Chromatography Forum

Trying to improve on our vitamines A and E determinations, we tried an Agilent ZORBAX Eclipse Plus C-18 4.6 x 50mm column on a PerkinElmer 200 HPLC system.
Putting on a methanol/water buffer (90/10) the pressure goes over 400 bar, even without the tubing to the detector fitted.

Anybody there who has an idea about what is going wrong ?

What particle size / flow rate are you using? 90% MeOH has similar viscosity as 100% aqueous, so if you are using a sub 2um column -
the pressure could be very high.

You should also check to see if your instrument or column has gotton
clogged up. Use a zero dead volume connector (instead of a column)
and pump the mobile phase through the system. If the pressure is still
really high - you know instrument is clogged somewhere. If the pressure is low without the column - then either:

A. Switch to larger particle size (e.g. sub 2 to 3 or 5um)
B. Better job of filtering samples
C. Guard column
C. Combination of a,b, and c

If you are using the small particle columns, remember that most of them are designed to work at higher temperatures (50 C). That will reduce the pressure significantly.

Also, start at a slow flow first to verify that the system/column isn't plugged.

Thank you for replying.
The particle size is 1.8µm.
I tried to run at 50°C. However, I'm not sure the core of the column reached that temperature before the flow was turned on.
I hope the very high pressure has not ruined the column.

These columns are designed to operate at higher pressures so you have probably not damaged the column yet. You can try reversing the flow if you are concerned that you have compressed the bed, but that is rarely a problem.

For the temperature issue, remember that it may take up to 30 minutes to fully equilibrate the column. Depends on your system design. You could always maintain a slow flow until system heats up. If your temperature controller pre-heats the mobile phase, then this should help.

As for lifetime, in my limited experience with these columns, I think mobile phase and sample filtering is a major cause of early column failure. You should be filtering to 0.2 um or smaller.

Below is fat soluble vitamin data with 100% MeOH as the mobile phase using Cadenza CD-C18 (3um). The back pressure is only 67 Bar

http://www.silvertonesciences.com/files/TI121E.pdf