Investigation of pore size effects at separation of oligonucleotides using Ion-pair RP HPLC : Examining of how the particle pore size of the stationary phase affects separations of oligonucleotides in therapeutic range

Sammanfattning: Oligonucleotides may become a new class of therapies with the potential of curing many today untreatable diseases. Oligonucleotides becomes increasingly more difficult to separate with an increase in length since the relative difference in retention of these very similar compounds becomes increasingly smaller. Therefore, coelution of impurities formed during synthesis may result in insufficient purity, which is necessary for therapeutic treatments. Oligonucleotides are also relatively large biomolecules, possibly consisting of hundreds of nucleotides. As a result, oligonucleotides may have limited diffusion through the stationary phase pores which affects separation performance. Surprisingly few studies have be published in this research area and a wider knowledge in how this affects separation is needed. In this master thesis, separation of deoxythymidine oligonucleotides with 5-30 mers in length were separated with 60, 100, 200 and 300 Å pore size reversed phase C4 columns. It was concluded that pore size resulted in more restricted diffusion if insufficient pore size was used. Poor peak performance was also observed with too large pore sizes which lead to less efficient separations.