Optimization of Gradient Elution in UPLC: A Core Study on the Separation of Homoserine Lactones Produced by Bukholderia Ubonensis and Structure Confirmation with Ultra High Resolution Mass Spectrometry

Abstract

Speed of analysis is a key factor in liquid chromatography. One possible way is to decrease the particle size and thus increase the pressure (ultra high pressure liquid chromatography, UHPLC). For the separation of a complex mixture, the application of gradient elution techniques is expedient. From a practical point of view, it has to be questioned how the high pressure influences the separation parameters at gradient mode. In our study we investigated the following chromatographic parameters: peak width versus temperature, peak width versus gradient time, retention time versus temperature, and retention factor versus flow rate. It was shown that even in sample volume overload conditions the gradient system worked in a reproducible way; the maximum possible sample volume (20 µL) was injected to get the lowest possible limit of detection. Increasing the flow rate, the peak width increased only moderately, and the same was found when the temperature was decreased. However, at 60°C the signal to noise ratio decreased in a small compass showing the thermal disequilibrium between the column outlet and the detector cell, which might have caused extra noise. Finally, a practical separation was presented in which the UHPLC can be applied for the determination of N‐acylhomoserine lantones (AHLs), which have a relatively low UV absorption at low wavelength. The detection limit was low enough for practical applications because of the low dispersion due to a small diameter stationary phase and the low dead volume in the column. The UPLC method was applied as a first screening for AHLs and the results were confirmed by fourier transformation ion cyclotron resonance mass spectrometry (FTICR‐MS), which has a high selectivity.

Keywords:

• Ultra high pressure liquid chromatography
• Gradient elution
• Overloading
• Separation parameters
• FTICR‐MS

Acknowledgments

All work was done at the GSF National Research Center for Environment and Health with financial support of the Chinese Scholarship Council (CSC) of X. L. as guest scientist. The authors want to thank Dr. Michael Rothballer, ATP/GSF for preparing the Burkholderia ubonensis real samples. Special thanks to Brigitte Look and Heidi Neumeir for their precise technical assistance and for their always encouraging words.