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Custom DNA Oligos – Purity Verification

In many cases oligonucleotides require quantitative analysis to verify specifications have been met. MALDI-TOF and ESI-MS can provide qualitative-quantification for purity. Preferable methods for determining purity are ion exchange (IE) or reverse-phase HPLC. In addition, these methods may be necessary to complete the characterization process. The method of choice depends on the nature of the oligonucleotide and in part to the final specifications required.

IE-HPLC (Ion Exchange, High Performance Liquid Chromatography).
The separation of the different components (n-1 truncations, deletions, etc.) of a sample via differential exchange of ionic species (such as negatively charged oligonucleotides) with ions bound to the separation phase is known as Ion Exchange chromatography. This technique enables the analysis of oligonucleotides up to 40-50 bases with single nucleotide resolution. When coupled to UV-Vis or Photodiode array (PDA) detectors, samples containing modifications with absorbance different from that of the oligonucleotides (260 nm) can be identified and thus easily quantified. However, the limited resolution obtained for oligonucleotides greater than 40 bases in length requires the utilization of a different technique: Capillary Electrophoresis (CE).

RP-HPLC (Reverse Phase, High Performance Liquid Chromatography)
Reverse phase chromatography results from the adsorption of hydrophobic molecules onto a hydrophobic solid support in a polar mobile phase. Decreasing the mobile phase polarity by using organic solvents reduces the hydrophobic interaction between the solute and the solid support resulting in selective elution. The more hydrophobic the molecule, the stronger it will adsorb onto the solid support (stationary phase), thus requiring more organic strength for elution of the molecule. Reverse phase systems can be configured with a wide variety of detectors, including UV, PDA and MS. Reverse phase chromatography is a powerful technique for achieving separation and is effective for the separation of a wide range of molecules and is complementary to IE-HPLC.

LC-MS (Liquid Chromatography Mass Spectrometry)
For applications or commercial products requiring specific purity profiles, or identification and quantification of by-products, we routinely develop extended LC-MS methods. Unlike simple mass spectrometry measurements, the extended LC-MS methods combine the strengths of RP-HPLC with the precision of MS to fully characterize oligonucleotides and identify by-products. For complex mixtures, Selective-Ion-Monitoring (SIM) provides unequaled sensitivity for ultra-low level detection limits.