On-line desalting platforms have recently been shown to provide substantial utility in high-throughput analysis of both native proteins and nucleic acids, albeit utilizing different solvent systems. Typical desalting workflows for both classes of analytes have employed microflow chromatography systems, with flow rates of tens to hundreds of microliters per minute and sample usage of about 50 pmol (1.6 µg) per injection. For desalting nucleic acids, piperidine mobile phases have been shown to produce high-quality MS1 spectra with minimal adduction. Translation of this method to a nanoflow system leveraged the same basic principles as the microflow setup -in which the highly polar nucleic acids do not interact extensively with a hydrophobic stationary phase, essentially resulting in size-exclusion functionality, represented in Figure . Piperidine additives in the mobile phases minimize salt adduction to the phosphate backbone of the nucleic acid, thus yielding much cleaner MS1 spectra. The desalted nucleic acids elute with the dead volume of the column, and salts are diverted to waste. Various oligonucleotides were desalted using this nanoflow method, and examples of the averaged MS1 spectra and corresponding chromatograms of four single-stranded nucleic acids are shown in Figures and. Their sequences and monoisotopic masses are summarized in Table . The observed elution times from each chromatogram Figure S2 were consistent with elution during the exclusion volume, indicating that the oligonucleotides were not retaining on the stationary phase regardless of their size. Desalting performance obtained using this nanoflow setup was comparable to that observed using a microflow system, offering similar throughput at 15 minutes per run, but 10 times lower sample consumption per acquisition.