The Chromatography Forum

of Delaware Valley

Liquid chromatography-mass spectrometry (LC-MS) has been the gold standard in bioanalytical chemistry for nearly three decades. Metabolomics research typically focuses on improving MS detection, and LC separations are often neglected by comparison. More often in metabolomics literature, LC is seen as a ponderous, antediluvian technique better replaced than improved.

Separations in metabolomics frequently utilize long, shallow reversed-phase (RPLC) gradients to achieve high peak capacity for the very complex samples analyzed. Very rarely, however, is attention paid to gradient elution theory and its utility in resolution optimization. Many misconceptions still abound regarding the now more common type of separation in modern bioanalytical chromatography. Although RPLC has historically dominated the bioanalytical separation space, interest in hydrophilic interaction chromatography (HILIC) as both an alternative and a complementary separation mode has grown. Regardless of separation mode, external influences on separations can often be as important as on-column considerations in high-efficiency chromatography.

Extra-column band broadening (ECBB) is very rarely discussed in metabolomics, but it is a critical factor in maximizing resolution, especially when small-bore columns are used. Dispersion due to improperly made connections and unnecessary extra-column volume widens peaks and depletes available separation power. Seemingly magical gains in signal intensity and resolution can be achieved by simply reducing the internal diameter and length of pre- and post-column tubing and making careful connections.

In this talk several topics will be covered in the hope of introducing both metabolomics scientists to chromatographic fundamentals and chromatographers to the field of metabolomics. Particular attention will be paid to the linear-solvent strength model and its application to RPLC gradient optimization. HILIC will also be discussed, as will the importance of ECBB in high-efficiency separations. Finally, examples from the presenter’s own research will be used to demonstrate practical applications of the topics discussed.