Principles and methods for the analysis and purification of synthetic deoxyribonucleotides by high-performance liquid chromatography

Abstract

The need for high-purity oligodeoxyribonucleotides for various applications has resulted in the development of novel synthesis, purification, and analytical techniques. A diversity of methods, including polyacrylamide slab gel electrophoresis, capillary gel electrophoresis, as well as high-performance liquid chromatography (HPLC), have been successfully used to aid in the characterization and isolation of these synthetic compounds. The information contained in this review article primarily details both the theoretical and practical aspects related to the use of HPLC for the analysis and purification of synthetic DNA. In addition, a variety of postsynthesis sample preparation protocols, commonly employed prior to and after HPLC, are described.

Study on Retention Behaviour of Homo-Oligonucleotides in IP-RPLC Using Dual-Point Retention Time Correction on “Standard Column” with Internal Standards

“…However, there is no interaction between the stationary phase and DNA fragments, therefore, nucleic acids with the same length but different base composition can not be resolved in SC just as in GPC. Even in AEC that separates oligonucleotides according to the number of charged groups (phosphate linkages) [9,13], abnormal phenomena have occurred sometimes, e.g., the 123, 192, 504 and 458 bp fragments were retained longer than expected [8,14], indicating that retention mechanisms other than ion-exchange were effective. In IP-RPLC, the retention sequence is controlled by the charge of polynucleotides, thus resting with the number of ion-pairs formed.…”

Characterization of Therapeutic Oligonucleotides Using Liquid Chromatography with On-line Mass Spectrometry Detection

“…Analytical techniques traditionally used for the analysis of oligonucleotides include polyacrylamide gel electrophoresis (PAGE), capillary gel electrophoresis (CGE) Gilar et al, 1997), and anion-exchange high performance liquid chromatography (AX-HPLC) (Bourque and Cohen, 1994;Srivatsa et al, 1997;Warren and Vella, 1995). Mass spectrometry (MS) is becoming a popular tool for analysis because of its capability to identify oligonucleotides.…”

Analysis and Purification of Synthetic Oligonucleotides by Reversed-Phase High-Performance Liquid Chromatography with Photodiode Array and Mass Spectrometry Detection

“…Nonporous ion-exchange HPLC sorbents have been successfully used for synthetic oligonucleotide cleanup (22,23). The disadvantage of ion-exchange HPLC is the contamination of oligonucleotides with inorganic salts.…”