Separation of oligonucleotides by ion-exchange and ion-pair chromatography

Oligonucleotides are a key class of molecules possessing many unique features that present both challenges and opportunities when employing charged-based chromatographic techniques. This chapter covers the physiochemical properties of this class of molecules and also the major types of endogenous and therapeutic oligonucleotides. The major modes of ionic separations are ion-exchange and ion-pair chromatography. These techniques will be discussed in detail including their mechanisms, which are quite complex. These discussions will also include mixed-mode separations that employ elements of both techniques. Several specific challenges are discussed including nonspecific adsorption and key considerations with sample preparation. Mass spectrometry is a popular detector used in the determination of oligonucleotides. However, modifications are needed for both ion-exchange and ion-pair chromatography when used with this detector. Finally, several applications of ion-exchange and ion-pair chromatography are critically evaluated to demonstrate the effectiveness of these techniques for a wide-range of applications.