A HPLC column is the most critical component of the HPLC system. Quality of your results will greatly depend on its performance. Though it is possible to monitor the HPLC column condition by keeping track on number of theoretical plates you are advised to look for other tell-tale signs such as :
- Degradation of peak shapes
- Variations in retention times
- Sudden increase in backpressure
- Poor resolution between peaks
- Appearance of ghost peaks
It is possible to extend the useful life of your HPLC column by adhering to recommended guidelines in handling, use and storage
- Avoid dropping and other mechanical shocks. Shocks often lead to channeling which can contribute to peak tailing
- Operate the HPLC column within recommended pressure limits. Excessive pressure leads to same problems as mechanical shocks
- Use only compatible nuts, ferrules and and fittings. Incompatible fittings can lead to leaks and damage to column threads
- Never over tighten the end fittings
- Never get tempted to use the column as a stirring rod for mixing of mobile phases and buffers.
- Install the HPLC column so that the flow direction matches the direction of the arrow
- Mobile phase should be the degassed prior to use and filtered using a 0.45 µm filter. Additionally, use in-line filter in the mobile phase reservoir.
- Equilibriate the column with the mobile phase which is compatible with the manufacturer storage solvent (iso-propylalcohol). Use at least 5– 10 volumes of equilibriating mobile phase
- Before storing rinse the column to remove any residual buffer or ion- pairing agents. This is essential to prevent degradation or buffer salt deposition on drying.
- Recommended pH range for silica-based columns is 2.0 – 7.5. Some columns permit range from pH 1- 11 but for safe operation and long column life you should use it as far as possible between 2.0-7.5 pH range
- When changing mobile phase the selection of solvents should be gradual in terms of polarity . Avoid drastic changes or extreme gradients.
- Reverse flush can be used if excessive back pressure develops
- Filter all samples through 0.45 or 0.2 μm filter prior to injection
- User guard column between the injector and analytical column to prevent contaminants and particulates from reaching the column
- Use mobile phase for dissolving the sample or if it that is not possible then another solvent or a mixture of solvents of compatible polarities should be used
- Never exceed the prescribed temperature limits and for consistency of results use a column oven
- HPLC Columns should be stored under ambient temperature conditions and never in a refrigerator or oven
- Storage conditions will depend on time interval between use:
Short storage of a few hours to overnight- same solvent as last analysis can be used.
Medium interval storage (two days to weekend ) – flush with pure water to prevent microbial or mould growth.
Long-term storage – use eluent which should not contain more than 50% water.
- Use of soap or detergent solutions is not recommended for cleaning columns after use.
- Strong acids or alkalis cannot be used as they will result in total irreversible damage.
- During storage end plugs should be tightly fitted to prevent solvent evaporation leading to drying of stationary phase
We hope you found the guidelines useful for getting good quality results and increasing useful life of your column. Please share any experiences that you may have by leaving your comment.