Our Support for HPC/UHPLC Method Development
Today almost every analytical methods is strongly dependent of a chromatographic or electrophoretic separation step, HPLC or UHPLC or UPLC, prior to the detection, in many cases mass spectrometry. Even mass spectrometry allows the detection of many compounds in a sample side-by-side, the quality of the analysis is dramatically enhanced when the compounds are separated and only very few are coeluting. Not using mass spectrometers as the detector, but UV-VIS, fluorescence or any other not mass based method does not work without a good separation.
HPLC/UHPLC is a science, separation science, not simply an out-of-the-box method, deep knowledge in the parameters is required to develop stable and reliable methods.
Often the development of a stable and reproducible separation method is an essential, if not the essential step in any analytical process. The most used separation methods are based on reversed-phase C18 materials, less important are C8 or C4. But other methods, like hydrophilic interaction liquid chromatography [HILIC] helps to overcome some problems.
Many mistakes when developing a separation method for new compounds can be made here:
This may work, but more probably it will fail and some observation are made:
One of the major mistakes is taking all available C18 materials as equal - they are very different in selectivity and specificity. There are more than 600 different C18 materials available and the number is increasing. Separation is strongly dependent on the solvent or buffer composition and the pH, minor changes can change the whole separation to worse.
There are many explanations for every of these observations, as some listed here, there are many more:
To find the optimal conditions for the separation problem, we will analyse your requirements, look at the structure of your compounds to be separated and the composition of your matrix. Out of these information we can suggest a combination of C18 material, the solvent/buffer system and the other conditions.
The final fine-tuning must be done in the lab.
Even is your lab does not work under regulatory-controlled conditions, like most of the research labs, it is worth to follow some of the rules as recommendations to generate a stable and reliable separation method.
HPLC/UHPLC is a science, separation science, not simply an out-of-the-box method, deep knowledge in the parameters is required to develop stable and reliable methods.
Often the development of a stable and reproducible separation method is an essential, if not the essential step in any analytical process. The most used separation methods are based on reversed-phase C18 materials, less important are C8 or C4. But other methods, like hydrophilic interaction liquid chromatography [HILIC] helps to overcome some problems.
Many mistakes when developing a separation method for new compounds can be made here:
- Just take the column which is in the HPLC/UHPLC already
- Or just take the column which is in the drawer below
- Just take the same solvent/buffer system which is already on the HPLC/UHPLC
- Just run the gradient the college has run before
- Just use all the setting which had been used before
This may work, but more probably it will fail and some observation are made:
- Peaks are not separated
- Peaks are tailing or elute very broad
- No peak is detected
One of the major mistakes is taking all available C18 materials as equal - they are very different in selectivity and specificity. There are more than 600 different C18 materials available and the number is increasing. Separation is strongly dependent on the solvent or buffer composition and the pH, minor changes can change the whole separation to worse.
There are many explanations for every of these observations, as some listed here, there are many more:
- The column used is not suited for the compounds
- The solvent/buffer system used is not suited for these compounds
- The solvent the compounds are dissolved in is not suited for the separation applied
- The detection system can not detect the compound
- The compound is not suited for HPLC/UHPLC separation
To find the optimal conditions for the separation problem, we will analyse your requirements, look at the structure of your compounds to be separated and the composition of your matrix. Out of these information we can suggest a combination of C18 material, the solvent/buffer system and the other conditions.
The final fine-tuning must be done in the lab.
Even is your lab does not work under regulatory-controlled conditions, like most of the research labs, it is worth to follow some of the rules as recommendations to generate a stable and reliable separation method.