HPLC Column for Retention and Resolution of Very Polar Compounds
The HPLC biopharma application discussed in this blog post was developed on request of one of our customers, AstraZeneca Inc., for a drug compound that is currently under development and hence cannot be named!
The challenge faced by the customer was that their existing method exhibited poor retention of the compounds, was eluting near the solvent front, and showed poor resolution of some components. In addition, the choice of mobile phase and buffer was limited by the fact that UV detection at very low wavelengths was required.
Developed at our Runcorn Applications Lab in the UK, Application Note 20710, HPLC for the Retention and Resolution of Very Polar Compounds, (downloadable PDF) describes the use of our porous, graphitic carbon HPLC column (Thermo Scientific Hypercarb HPLC column), designed specifically for the analysis of difficult polar compounds, in two versions of the method:
- Method 1 is suitable for systems with low dwell volume (less than 100 µL) and optimized (low) dead volume. This combination allows the use of narrow bore columns and fast gradients, resulting in faster methods using approximately 11x less solvent.
- Method 2 is suitable for systems with higher dwell volume (approximately 1100 µL) and higher dead volume, which means a 4.6 mm diameter column is required. (This method was developed on a competitor's HPLC system based on the customer's request.)
For Method 1, all four compounds of the drug were separated in less than 4 mins and the total analysis time was 9 mins! Analysis time for Method 2 was 27 mins because the customer added 2 more compounds to the original list of 4. Both methods were tested for robustness by performing 40 runs.
Our graphitic carbon HPLC column is intended for difficult separations in reversed phase and normal phase HPLC and LC/MS applications as it offers retention mechanisms that are different from those observed on silica-based bonded phases. In case you are interested, here is a free, downloadable PDF of a guide addressing the challenges for method development for very polar compounds by using this HPLC column: Method Development Guide for Hypercarb Columns. The guide provides many tips and tricks, and covers many topics, such as Very Polar Analytes, High Temperature, Acidic Analysis, Non-Acidic Analysis, and more.
Also, don't forget to check other biopharma applications on this blog.
Do let us know if you have any questions in the Comments box; we look forward to hearing from you.