Chromatography Forum

Hello everyone

I am currently working on carotenoids. I am using a 1200 series HPLC witha a pressure limit of 400 bar and a YMC carotenoid column (150 X 4.6mm S-5) to separate my carotenoids. I have a total of 300 samples to run.

For my extraction, I used acetone:chloroform (1:1).

The gradient I use is as follows:
mobile phase A: TBME
mobile phase B: methanol
Gradient: 0 min A is 15%, 0.5 A is 15%, 1.5 A is 35%, 3.0 A is 50%, 4.5 A is 80%, 9.0 A is 80%, 13 A is 15%.

The flow rate is 0.8ml/min

The injection volume is 10 microliters.

Now the problem

I first used an old column and discovered that whenever I completely run my 100 samples, the column generates a high pressure in the HPLC system, reaching about 250 bar and above.

Upon troubleshooting, I found that the pressure was coming from the column and not due to tube clogging or any other system-related issue.

Afterward, we decided to buy a new column, and after 100 samples, the same problem occurred. We experienced rapid pressure increase.

Using a flow rate of 0.8 ml/min, the ideal pressure is usually about 60-80 bar. However, after 100 samples run, there is a sharp rise in pressure generated from the column

Procedures to correct this problem

I carried out a reverse wash using the following reagent:

Methanol and TBME (to remove polar compounds in the column)

100% TBME (to remove polar compounds in the column)

100% isopropanol

Water coupled with increasing the temperature of the column to 50 degree.

I Carried out overnight wash using TBME and methanol
All above steps were done with a low flow rate of 0.3ml/min.

While sometimes the column pressure drops, it only takes a while before it spikes again.

Please, what can I do in this case, and what could be going wrong, as it is non-economical to keep purchasing new columns?

In short, the problem you are experiencing is certainly due to what you inject in it. Things you may consider if not yet done:
-centrifuge twice or filter to make sure the extracts are totally clear and particle-free.
-add a guard column before you column (cheaper to replace than a column itself).
-decrease the hydrophobicity of your extraction solvent so it becomes more compatible with the intital mobile phase composition.

I would add to Gaetans post:

Possibly you have some compounds precipitating from yourr aceton/CHCl3-extract when it comes in contact with the initial mobile phase that is much more polar.

You could check this in a test tube by diluting some of your extract with the mobile phase.

If you do not want to change your extraction procedure, you'll have to dilute your extract with methanol/TBME and filter. You also need to check how this influences the recovery of your analytes.