Purification of Synthetic OligonucleotidesUtilizing Denaturing High Performance
Liquid Chromatography (dHPLC)
S. Sheikhavandi1, J. Riviello1, L. Bao2, J. Adams3, M.A. Marino4
1Applied Genomics and Molecular Genetics, Transgenomic, Inc.,San Diego, CA;

2Separations Research and Development, Transgenomic, Inc., San Diego, CA;
3Market Development, Transgenomic, Inc., San Diego, CA;
4Applied Genomics and Molecular Genetics, Transgenomic, Inc., Gaithersburg, MD.

The genomics revolution has rapidly increased the demand for highly
purified synthetic oligonucleotides for PCR, cloning, genotyping, chipbased technologies, and various applications in the field ofpharmacogenetics. Gel electrophoresis and high performance liquid
chromatography (HPLC) have previously been the most commonly usedtech niques for oligo purification. Compared to both gel electrophoresis
and typical IP-RP HPLC, dHPLC offers the advantages of higher resolution,
larger size range for separation, purification chemistries, reproducibility,automation, and convenient data archiving.
We describe the use of dHPLC for the analysis and purification ofsynthetic oligonucleotides. The u nique separation chemistries of theOligoSEP® cartridges combined with elevated temperatures (80°C) offers
rapid and highly efficient separation and purification. The WAVE® OLIGO
System is a fully automated dHPLC system specifically designed for theanalysis and purification of synthetic oligonucleotides. Using thermallystable polymeric reversed phase columns in the ion-pairing mode withfully denaturing conditions, a broad range of synthetic oligonucleotides
can be purified. In addition, by changing the eluent chemistry, separations
can be performed based either on oligo length alone, or a combination oflength and base pair composition. The purification of unmodified, dyelabeled, and biotinylated oligonucleotides has also been accomplishedusing this technique.

OligoSep – A 4.6 x 50 mm cartridge containing 2.2µ microporous
polystyrene/divinylbenzene particles which are octyldecylated. Primarily
used for analytical work

Purification of 1µmol of a 48 mer using theOligoSep Prep HC with TEAAGenentech 116006 Crude and WAVE Purified
3230
282624

Absorbance (mV)

Cartridges for Oligonucleotide Analysisand Purification

Abstract

22Purification

20181614(fraction collect ed)

12108OligoPrep HC – A 7.8 x 50 mm cartridge containing 5.6µ macroporous
divinylbenzene particles which are octyldecylated. Used for both analyticaland purification. Loading capacity is 1 µmol

6420-2-40

1234

5678910Tim e (Minut es)

11121314151617

Chromatographic
Conditions

All separations are run at 80°C

Denaturing Separation Chemistries for
Oligonucleotide Analysis and Purification

Cartridge : OligoPrep HC
Eluent A: 100 mM TEAA
Eluent B: 100 mM
TEAA/75% ACN
Gradient: 10% to 35% B in
14 minFlow rate: 0.9 mL/min
Sample : 1500 µL➤ Triethylammonium acetate● Cationic ion pairing reagent which results in separations which aredependent on both length and composition of the oligonucleotide

T emperature: 80°C
D et ection: UV@290nm● Addition of hydrophobic groups, such as dyes, minimizes

compositional differences

Synthesis of Oligonucleotides Using
Phosphoramidites

Wavemaker Fragment Collector Screen➤ Tetrabutylammonium bromide● Cationic ion pairing reagent which results in separations remove are

A sequential andcontinuous cycle

dependent primarily on the length of the oligonucleotide● Addition of hydrophobic groups, such as dyes, increase retention

time but length dependency is maintained

1. Remove DMTAll separations are run at 80°C

2. Cap with OAc
3. Couple Amidite
4. Oxidize triester
5. Repeat
Final productdeprotected and cleaved
from synthesis support
using NH4OH

Separation of a Poly dT Size Ladder Using
TBAB
Chromatographic
Conditions
Cartridge: OligoSep
Eluent A: 30 mM TBAB/10%
ACN

Purity vs Oligonucleotide Length

Eluent B: 30 mM TBAB/65%ACN

Length20 mer

Theoretical yields (%)
based on coupling efficiency

90.0%

95.0%

98.0%

99.0%

99.5%

12366782

9040 mer

1.513456782

60 mer

0.18

4.6

3055

74

80 mer0.02

1.7204567

100 mer

0.003

0.6

133761Comparison of Crude and Purified 72 mer

Gradient: 34% B-2 to 55%in 2 min, 55% to 68% in 4min, 68% to 77% in 3 min,77% to 95% in 5 min
Flow rate: 0.9 mL/min
Sample : 10µL of 1.2
pmol/µL

Chromatographic
Conditions
Column: OligoPrep HC
Eluent A: 100mM TEAA
Eluent B: 100mM
TEAA/25% ACN
Gradient: 5% B for 0.5min, 20% to 40% B from
0.6 to 15 min
Flow rate: 0.9 mL/min
Sample : 25 µL
Temperature: 80°C

T emperature: 80°C
D et ection: UV@260 nm

Comparison of TEAA and TBAB for the
Separation of Five 20 mers

Detection: UV@260 nm

Conclusion: Oligon ucleotides directly from a synthesizer always containsignificant impurities

TEAA

TBAB

size and composition
based separation

size basedseparation

Comparison of a Crude and Purified
36 mer Taqman ProbeWAVE®

Analysis and Purification ofOligonucleotides

ChromatographicConditions
Cartridge : OligoPrep HC
Eluent A: 100 mM TEAA
Eluent C: 100 mM
TEAA/25% ACN
Gradient: 5% to 30% B in
16 minFlow rate: 1.0 mL/min
Sample : 25 µL➤ The Transgenomic WAVE® System offers high performance separations

and purification using rugged polymeric based columns➤ Separations are performed under fully denaturing conditions (80°C)➤ Automated fragment collection is used in the purification mode
➤ Fast analysis and purification run times (<15 minutes)➤ No desalting required (TEAA)➤ Applicable to all classes of oligon ucleotides

Separation of Failure Sequences for a 26
mer Using TBAB (Crude and Purified)Temperature: 80°C
Detection: UV@300 nm
Purity assay: 93% byanion exchange

Wave Oligonucleotide System
Chromatographic
Conditions
Cartridge: OligoPrep HC
Eluent A: 30mM TBAB/10%
ACN
Eluent B: 30mM TBAB/80%
ACN
Gradient: 10% B for 2 min,
60% to 80% B from 2 to 14min
Flow rate: 0.9 mL/min
Sample : 50 µL
T emperature: 80°C
D et ection: UV@260 nm® assay -Purity by WAVE
92%Summary➤ High resolution assay and purification of synthetic oligonucleotides isachieved using the WA VE® System in the dHPLC mode
➤ The system is fully automated with preloaded methods for a wide rangeof oligonucleotides➤ Rugged polymeric cartridge operates under fully denaturing conditions

to assure high resolution and purification➤ Size or size/composition based separations accomplished by changingeluents➤ For most applications, no desalting is required with the TEAA chemistry