and HPLC grade solvents sourced from reliable sources.
- Mixing of solvents is not necessarily additive. Use same mixing procedure every time to prevent errors due to heat of mixing.
- After mixing solvents vacuum filter the mobile phase using a 0.45 µ filter to remove any suspended impurities that can result in blockages of the column thereby affecting flow rate of mobile phase.
- Degass the HPLC mobile phase before use as otherwise dissolved air bubble formation can result in blockages down the line and also contribute to detector signal noise. Avoid excessive degassing as this can result in loss of volatile components of mobile phase mixture.
- HPLC mobile phase should be prepared fresh before analysis as long storage can lead to microbial growth and other suspensions which can lead to complications in the flow during analysis. In case such suspensions are seen the mobile phase should be discarded and freshly prepared.
- HPLC mobile phase should be stored in borosilicate glass bottles as in plastic containers there can be leaching of plasticizers
- Glass containers are not suitable for HPLC mobile phases with pH above 8.0 as metal ions will leach from containers. In such cases stainless steel containers are recommended.
- The HPLC mobile phase container should have narrow opening which should be covered to prevent intake of oxygen from air and also to prevent evaporation of volatile compounds.
- Use on-line filters in mobile phase bottles for removal of any residual solid suspensions.
- Oxidizable solvents such as chloroform or THF should be protected with cover of inert gas such as nitrogen.
- Photosensitive mobile phases should be kept in amber coloured bottles. In case of non-availability of such bottles these can be covered using aluminium foil.
- HPLC mobile phase changeover should be gradual and intermediate polarity solvents should be introduced in between
- Remember to wash column and pump with water to prevent deposition of solid crystalline deposits which can cause column blockages and lead to faster wear of pump components
Precautions exercised during preparation and handling of mobile phase are bound to ensure reproducibility of results run after run and save you from avoidable work stress.
Please do offer your comments and suggestions on the article.