Clogging guard (Colistin sulfate on UHPLC)

[artsjeroen]

I'm trying to transfer our Collistin method from HPLC to UHPLC. Everything looks promising. The run time was decreased from 30 minutes to 7 minutes for all peaks of the analyste and the excipients (vitamins in the finished product) to elute.

The only problem is that after each injection the pressure increases about 10-20 PSI (0.7 - 1.4 Bar) on the UHPLC, flushing the column with a gradient 20 - 80% acetonitrile caused the guard column to clog completely. I have replaced the guard column, and after 20 injections I flushed the column with 20/80 Water/ACN, the clogging does not appear but there is a pressure increase when running with mobile phase again.

The mobile phase is 79/21 - 30 mM Na2SO4 ph 2.4/ACN.
The column is Kinetex C18 2.6 µm, 3.0 x 100 mm with guard.

The column (HPLC) is symmetry C18 3.5 µm, 4.6 x 150 mm without guard, no problems with this column.
The injection volume on UHPLC is decreased from 20 µl to 5 µl.

The sample is 100 mg in 50 ml, diluted 10x (all in ultrapure water). Both solutions are clear as Colistin dissolves freely in water.

Anyone some idea what is happening, why my column is clogging?

In the EP monograph the samples are dissolved in 80 ml water and diluted to 100 ml with ACN. I don't see why they would do this? Colistin is a mixture of (relatively small) polypeptides, which doesn't dissolve in organic solvents. Why would they add acetonitrile to the solvent?

I've read something about antisolvents for peptides, but I think that is only relevant when extracting the peptides from plasma?

[Gerhard Kratz]

The mobile phase is 79/21 - 30 mM Na2SO4 ph 2.4/ACN and you wash with 20/80 Water/ACN there will be precipitation of Na2SO4 especially on a 2,6µm particle size column and can clog the column. I would recommend to wash first with 80%water 20%ACN for at least 4 column volume.

[artsjeroen]

Sorry my fault, I washed with 80/20 Water/ACN for a few hours. Not 20/80 Water/ACN.
Still the pressure increased.

As if the analyte sticks to the column head.
I've been rinsing the column with 0.1% TFA in ACN / 0.1% TFA in water with a gradient 10 - 90% for one hour (to remove peptides) but this did not decrease the backpressure. I've three guards clogged in two days now

[Gerhard Kratz]

If possible try a guard column with a larger particle size, 5µm for example. should not have an impact on the performance of your method.
What are the dimensions of your guard column?

[artsjeroen]

I am using these guard cartridges:
http://www.phenomenex.com/Products/Part/AJ0-8775

l = 2.0 mm, id. = 3.0 mm

[Gerhard Kratz]

SecurityGuard™ ULTRA cartridges for C18 UHPLC, sub-2µm and core-shell columns with 3.0mm internal diameters (ID), 3/Pk
So these particles are smaller than in your analytical column. Try a 5µm Luna C18 and check if the connection Security guard holder to Symmetry column is correct.

[artsjeroen]

SecurityGuard™ ULTRA cartridges for C18 UHPLC, sub-2µm and core-shell columns with 3.0mm internal diameters (ID), 3/Pk
So these particles are smaller than in your analytical column. Try a 5µm Luna C18 and check if the connection Security guard holder to Symmetry column is correct.

The security guard holder is connected to the Kinetex not the Symmetry.
But I will try a larger particle size guard connected to the Kinetex, to see if that resolves my problem.

[Klaus I.]

Is your pressure increase really caused from the injections of your analyte or is the origin a contamination of your buffer salt / mobile phase?

[artsjeroen]

To check that, I've done 20 injections of 5 µl ultrapure water. I saw no pressure increase during this run. So my guess is still that the polypeptides stick to the column.

The first guard column was completely clogged after rinsing with a gradient ACN/Water 20/80 to 80/20. When I returned to mobile phase the pressure increased above maximum pressure (>8000 PSI).

Rinsing only the guard with 90/10 UPW/ACN did not lower the back pressure after several hours. So I don't think salt precipitation of Na2SO4 is the problem since this would easily dissolve in water.

So my theory is that the polypeptides are locked on the column. And I have no idea how to get rid of them. I've read on several places that it might be impossible to get them off.

I don't know why this doesn't happen on the HPLC (Symmetry C18) column. The pore size of both columns is 100 Å. Maybe the ultra-guards have smaller pore sizes, but this I don't know.

[Klaus I.]

Rinsing only the guard with 90/10 UPW/ACN did not lower the back pressure after several hours. So I don't think salt precipitation of Na2SO4 is the problem since this would easily dissolve in water.

Correct, I was thinking about the impurities in the salt. But when you are sure that the problem comes due to the injections my idea is wrong.

Unfortunately I have no personal experience with your analytes. But I can remember a discussion with my college. He has preferred a special column manufacturer when he was working in this area to avoid clogging of the column. Most probably this manufacturer has used different frits at column-inlet (and outlet). So maybe it is a problem of the guard column hardware?

[artsjeroen]

I think the hardware of the guard was indeed different. I started injections without the guard, and the pressure kept decreasing for the first 10-20 injections and can now be kept at a constant back-pressure.