Chromatography Forum

Greeting again, it's been at least 8 years since my last post and my original user name was charles a burger, but me move on.

My issue, in the simplest words;

I have an Main peak that is eluting at 3.89 min. However, with the conditions of my method, the void peak comes out at 6.90 min.

NEVER have I seen something come out that fast.

YMC-Pack ODS (A) 250 mm x 4.6 mm 5um (200A pore size)
95% Ammonium Acetate pH 7.0
5% Methanol
@ 0.4ml/min flow rate

Compound is a synthetic polynucleotide

It's almost like something is pushing it through. Has anyone experience something like this? I really want to know how this is even possible.

Thank you

Charles

There may be two possibilities.

1. It's a late eluter, coming from a previous injection.
Check if it's much broader than your normal peaks. Also if it is observed only after some injections, not your first one.
e.g. inject your sample once, followed by several blank injections or even better, extend your runtime to about 3-5x your normal runtime.

2. It's by size-exclusion, so it's a molecule too large to get access to the pores and therefore only sees a fraction of the void volume (only has access to the inter-particle volume but not to the intra-pore volume; whereas the common void volume is the sum of inter-particle and intra-pore volumes)

Hollow wrote:
It's by size-exclusion...

...and/or by ion exclusion (via electrostatic repulsion from the charged surface of the stationary phase). The silica-based stationary phase has negatively charged residual silanol groups (at pH 7.0 they dissociate well) on the surface. They can repel the negatively charged molecules of the polynucleotide. The quantity of these residual silanols on the ODS is sufficient to cause ion exclusion. However, the ion exclusion is less effective when the ionic strength of the mobile phase is high (>50-100 mM).

Vcolumn = 0.001*3.14*(4.6^2)*250/4 = 4.16 ml.
VR = 3.89*0.4 = 1.56 ml.
VR/Vcolumn = 1.56/4.16 = 0.38. This value is in the usual range (0.36 to 0.42) of the interparticle volume fraction in the column. The polynucleotide is virtually totally excluded from the pores of the ODS particles.

Vvoid = 6.90*0.4 = 2.76 ml.
Vvoid/Vcolumn = 2.76/4.16 = 0.66. This is a reasonable value for the column void volume (or hold-up volume = interparticle volume + intraparticle pore volume) fraction.

At pH 7 your residual silanols are ionized and your Oligo is ionized. Oligo elutes from the column before void due to size-exclusion and anion-exclusion. Drop pH to 2-3 and see if peaks retains better.
You can also look at our approach here:

https://www.linkedin.com/posts/vlad-orl ... esktop_web