Clarity Oligo-RP

The void volume corresponds to the amount of mobile phase in an LC system. In most separation modes the void volume would be equivalent to the amount of mobile phase needed to elute a compound that doesn't interact with the stationary phase and is hence unretained. In size exclusion, analytes are not separated based on chemical interactions with the stationary phase but rather their ability, based on size, to enter pores in the stationary phase. Therefore, in size exclusion the terms total permeation and total exclusion volume have greater significance than void volume. The total permeation volume corresponds to the amount of mobile phase in the system, including that in the pores of the stationary phase, whereas the total exclusion volume does not include the mobile phase within the pores of the stationary phase. Consequently, the total permeation volume corresponds to the amount of mobile phase needed to elute a compound that is small enough to completely enter into the stationary phase pores and the total exclusion volume is the amount of mobile phase needed to elute a compound that is entirely excluded from the stationary phase pores.

The following loading values are consistent with the specified internal diameter for a 20-40mer oligonucleotide: 4.6 mm/250 nanomol 10 mm/1 µmol 21.2 mm/5-10 µmol When considering the mass of the oligonucleotide being loaded onto the column in nanomole values, we can convert from nanomole to milligrams by considering that one deoxyribonucleotide monophosphate is equivalent to approximately 300 Daltons. Therefore, when 250 nmol of a 20mer oligo is loaded onto a 4.6 mm ID column, the loading mass is about 1.5 mg.

Typically ion-pairing reagents are used to increase the retention and resolution of oligo’s separated by reversed-phase HPLC. High molarity amounts (100 mM triethylamine acetate [TEAA]) are typically added when analyzing oligonucleotides, however the high level of TEAA leads to ion suppression that makes MS detection of oligonucleotides difficult at therapeutic and reagent concentrations. With LCMS we recommend hexafluoroisopropanol (HFIP) as a mobile-phase additive which has enabled reasonable sensitivity by MS. Our standard recommendation is 100mM HFIP and 4mM TEA in both A and B of a gradient mixture however more recent studies have shown some oligonucleotides work well even at concentrations as low as 10mM HFIP.

The alkylamine used directly affects retention time, with longer chains leading to increased retention, e.g. hexylamine will show longer retention times than triethylamine. There is also a difference in solubility so generally gradients need to be adjusted to allow for this. Generally, the effect of ion-pair is oligo-specific; for example, Gapmers longer amines offer better resolution whereas for siRNA shorter amines offer an improvement in resolution

Phosphorothioate (PS) can frequently desulfurize in to Phosphodiester (PO) and can result in reduced recoveries during solid phase extraction and sample clean-up step. To abstain PS in oxidizing back to PO we can add anti-oxidants like Cysteine in to the loading buffers. For e.g. in Clarity OTX Loading / Lysis buffer add approx. 1gm Cysteine in 1L bottle of the buffer.