Chromatography Forum

I am using a gradient method to test a small molecular.
mobile phase A: Water
mobile phase B: ACN
as the injections goes, the system pressure always increase gradually. the peaks are still very good. usually it takes 50 injections to get 3000psi from 1200 psi. I took off the in-line filter and column to clean (sonicated by ACN, IAP, Hexane, MeOH etc. ), but no much help. The sample was disolved in potassium phoshate buffer and saline.

If mix of mobile phase Water and ACN can generate high pressure?

If you just run ACN and Water at different % you will see that back pressure will change. But more important is the question why you don`t use same buffer in your mobile phase, and what is the concentration of your buffer? Usually you can kill a column if you have a non volatile buffer at high concentration in your mobile phase and flush the column with 100% ACN. So what is your injection volume and the concentration of your buffer and what is the initial ammount of ACN in your mobile phase?

I am tesing the dosing fomulation which prepared with buffer.

The run starts and ends with 95%ACN. The injection volumn is 10ul. Do you think the buffer will percipitate in ACN during the run? I thought it was, but I am using water to flush/back-flush, it did not work much.

Thanks

If you just run ACN and Water at different % you will see that back pressure will change. But more important is the question why you don`t use same buffer in your mobile phase, and what is the concentration of your buffer? Usually you can kill a column if you have a non volatile buffer at high concentration in your mobile phase and flush the column with 100% ACN. So what is your injection volume and the concentration of your buffer and what is the initial ammount of ACN in your mobile phase?

If I remember correctly there is no max viscosity with mixtures of water and ACN (the max. being at 100% water, if someone has other data handy, plese comment). In any case a builtup over many injections would not occur even if viscosity increased when mixing the two solvents.
Since buffer precipitation is apparently not a problem I can only think of the cause being the injection of crud, or part of the analyte itself adsorbes where one would not want it.

If your run starts at 95% ACN (do I understand this correctly?) the phosphate and NaCl in your sample will precipitate.

I just tried filtered the injection sample, the pressure still builded-up.

If I remember correctly there is no max viscosity with mixtures of water and ACN (the max. being at 100% water, if someone has other data handy, plese comment). In any case a builtup over many injections would not occur even if viscosity increased when mixing the two solvents.
Since buffer precipitation is apparently not a problem I can only think of the cause being the injection of crud, or part of the analyte itself adsorbes where one would not want it.

The v/v% viscosity maximum for ACN/H2O is ~24% ACN. What is the intial %ACN in your mobile phase?

If your run starts at 95% ACN (do I understand this correctly?) the phosphate and NaCl in your sample will precipitate.

Sound like you are precipitating the buffer as Carls has said and your water flush is insufficient to redissolve and clear the buffer from the column before the next injection.

Consequently, you see a gradual increase in pressure with each subsequent injection.

If you cannot rempve the phosphate/ saline from the sample, I would run some tests and increase the time period of the water flush to see how long it needs to be to avoid the pressure rise.

If you can monitor the pressure over the run do it

there are 3 main behaviors of pressure build up

one hump at the beginning of the run-then your samples as dirty
in the run at a certain RT, mostly a precipitation of the salt
from beginning to end always building up in a shallow upward fashion- that is mainly your mobile phase.

flora0975, I am not talking of crud which you can filter out!!

carls, do you have a ref. for this viscosity change?

unmgvar, I don´t follow your "3 main behaviors", could you elaborate?

thank you, Carls.

Based on your clue, I am trying to modify the gradient method with high %water in the begin. Let me see how if it works.

If your run starts at 95% ACN (do I understand this correctly?) the phosphate and NaCl in your sample will precipitate.

I can see the first and third behaviors of pressure build-up in my run.

If you can monitor the pressure over the run do it

there are 3 main behaviors of pressure build up

one hump at the beginning of the run-then your samples as dirty
in the run at a certain RT, mostly a precipitation of the salt
from beginning to end always building up in a shallow upward fashion- that is mainly your mobile phase.

carls, do you have a ref. for this viscosity change?

Ref: J.W. Dolan, L.R. Snyder Troubleshooting LC Systems, Humana Press, Clifton, 1989, p. 85.

We have also empirically determined this using an LC and an RPLC column as well. We see maximum backpressure around 23-24 %ACN.

intial %ACN is 95%.

The v/v% viscosity maximum for ACN/H2O is ~24% ACN. What is the intial %ACN in your mobile phase?

My initial gradient is:
min %ACN
0 95
5 95
20 65
24 50
25 95
33 95
The main peak RT is around 16min which comes with around 30% Water/70% ACN flow.

I tried to start at high % water, the main peak showed up in very early RT (less than 5 min). The pressure is consistent now!

How can I change the gradient with high %Water but not have so early RT for main peak?

Thanks a lot

thank you, Carls.

Based on your clue, I am trying to modify the gradient method with high %water in the begin. Let me see how if it works.

If your run starts at 95% ACN (do I understand this correctly?) the phosphate and NaCl in your sample will precipitate.