Hydrophilic interaction liquid chromatography (HILIC) is one of the fastest growing modes of separation, in which any polar chromatographic surface can be used. Chemically bonded diol coated phases, as found in TSKgel SW size exclusion chromatography (SEC) columns, demonstrate high polarity and hydrogen bonding properties and do not contain ionizable groups other than the unreacted residual silanols, making them appropriate for HILIC mode.
For many years, SEC columns have been used to separate various nucleic acid species such as DNA, RNA, and tRNA as well as their constituent bases, adenine, guanine, thymine, cytosine, and uracil. In medicine, several primary nucleobases are the basis for the nucleoside analogues and other synthetic analogs which are used as anticancer and antiviral agents. Nucleobase modifications are the basis of oligonucleotide-based therapeutics, making their purification very important.
The TSKgel SuperSW mAb HTP column is a newly introduced SEC column designed for the high throughput separation of monoclonal antibodies from their high and low molecular mass variants. TSKgel SuperSW mAb HTP has a diol coating to minimize secondary interactions which may occur in SEC separations. This note demonstrates the benefits of using a TSKgel SuperSW mAb HTP column in HILIC mode for the superior resolution of four nucleobases, as opposed to using the column in SEC mode or using a HILIC column.Materials and Methods
Results and Discussion
This work illustrates the novelty and utility of the TSKgel SuperSW mAb HTP column as a diol-functionalized HILIC column for the high resolution separation of nucleobases on the basis of their relative hydrophilicity, rather than differences in their relative molecular mass. As shown, markedly different separation profiles are observed with the use of the TSKgel Amide-80 HILIC column under identical chromatographic conditions. Additionally, nucleobase separation using the TSKgel SuperSW mAb HTP under conventional SEC conditions yielded poor resolution of all components, making it an ineffective mode of separation for this application. The TSKgel SuperSW mAb HTP column, while designed for SEC separation of monoclonal antibodies, is an extremely effective tool in HILIC mode that should be considered for the fast separation of nucleobases.
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