Interchim® offers in its puriFlash® range two devices dedicated to the purification of peptides and oligonucleotides, the 5.125P (250mL/min @ 125bar) and 5.250P (125mL/min @ 250bar). These two devices differ from the others with 2 factors:

  • A UV cell with a 1.3mm optical path, allowing the acquisition of a higher signal
  • Their tubings have reduced internal diameters (ID) of 1mm for 5.125P and 0.75mm for 5.250P

In this article, we will focus on the 5.250P purification system. Indeed, with an internal diameter (ID) of 0.75mm, this device offers a range of applications particularly adapted to the purification in the reverse phase of molecules such as peptides and oligonucleotides.


Higher UV flow cell

The most obvious point concerns the UV cell. With a light path of 1.3mm (compared to 0.3mm on other purification systems) allows an increase in signal level.

According to Beer-Lambert’s law (A=ε l c, A : absorbance, l : optical path length, ε : extinction coefficient), by multiplying the optical path by 4, the absorbance will be increasing. We therefore have a first element that allows a better observation of impurities because their signal will be more important.


Decrease dead volume

We will, through an application, show another advantage related to the reduction of the internal diameter and a higher optical path.


Products: Uracile, Toluene, Naphtalene
Column: PF-5C18HP-150/212
Injected quantity: 100µL
Solvent: Water/Acetonitrile (30/70)
Method time: 9min
Detection: 254nm

Application on system with tubing ID 1.6mm and UV flow-cell 0.3mm

Système avec tubing ID 1.6mm et cellule UV 0.3mm

Application with puriFlash®5.250P (tubing ID 0.75mm, UV flow-cell 1.3mm)

PuriFlash® 5.250P (tubing ID 0,75 mm, cellule UV 1,3 mm)


Comparison of the two chromatograms

Intensity level comparison on column 5µm, 150×21.2mm
So, in orange the results obtained with the puriFlash® 5.250P.

Comparaison intensité de signaux sur colonne 5 µm, 150 x 21,2 mm

There is a significant difference between the two chromatograms. With the same method, two advantages can be noted:

  • The height of the peaks is higher with puriFlash®5.250P
  • Peak diffusion is reduced with puriFlash®5.250P

Both elements show us that it is easier to visualize impurities in small quantities and to separate them. This will make it easier to separate an impurity, in small quantities, very close to an intense compound.


Other benefits: use analitical columns (4.6mm ID)

Another advantage can be taken into account. The use of columns with an internal diameter of 4.6mm.

We then have a system to develop an analytical method, then to transpose directly by freeing ourselves from the problems related to the different dead volumes between machines.

Application :

Products: Uracile, Toluene, Naphtalene
Column: PF-5C18HP-150/P46
Injected quantity: 10µL
Solvent: Water/Acetonitrile (30/70)
Method time: 15min
Detection : 254nm

1.6mm ID tubing and 0.3mm Flow-cell on 250×4.6mm, 5µm column

ID Tubing 1.6 mm, cellule UV 0,3 mm sur colonne PF-5C18HP 250 x 4,6 mm


0.75 mm ID tubing and 1.3mm Flow-cell on 250×4.6mm, 5µm column

ID tubing 0,75 mm, cellule UV 1,3 mm sur colonne PF-5C18HP 250 x 4,6 mm


Comparison of the two chromatograms

Intensity level comparison on column 4.6mm

Comparaison intensité signaux colonne 4,6 mm


Similarly, the intensity of the detected peaks is higher on a system with reduced dead volume, but the most important difference is the diffusion of the peaks. With ID=0.75mm tubing, the two peaks at 9min and 11min are better separated.

The 5.250P system therefore allows to consider an analytical column development with an ID of 4.6mm.


Analytical development and purification on the same system

Since it is possible to develop an analytical method, the transposition on preparative or Flash column can be done easily. The advantage is therefore to be able to develop and purify on the same system.

Column 250×4.6mm, 5µm, C18HP

Colonne 250 x 4,6 mm, 5 µm, C18HP

Column 150×21.2mm, 5µm, C18HP

Colonne 150 x 21,2 mm, 5 µm, C18HP


The puriFlash 5.250P therefore seems to be a perfect device for difficult purifications such as peptide and oligonucleotide purifications.

This device will allow you to:

  • obtain an intense signal thanks to a combination of UV cell, with a larger optical path, and a dead volume reduction (ID=0.75mm) to easily identify impurities in small quantities.
  • use columns filled with a 5µm phase for difficult purifications
  • use analytical columns
  • transpose directly from an analytical column to a preparative or Flash column directly on the same device.


Know more: