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Biochromatography - Oligonucleotide Analysis

Oligonucleotides are among the most widely used reagents in modern molecular biology, where they participate in a variety of applications, from primers for DNA sequencing and PCR to therapeutic agents and tools to study gene function.

Becauese of the nature of these applications, the quality of synthesised oligonucleotides can be of vital importance. Demonstration of purity, especially of clinical grade oligonucleotides is a critical step that ensures the efficacy and safety of these reagents.

Analysis of oligonucleotides can be very demanding often requiring separation based on small changes in size or sequence. Separations become increasingly difficult as the size of the oligonucleotide increases.

There are three UHPLC/HPLC techniques that are routinely used for oligonucleotide separations:

Trityl-on: This procedure is relatively simple to perform and separates the full-length target oligo, which still has the DMT group attached, from the deprotected failure sequences. The analytical information obtained is limited and this is generally considered to be a purification method.

Ion-exchange separations of the trityl-off, deprotected oligos: This method uses the negative charge on the backbone of the oligo to facilitate the separation. Resolution is good for the shorter oligos but decreases with increasing chain length. Aqueous eluents are used but oligos are highly charged, and high concentrations of salt are needed to achieve elution from the column.

Ion-pair reversed-phase separation of the trityl-off, deprotected oligos: This technique uses organic solvents and volatile ion-pairing agents and is suitable for LC/MS. The technique is best performed with high efficiency particles. Conditions that fully denature the oligos and prevent association with complimentary sequences are required. Thus, the separation is best performed at elevated temperatures.


Product Information

Crawford Scientific offers several specialty columns for oligonucleotide anaysis. For more product information see the links below;

AdvanceBio Oligonucleotide Columns

Agilent Eclipse Plus C18

Agilent PL-SAX

Agilent Polaris C18-A

Agilent PLRP-S

Thermo Scientific DNAPAC PA100

Thermo Scientific DNAPAC PA200

Thermo Scientific DNASwift Monolith



Please contact us to discuss your specific biochromatography needs

contact analytical services

Application notes

Here at Crawford Scientific we have also gathered all of the most relevant and useful documentation, including application notes, relating to oligonucleotide analysis, see below;


Fast Oligonucleotide Impurity Profiling

Polar Modified Stationary Phases for the analysis of Nucleotides

High performance Oligonucleotide Purification using Agilent TOP-DNA

Improved Column Lifetime with Thermally Stable Polymer Columns for Oligonucleotide Ion-Pair RP HPLC

Use Temperature to Enhance Oligonucleotide Mass Transfer and Improve Resolution in Ion-Pair RP HPLC

Oligonucleotides—Analysis of Antisense Therapeutics


DNAPAC PA200 Oligonucelotide Column

DNAPAC PA100 Oligonucelotide Column

DNASwift Monolith for DNA RNA



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