Let’s Take a Closer Look to Biotechnology

Oligonucleotide Separation October 24, 2007

Posted by yepyhardi in purification, technique.
Tags: hplc, method, oligo, purification
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Full article at Waters Website

Separations of detritylated synthetic oligonucleotides on an XBridge™ OST C18 column are based on ion-pair, reversed-phase chromatographic principles (IP-RP-LC). As shown in Figure 1, the ion-pairing additive in the mobile phase is adsorbed on a hydrophobic sorbent and provides for charge-to-charge interactions with negative charges contained on the oligonucleotide backbone (e.g., phosphate groups).

As a result, an efficient charge-based (length-based) oligonucleotide separation is achieved (Figure 2). Gradient elution using an acetonitrile or methanol eluent displaces both ion-pairing agent and the oligo-nucleotides from the sorbent surface.

Figure 2: Separation of a 15 – 60mer Deoxythymidine Ladder on XBridge™ OST C18

HPLC Condition

• HPLC system: Waters BioAlliance™ 2796, PDA Detector with micro UV cell

• Sample Injected: Approximately 100 pmoles of a detritylated 15 – 60mer oligonucleotide ladder diluted in 0.1 M TEAA

• Column: Waters XBridge™ OST C18, 2.5 μm (2.1 x 50 mm)

• Mobile Phases: A: 0.1 M TEAA; B: Acetonitrile / 0.1M TEAA, 20/80, v/v

• Flow rate: 0.2 ml/min

• Gradient Delay: 0.45 mL

• Gradient: 40 to 62.5% B in 30 minutes (8-12.5% acetonitrile, 0.15% acetonitrile per minute)

• Detection: 260 nm, 5 scans per second

Two commonly used ion-pairing agents for oligonucleotide applications are triethyl ammonium and dimethylbutyl ammonium ions. The final pH of these mobile phases containing either of these ion-pairing reagents is adjusted by the addition of Acetic Acid, or in some cases, Hexafluoroisopropanol (HFIP). These mobile phases are volatile making them suitable for LC-MS applications.

Sample Preparation

1. Dissolve the detritylated synthetic oligonucleotide sample in Mobile Phase A (e.g., 0.1 M TEAA). For example, a 0.05 – 0.2 μmole scale synthesis can be prepared in 0.1 mL of 0.1 M TEAA. Proportionately larger or smaller volumes of 0.1M TEAA are required when dissolving samples from different scale syntheses. Due to the nature of gradient separations, relatively large volumes of sample (in low organic strength eluent) can be injected and concentrated onto the head of the column before beginning the gradient elution program.
2. Samples must be completely in solution and free of particulates before injecting onto the column. Remove all particles from the sample (Controlled Pore Glass Synthesis Support, etc.), which may block the inlet column frit, increase the operating pressure, and shorten the column life time. Sample contamination with high concentration of salts and/or detergents may also interfere with analysis.
3. To remove particulates the sample may be filtered with a 0.2 m membrane. Be sure that the selected membrane is compatible and does not dissolve with the selected Mobile Phase diluent. Contact the membrane manufacturer with solvent compatibility questions. An alternative method of particulate removal involves centrifugation for 20 minutes at 8,000 rpm, followed by the transfer of the supernatant liquid to an appropriate vial.

Recommended Mobile Phase 
The most common ion-pair mobile phase for synthetic oligonucleotide separations is based on Triethylammonium Acetate (TEAA). This mobile phase can be prepared by titrating Glacial Acetic Acid aqueous solution with Triethylamine (TEA).
Note: To maximize column life, it is ESSENTIAL that all prepared OST Mobile Phases be fi ltered through a solvent compatible, 0.45 μm membrane and contained in bottles that are clean and particulate free.
TEAA
1L of 0.1 M TEAA may be prepared as follows:

1) Perform work in a hood.
2) Add 5.6 mL of glacial Acetic Acid into 950 mL of water and mix well.
3) Slowly add 13.86 mL of TEA.
4) The pH should be adjusted to pH 7 +/- 0.5 by careful addition of Acetic Acid.
5) Adjust final volume to 1 L with water.

Alternatively, premixed TEAA can be used [(e.g., Sigma 1 M TEAA (part no. 90357)]. Mix 100 mL with 900 mL of water to prepare 1 L of 0.1 M TEAA mobile phase.

Recommended Injector Wash
Between analyses, the HPLC system injector seals should be washed. A 90% Water / 10% Acetonitrile injector wash solvent is recommended.

General Consideration in Developing Separation
Separation of detritylated synthetic oligonucleotides by ion-pair, reversed-phase chromatography uses very shallow gradients. With both TEAA and TEA-HFIP ion-pairing systems, a rate of strong eluent change between 0.1-0.25 % Acetonitrile (or Methanol) per minute is recommended. However, the formation of shallow gradients can place performance demands on LC pumps and mixers that can compromise the quality of the separation. Consequently, it is strongly advised that Mobile Phase B formulation contain a premix blend of aqueous and organic solvents (e.g., Mobile Phase A= 0.1 M TEAA and Mobile Phase B = Acetonitrile / 0.1M TEAA, 20/80, v/v) to minimize potentially inadequate solvent mixing that can compromise component resolution.