Use of Reversed phase chromatography |
High resolution mode
(gradient elution) | Group separation mode
(step elution) |
| Separates peptides, proteins and oligonucleotides according to net hydrophobicity. | Concentrates dilute oligonucleotide and peptide samples. |
| Suitable for intermediate steps and polishing in multi-step purification protocols. | Suitable for so called solid phase extraction. |
| Main technique for the purification of synthetic peptides. | Suitable for desalting of peptide and oligonucleotide samples. |
| Main technique for the analysis of peptides and for peptide mapping. |  |
Experimental |
| High resolution mode
(gradient elution) | Group separation mode
(step elution) |
Reversed phase chromatography medium | Use 10 - 30 mm media for purification work.
Use 5 - 12 mm media for analytical work.
Use polymer-based media for protein separations (allows cleaning under alkaline conditions). | Use media with 30 mm beads or larger to allow higher flow rates. |
Column | Typical column lenghts are 1 - 10 cm. | Column length is less important. Short and "fat" columns, however, will allow higher flow rates. |
Eluents | Oligonucleotides are separated under alkaline conditions
Standard conditions for peptides are
ACN; TFA at pH ~ 2. | Eluent optimisation is not necessary for desalting and concentrating the sample.
Sample salts will elute during sample application. |
Sample volume | Sample volume restricted to 0.5 - 5% of column volume in isocratic mode.
No sample volume restriction in gradient mode. | The sample volume is not important since the sample will bind to the column. |
Sample amount | 5 - 10 % of the total loading capacity of the column used can be applied without loss of resolution. | Around 40 % of the total loading capacity of the column used can be applied. |
