In chromatography, we often focus on mobile phases, detectors, columns, and gradients.
But there's a silent troublemaker that can ruin your run before it even begins: the sample solubility!
What Happens When Your Sample Isn’t Fully Soluble?
Presence of particulate material in the sample can clog the:
- Injector
- Column
- Tubing
Resulting in high backpressure and potential column or system damage.
If only a part of your sample dissolves, you may observe:
- Tailing
- Split peaks
- Inconsistent retention times.
If the full amount of your sample isn’t in solution, your peak area won’t reflect the real concentration = inaccurate results. (It can be due to other reasons as well)
Insoluble compounds may:
- Stick to the injection system
- Carryover in the column
Cause bleeding into subsequent runs or long-term column degradation.
What Should You Do?
Dissolve your analyte in a solvent that fully solubilizes it.
The solvent can be:
- Your mobile phase, or
- Compatible with your mobile phase.
This improves miscibility and reduces peak distortion due to solvent effects.
Filter your sample with a 0.22 µm filter to remove any remaining particulates.
Adjusting the pH of your sample slightly can improve solubility without compromising compound stability.
Store samples properly and check its solubility over time.
(especially if samples will stay for a long period in autosampler).
A perfectly designed method won't matter if your sample isn't fully in solution. Solubility is Step 0 of good chromatography.
Solubility issues are one of the most preventable causes of bad data and column damage in HPLC.
Taking the time to dissolve, filter, and validate your sample prep can save hours of troubleshooting and avoid system and column damage.
Read also:
Resource Person: Abanoub Efraim
