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Michael G. Bartlett, Ph.D., FAAPS
University of Georgia
Oligonucleotides have unique challenges relative to the other classes of biomolecules. They are hydrophilic, have a high number of negative charges, and may have complex secondary structure. Analysis of oligonucleotides has generally focused on the use of ion-pair liquid chromatography. This solvent system has proven to be more complex than originally believed showing great variability in response when using different components. In addition, the formation of colloids in the mobile phase can dramatically reduce the effectiveness of these mobile phases, if not properly controlled.
As a result of these studies, it is apparent that there is no single combination of ion-pairing agent, fluorinated alcohol and organic solvent that is optimal for LC-MS of oligonucleotides. There is a complex relationship between the composition of the oligonucleotide and the solution components involved in promoting efficient electrospray desorption and chromatographic performance. In addition, colloids were noted to form when using particular concentrations and combinations of mobile phase components. The formation of these colloids was found to be pH dependent.
Another significant challenge with oligonucleotide LC-MS is non-specific adsorption. The loss of analytes can occur during both sample preparation and chromatographic analysis and different approaches are needed to address both of these situations. More recently we have been exploring microflow LC, nanoelectrospray and hydrophilic interaction liquid chromatography as ways to improve both separations and detection limits for oligonucleotides.