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Abstract
Oligonucleotides are short nucleic acid polymers used in research, genetic testing and
forensics. When used for these applications, they are generally made up to 13 to 25 nucleotides
and are designed to hybridize specifically to DNA or RNA sequences. These small bits of
nucleic acids are used in polymerase chain reaction (PCR) and as therapeutic agents that are
prepared by solid-phase clinical synthesis. Chromatographic separation methods have been
widely used to analyze oligonucleotides. The backbone of oligonucleotides is highly charged and
hydrophilic. Due to this the oligonucleotides do not retain well on the typical reversed-phase
HPLC columns as it is hydrophobic. However, with the aid of an alkyl amine ion-pairing (IP)
reagent in the mobile phase, the oligonucleotides retain to the column well. This success of alkyl
amine based chromatographic approaches has led to the development methods in the field of
therapeutic oligonucleotides. In this experiment, systemic evaluation of the combined impact of
alkyl amines along with HFIP, on the separation of DNA (both the n and the n-1 analogs) under
various conditions such as pH of the mobile phases, column temperature, concentrations and
percentages of the solvents in the mobile phases, and the sequences of the oligonucleotides was
assessed. Concentration and solubility have been found to be the key factors, which influenced
the retention and separation. Alkyl amines form micelles in the MP at higher concentrations and
higher pH values.
forensics. When used for these applications, they are generally made up to 13 to 25 nucleotides
and are designed to hybridize specifically to DNA or RNA sequences. These small bits of
nucleic acids are used in polymerase chain reaction (PCR) and as therapeutic agents that are
prepared by solid-phase clinical synthesis. Chromatographic separation methods have been
widely used to analyze oligonucleotides. The backbone of oligonucleotides is highly charged and
hydrophilic. Due to this the oligonucleotides do not retain well on the typical reversed-phase
HPLC columns as it is hydrophobic. However, with the aid of an alkyl amine ion-pairing (IP)
reagent in the mobile phase, the oligonucleotides retain to the column well. This success of alkyl
amine based chromatographic approaches has led to the development methods in the field of
therapeutic oligonucleotides. In this experiment, systemic evaluation of the combined impact of
alkyl amines along with HFIP, on the separation of DNA (both the n and the n-1 analogs) under
various conditions such as pH of the mobile phases, column temperature, concentrations and
percentages of the solvents in the mobile phases, and the sequences of the oligonucleotides was
assessed. Concentration and solubility have been found to be the key factors, which influenced
the retention and separation. Alkyl amines form micelles in the MP at higher concentrations and
higher pH values.