Desalting is a basic purification method in oligo synthesis that removes small impurities like salts and synthesis by-products. It’s a quick, cost-effective process ideal for applications where ultra-high purity isn’t essential, delivering oligonucleotides ready for routine assays.

Reversed-phase chromatography is a separation technique that uses a hydrophobic stationary phase to purify and analyze synthetic oligonucleotides. The method is based on the principle of separating molecules according to their hydrophobicity, with oligonucleotide separation primarily determined by length and the presence of the temporary, highly hydrophobic 5′ DMT group and other hydrophobic groups. Different molecules interact with the stationary phase and mobile phase to varying degrees, resulting in different elution times.

At Ella Biotech we use RP-HPLC for the purification of oligonucleotides as well as for quality control.

Ion exchange chromatography (IEX) separates oligonucleotides based on their negative charge, which depends on the number of phosphodiester groups in their backbone, and the charge of modifications. The method uses a cationic stationary phase, and separation improves with increasing ion strength of the mobile phase. This method is ideal for separating full-length oligonucleotides from impurities, but is only applicable to shorter sequences.

We use IEX-HPLC as a complementary method to reversed-phase chromatography for both purification and quality control.

Polyacrylamide gel electrophoresis (PAGE) is a technique we use to separate oligonucleotides based on their electrophoretic mobility, which is determined by their length, conformation, and charge. The technique works by moving molecules through a polyacrylamide gel within an electric field, where the charge-to-size ratio determines migration speed.

PAGE is primarily used for quality control purposes and is offered as a purification method when specifically requested by customers.

The above described methods can be adjusted to fit your demand.

Electrospray ionization (ESI) mass spectrometry uses a small amount of the synthetic oligonucleotide that is ionized to multiple negatively charged ions/species that are propelled into a mass detector/analyzer. The ESI-MS technique has the advantage that the oligonucleotide is usually not fragmented during the ionization process and is suitable for the analysis of longer oligonucleotides (up to ~150 bases in length).

At Ella Biotech, ESI-MS is primarily used for analyzing oligonucleotides longer than 30mers, as well as for all oligos produced in accordance with ISO 13485:2016 standards.

Matrix-assisted laser desorption/ionization (MALDI) is a mild ionization technique used in mass spectrometry to analyze oligonucleotides, typically up to 50-60 bases in length (around 18,000 Daltons). The MALDI-TOF (Time of Flight) method is ideal for rapid identity control of oligonucleotides, providing fast analysis times and high throughput.

MALDI-MS is our preferred method for internal high-throughput quality control. We perform quality control checks on oligos from each production run, as well as all modified oligonucleotides and dual-labeled probes produced.

Ion-exchange HPLC is an extremely effective method for separating oligonucleotides based on their charge. It employs a salt-gradient mobile phase on stationary phase, offering a highly efficient solution for oligonucleotide analysis. This method is particularly well-suited to the analysis of up to 40-base sequences, including those with significant secondary structure, such as high-GC content oligonucleotides.

We also use IEX-HPLC for the purification of the oligonucleotide as well as for quality control.

At Ella Biotech we use RP-HPLC for both purification and quality control of oligonucleotides.

RP-HPLC is based on the difference in hydrophobicity between the full-length oligonucleotide sequence, which contains more hydrophobic groups, and shorter sequences. This technique allows for efficient separation, providing quantitative as well as purity assessments of oligos. It is especially effective for analyzing oligonucleotides up to 50 bases in length.

Ultraviolet (UV) spectrometry is a technique that measures the absorbance of ultraviolet light at 260 nanometres (nm), which correlates with the concentration of nucleotides in a solution. This technique offers a rapid and reliable method for determining the final amount/concentration of oligos by evaluating the optical density (OD) of the sample.