PRP-1 and PRP-3 Reversed-Phase DNA Columns
Reversed-Phase HPLC Purification of DNA
Hamilton polymeric reversed-phase columns are copolymers of styrene and divinylbenzene. PRP-1 and PRP-3 columns provide a number of benefits for purifying synthesized oligomers by reversed-phase HPLC:
- Excellent sample recovery, of greater than 95%.
- High sample capacity, 10 mg or more for a 4.1 x150 mm column.
- pH stability for purification of DNA with secondary structure at pH 12.7.
- PRP-1 and PRP-3 are stable over a wide range of solvents and mobile phases.
Wide DNA Application
PRP-1 and PRP-3 reversed-phase columns are a great choice for purification of synthetic DNA, RNA,and chimeric DNA-RNA oligomers. Purifications with excellent recovery are possible with molecules like (5’-monomethoxytrityl1, 5’-dimethoxytrityl1,2, fluorescein, phosphorothioate4, tert-butyldimethylsilyl5,7, and N-Acetyl-2-aminofluorene3,10) protected and deprotected oligomers. Easily purify oligomers with secondary structures like hairpin loops and guanine rich sequences.
Excellent DNA/RNA Recovery
Recovery of trityl-on DNA1 and chimeric DNA-RNA oligomers is greater than 95%7 and recovery of oligomers containing secondary structure is greater than 90%2 with PRP-1 columns. This is possible because the polymeric (poly(styrene-divinylbenzene)) packing in PRP-1 and PRP-3 columns are free from acidic silanol groups. C18 columns typically recover only 50-80% of protected DNA because the silanol groups on the column irreversibly bind the protected DNA or hydrolyze the dimethoxytrityl (DMT) protecting group during purification.
High DNA Sample Capacity
The sample capacity of a 4.1 x 150 mm PRP-1 column is approximately 10 to 25 mg of DMT protected DNA per run by Ikuta et al.1. When compared to 1 mg/run for a silica C18 column of the same size, it is readily apparent that PRP-1 and PRP-3 are the columns of choice for protected oligodeoxynucleotide purification. Loading capacities up to 500 mg have been obtained on a 21.5 x 250 mm column. A column’s sample capacity is directly proportional to its volume. For example, a 4.1 x 150 mm analytical column has approximately 2.0 mL of volume, while a 21.5 x 250 mm preparative column 90.8 mL. If 10 mg of sample can be purified on the analytical column, the preparative column can purify 45 times that amount or 450 mg.
Column Dimension | Loading Capacity |
| 4.1 x 150 mm | 10 mg |
| 4.1 x 250 mm | 17 mg |
| 10 x 250 mm | 99 mg |
| 21.2 x 250 mm | 446 mg |
| 30 x 250 mm | 892 mg |
| 50 x 250 mm | 2,479 mg |
| 100 x 250 mm | 9,915 mg |
Easy Purification Scaleup
Scaling up from an analytical column to a semiprep or preparative column is easy. The runs below illustrate the reproducible scaleup from a 4.1 mm column to a 100 mm ID column with Cytosine, Uracil, and Uridine.
PRP-1 DNA Analytical Column
PRP-1 DNA Scaleup Column #1
PRP-1 DNA Scaleup Column #2
*The flow rate must be increased proportionally to the size of the column when scaling up to larger sizes to maintain analyte retention times of oligomers. The following table is a guideline to determine the appropriate flow rate for a given column hardware dimension.
Flow Rate Scaleup
| Column ID | Flow Rate (mL/min) |
|---|---|
| 1.0 | 0.05 |
| 2.1 | 0.22 |
| 4.1 | 0.80 |
| 4.6 | 1.06 |
| 7.8 | 3.04 |
| 10 | 5.00 |
| 21.2 | 22.50 |
| 30 | 45.00 |
| 50 | 125.00 |
| 100 | 500.00 |
*Column equilibration is key to reproducible chromatography: Flush with 10 or more column volumes of the starting mobile phase composition prior to sample injection and gradient elution.
Long Column Life
The poly(styrene-divinylbenzene) stationary phase in PRP-1 and PRP-3 columns is extremely robust and resilient to most HPLC method conditions. This stability prevents column degradation by any of the purification methods in DNA/RNA techniques, assuring reproducible separations and long column life. The inertness of the stationary phase also contributes to the exceptional recovery (>95%) of protected and deprotected oligomers.
Wide DNA/RNA Mobile Phase Selection
A wide variety of mobile phases and sample preparation conditions can be used with PRP-1 and PRP-3 reversed-phase columns for purification of protected and deprotected DNA oligomers.
Purification of DMT-on and DMT-off DNA
| A) 100 mM Triethyammonium Acetate pH 7.5 Buffer | B) Acetonitrile |
Purification of DMT-on DNA with Secondary Structure
| A) 10 mM Ethylenediammonium Acetate pH 7.6 | B) 1:110 mM Ethylenediammonium Acetate pH 7.6 :Acetonitril |
| A) 50 mM Sodium Hydroxide pH 12.7 | B) 1:1 50 mM Sodium Hydroxide : Acetonitrile |
| A) 10 mM Potassium Phosphate pH 7 | B) 7:1:2 V:V:V Acetonitrile : Methanol : 10 mM Potassium Phosphate pH 7 |
| A) 50 mM Triethylammonium Acetate pH 7 :5% Acetonitrile | B) 50 mM Triethylammonium Acetate pH 7 : 50% Acetonitril |
Purification of DMT-on DNA for NMR
| A) 10 mM Potassium Phosphate pH 7.0 | B) 7:1:2 V:V:V Acetonitrile : Methanol : 10 mM Potassium Phosphate pH 7.0 |
Purification of DMT-on DNA with Secondary Structure and On-Column DMT Deprotection
| A) 15% Acetonitrile in 100 mM Tetraethylammonium Hydroxide | B) 15% Acetonitrile in 100 mM Triethylammonium Bicarbonate pH 7.0 | C) 0.5% Trifluoroacetic Acid | D) Acetonitrile |
Purification of Phosphorothioate modified DNA
| A) 100 mM Potassium Phosphate with 2 mM Tetrabutylammonium Phosphate, pH 7.0 Buffer | B) Acetonitrile |
Purification of 2-(acetylamino) fluorene Modified DNA
| A) 10 mM Potassium Phosphate pH 7.0 | B) 7:1:2 V:V:V Acetonitrile : Methanol : 10 mM Potassium Phosphate pH 7.0 |
Purification of Fluorescein Modified DNA
| A) 20 mM Sodium Acetate pH 6.7 | B) Acetonitrile |
Purification of RNA and DNA/RNA Chimeras
| A) 10 mM Tetrabutylammonium acetate, 1 mM Tetrabutylammonium phosphate pH 7.5 | B) 8:2 (V:V) 10 mM Tetrabutylammonium acetate, 1 mM Tetrabutylammonium phosphate pH 7.5, Acetonitrile |
Purification of RNA
| A) 100 mM Trimethylammonium acetate pH 7.0 | B) Acetonitrile |
