Luna C18(2) reverse phase issue

[MB]

A recently bought Luna-C18(2) column, which is specifically used for final QC only, is giving me some problems.. The exact specifications of the column are Luna 3u C18(2) 100A - 100x4.6 mm.

The column is only used to determent the purity of a product, samples are generally organic compounds with molecular weights between ~300-500. Occasionally these samples are analyzed as their salt form e.g. HBr/HCl, tosylate, etc
The purity of the samples is already >95% before checked on this particular column.

The runs are generally isocratic with various rations of ACN/Water, ratios range from 30%/70% to 80%/20% ACN/Water. Occasionally 10%-20% aqueous buffer is used to improve peak shape and/or retention.
These buffers are 50 mM acetate buffers (either pH=4 or pH=8).

On arrival we have checked the column by injecting a sample containing benzene/toluene (sadly I did not have the other 3 analites used in the attached test run). At Identical conditions (65%/35% ACN/Water;flow:0.75 ml/min; RT) identical retention times were observed for both benzene and toluene. The back pressure was also comparable (77 bars).

After having analyzed 12 samples (approximately 25 injections) the back pressure of the column has significantly increased.
Injection of a benzene/toluene sample at the above mentioned conditions still gave the same retention time but with a back pressure of ~140 bar.

I have attempted to clean the column using the cleaning method as described in the manual but to now avail. The back pressure seemed to have increased after cleaning.

I realize that using various different conditions (ratios and pH) could result in sight changes in column behavior, but back pressures that increase up to 80 bars seems extreme.

Within 2 weeks the back pressure has increased to point were it is hardly usable for isocratic runs with 50%/50% ACN/Water.

What could be the origin of this behavior. Previous luna columns have been used for much longer periods.

I am a bit in the dark at the moment how to attempt to clean/unblock the column. Or if the column is beyond salvation.

[tom jupille]

You have probably already checked for this, but just for completeness' sake, have you confirmed that the pressure drop is indeed due to the column? If you have an in-line filter mounted between the injector and the column, that may have plugged.

If it is, and (again) if you haven't done it already, try back-flushing the column. Check with Phenomenex to be sure you can do it with this column.

If the problem persists, then the column may, indeed, be dead.

The cause could be something as simple as bacterial growth in you mobile phase.

[Consumer Products Guy]

We use similar Luna column but 50mm for two of our assays, and we have backflushed occasionally to clean it out.

[MB]

You have probably already checked for this, but just for completeness' sake, have you confirmed that the pressure drop is indeed due to the column? If you have an in-line filter mounted between the injector and the column, that may have plugged.

If it is, and (again) if you haven't done it already, try back-flushing the column. Check with Phenomenex to be sure you can do it with this column.

If the problem persists, then the column may, indeed, be dead.

The cause could be something as simple as bacterial growth in you mobile phase.

I have back-flushed the column.

The bacterial growth might be a good point. Both buffers are generally just refilled instead of replaced and not used regularly.

I guess there is not easy way to test for that other then making a culture sample.

Going to replace the buffers and try back flushing again.

Menno