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RP-HPLC, IEX-HPLC and HPLC Purification & Dialysis

Purification
 
Oligonucleotides are assembled via solid-phase synthesis. In that way, nucleotides are attached according to the sequence of choice leading to chain growth. It is generally accepted that chemical reactions never reach 100% conversion. However, in state-of-the-art oligonucleotide synthesis, coupling yields of up to 99.5% are achieved. With the unreacted 0.5% of the strands, chain assembly stops and truncated sequences form as side products.
Depending on the intended application, it is advantageous to remove these shorter sequences from the full-length product. Therefore, Microsynth offers various types of purification methods.


Overview

RP-HPLC Purification

Oligos <50 nt in length can be well purified via RP-HPLC (reverse-phase high-performance liquid chromatography). Through this purification approach, preferably residual, n-x truncated oligos (lacking the hydrophobic DMT protection group at the 5’ end) are removed. This results in a ≥85% purity of the targeted oligonucleotide. RP-HPLC is useful for a higher level of purity that is required for more demanding applications, such as cloning, DNA fingerprinting, real-time PCR, FISH, etc.

Potential Applications:

  • Molecular cloning
  • DNA fingerprinting
  • Real-Time PCR and digital PCR*
  • FISH

* Microsynth primer and qPCR probes are compatible with all commercially available supermixes

IEX-HPLC Purification

IEX-HPLC (ion-exchange high-performance liquid chromatography) is a preferred purification method for longer oligonucleotides (40-80 nt). Through this purification approach, residual n-x truncated oligos are removed in an efficient way. Whereas RP-purification yields very good results for oligos < 50 nt, IEX purification is superior for longer oligonucleotides. (40-80 nt). For oligos with this length, IEX results in ≥85% purity of the targeted oligonucleotide. IEX is useful for a higher level of purity of long oligonucleotides required for more demanding applications, such as direct cloning or NGS applications.

Potential Applications:

  • Molecular cloning (direct cloning)
  • NGS Applications

HPLC Purification & Dialysis

Dialysis as an add-on to HPLC is recommended if oligos need to be present in a physiological state. This purity level is strongly recommended when performing in vivo experiments (e.g. in mice).

Potential Applications:

  • Antisense experiments
  • Cell culture studies
  • Physical Chemistry and Structure Analysis (NMR, MS, etc.)

DNA Yields

 
RP-HPLC Purified DNA Oligos 
Synthesis scale1 Length Restriction Guaranteed Yield2 Production Time [wd]
[OD260] [nmol]3
Genomics not available
0.04 µmol 13 - 50 1 5 2
0.2 µmol 6 - 50 3 15 2
1.0 µmol 6 - 50 15 75 2
15 µmol 6 - 50 300 1'500 3
 
IEX-HPLC Purified DNA Oligos 
Synthesis scale1 Length Restriction Guaranteed Yield2 Production Time [wd]
[OD260] [nmol]5
Genomics not available
0.04 µmol not available
0.2 µmol 40 - 80 2 3.3 3-5
1.0 µmol 6 10 3-5
15 µmol not available
 
HPLC Purified & Dialysed DNA Oligos 
Synthesis scale1 Length Restriction Guaranteed Yield2 Production Time [wd]
[OD260] [nmol]3
Genomics not available
0.04 µmol not available
0.2 µmol 8 - 50 3 15 3
1.0 µmol 8 - 50 15 75 3
15 µmol 8 - 50 200 1'000 4
 
 
 
1 The synthesis scale represents the initial amount of 3' bases (starting material).
2 Our guaranteed and average yields are measured in OD and are valid only for unmodified oligos >20mer.
3 Yields indicated in nmol represent an example calculation for a 20mer. For this calculation the following rule of thumb equation was applied: nmol of oligo = OD x 100/length of oligo. Please note that this calculation is based on sequences with virtually homogenous distribution of the 4 DNA bases; it may vary for sequences with high GC contents >70% etc.
4 Oligos longer than 150 DNA bases on request (we would like to discuss the proposed experiment/application with you beforehand in order to guarantee the best possible outcome)
5 Yields indicated in nmol represent an example calculation for a 60 nt DNA Oligo. Formula: nmol= OD*100/length of the Oligo

RNA Yields

 
HPLC Purified RNA Oligos 
Synthesis scale1 Length Restriction Guaranteed Yield2 Production Time [wd]
[OD260] [nmol]3
Genomics not available
0.04 µmol 10 - 30 1 5 2
0.2 µmol 10 - 50 3 15 2
1.0 µmol 13 65 2
15 µmol 10 - 40 300 1'500 4
 
HPLC Purified & Dialysed RNA Oligos 
Synthesis scale1 Length Restriction Guaranteed Yield2 Production Time [wd]
[OD260] [nmol]3
Genomics not available
0.04 µmol not available
0.2 µmol 10 - 50 2 10 4
1.0 µmol 9 45 4
15 µmol 10 - 40 200 1'000 4
 

 

1 The synthesis scale represents the initial amount of 3' bases (starting material).

2 Our guaranteed and average yields are measured in OD and are valid only for unmodified oligos >20 and <40 nucleotides.
3 Yields indicated in nmol represent an example calculation for a 20mer. For this calculation the following rule of thumb equation was applied: nmol of oligo = OD x 100/length of oligo. Please note that this calculation is based on sequences with virtually homogenous distribution of the 4 RNA bases.