Nucleic acid therapeutics such as antisense, siRNA and aptamers are expected to play an important role as next-generation pharmaceuticals together with antibody drugs. These drugs demand chromatographic purification and analysis that can recognize slight structural differences following synthesis.
On this page, we provide useful tips for optimization of ion-exchange chromatography methods for oligonucleotides.
Samples
| Single-strand DNA | 5’-TCATCACACTGAATACCAAT-3’ (DNA 20 mer) |
| 5’-GTCATCACACTGAATACCAAT-3’ (DNA 21 mer) | |
| Single-strand RNA | 5’-U(M)C(M)A(M)U(M)C(M)A(M)C(M)A(M)C(M)U(M)G(M)A(M)A(M)U(M)A(M)C(M)C(M)A(M)A(M)U(M)-3’ (2’-OMe RNA 20 mer) |
| 5’-G(M)U(M)C(M)A(M)U(M)C(M)A(M)C(M)A(M)C(M)U(M)G(M)A(M)A(M)U(M)A(M)C(M)C(M)A(M)A(M)U(M)-3’ (2’-OMe RNA 21 mer) |
|
| 5’-UCAUCACACUGAAUACCAAU-3’ (RNA 20 mer) | |
| 5’-GUCAUCACACUGAAUACCAAU-3’ (RNA 21 mer) |
N(M)=2’-OMe RNA
Reducing carry-over
Carry-over is observed on gradient with low initial concentration of NaCl. But good separation with virtually no carry-over can be achieved by increasing the initial concentration (e.g. 300-400 mM NaCl).
A) 20 mM Tris-HCl (pH8.1)
B) 20 mM Tris-HCl (pH8.1) containing 1.0 M NaCl
5-70%B (0-15 min), 74%B (15-18 min), 5%B (18-33 min)
Initial : 50 mM NaCl
A) 20 mM Tris-HCl (pH8.1)
B) 20 mM Tris-HCl (pH8.1) containing 1.0 M NaCl
40-70%B (0-15 min), 74%B (15-18 min), 40%B (18-33 min)
Initial : 400 mM NaCl
| Column | BioPro IEX QF, 5 µm, 100 X 4.6 mmI.D. |
|---|---|
| Flow rate | 1.0 mL/min |
| Temperature | 25°C |
| Detection | UV at 260 nm |
| Injection | 2 µL (10 nmol/mL) |
Improving peak tailing
By changing the buffer from 20 mM Tris-HCl (pH 8.1) to 10 mM NaOH, tailing factor of the oligonucleotide peak was improved. In addition, changing counter ion from NaCl to NaClO4 is effective.
→ It is important to optimize buffer and counter ion for excellent peak shape of oligonucleotides.
1) Influence of buffer type 
| Eluent | A) X B) X containing 2.0 M NaCl 15-100%B (0-30 min) |
|---|
2) Influence of counter ion type
| Eluent | A) 10 mM NaOH B) 10 mM NaOH containing 2.0 M Y 15-100%B (0-30 min) for NaCl 5-50%B (0-30 min) for NaClO4 |
|---|
Gradient profile is adjusted because eluting strength of NaClO4 is two to three times more than that of NaCl on ion exchange chromatography.
| Column | BioPro IEX QF, 5 µm, 100 X 4.6 mmI.D. |
|---|---|
| Flow rate | 1.0 mL/min |
| Temperature | 25°C |
| Detection | UV at 260 nm |
| Injection | 2 µL (10 nmol/mL) |
| Sample | RNA 20 mer |
Analysis examples with the optimized conditions
Good separation without carry-over and peak tailing of oligonucleotides was achieved by optimization of buffer/counter ion in the mobile phase and gradient profile, and by using BioPro IEX QF, non porous anion exchange column.
Single-strand DNA 
Single-strand 2’-OMe RNA
Single-strand RNA
| Column | BioPro IEX QF 5 µm, 100 X 4.6 mm.I.D. |
|---|---|
| Eluent | A) 10 mM NaOH B) 10 mM NaOH containing 1.0 M NaClO4 25-55%B (0-15 min), 100%B (15-20 min) |
| Flow rate | 1.0 mL/min |
| Temperature | 25°C |
| Detection | UV at 260 nm |
| Injection | 4 µL (5 nmol/mL each) |
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