UPLC Acquity BEH RP18 column lifetime

[kagliostro]

Hi to everyone,
I recently started to use an UPLC system with an Acquity BEH RP18 (1.7um, 2x100mm) and the column lifetime is ridicoulously short although I always clean the column with water acetonitrile after my analysis.
The eluent that I am using is a 20 mM Ammonium acetate mixed with acetonitrile (72:28) and column temperature of 40°C. Column use to last only 1000 injections and recently this has dropped to 500 or 600 inj.
What am I doing wrong and how can I extend column lifetime? Did anyone test Kinetex core shell columns from phenomenex? are those columns any better?
Thanks in advance for your help

[kagliostro]

Has anyone tested the cortec s column made by waters? are they any better?

[M_Farooq]

Could you elaborate more about the column performance? Does the retention time change or the peak shape change? Note that silica has very small finite solubility in water - more so at higher temperature. Hybrid materials like BEH silica are supposed to have higher stability than common silicas.

Could you open a dead column and see there is a void at the column head or there is discolouration. Discolouration is a clear indication of dirty samples.

[kagliostro]

Could you elaborate more about the column performance? Does the retention time change or the peak shape change? Note that silica has very small finite solubility in water - more so at higher temperature. Hybrid materials like BEH silica are supposed to have higher stability than common silicas.

Could you open a dead column and see there is a void at the column head or there is discolouration. Discolouration is a clear indication of dirty samples.

First of all thanks for your reply.

There isn't any change on the retention time while we observe an increase of the back pressure which goes from 10000 psi (let's say with a new column) to 13500 and more within 500 injections. To be honest I never opened a column to check if there is any dirty but I'll do it on Monday. However, we do filter solutions before we inject them in the UPLC.
I also tried to wash column with a gradient going from water acetonitrile (95:5) to 100 % acetonitrile and back to 70:30 at 60C but did not help to reduce the very high back pressure. Chromatographycally speaking we observe a decrease of number of plates which goes from 17500 (new column) down to 6000 or 5000.

[M_Farooq]

Thanks for the details. It is interesting to know that the retention time does not change in your case but your efficiency degrades. It is very likely that the frits and the column inlet get spoiled soon in your case. This is indicated by an increase in pressure. Do you see peak shoulders as well on dead columns?

BTW, the column care manual suggests that you use 1-10 mM ammonium acetate. Highly aqueous mobile phases will eventually dissolve silica.
http://www.waters.com/webassets/cms/sup ... 001371.pdf

If you have time and would like to play with the hardware, do open the column inlet and see how it looks like from inside.

[kagliostro]

Thanks for the details. It is interesting to know that the retention time does not change in your case but your efficiency degrades. It is very likely that the frits and the column inlet get spoiled soon in your case. This is indicated by an increase in pressure. Do you see peak shoulders as well on dead columns?

BTW, the column care manual suggests that you use 1-10 mM ammonium acetate. Highly aqueous mobile phases will eventually dissolve silica.
http://www.waters.com/webassets/cms/sup ... 001371.pdf

If you have time and would like to play with the hardware, do open the column inlet and see how it looks like from inside.

I will open the column tomorrow although I do not expect to see any dirt since sample solutions are always before injections. Do you think that the ammonium acetate is so crucial for the column lifetime? Ultimately it should a pH 7 buffer and columns are guaranteed on wide pH going from 2 to 11.
Thanks a lot, I will update this post as soon as I will open the column

[HPLCaddict]

The pressure increase that you are describing very much indicates column blockage as already pointed out. This is not a fault of the column itself. It just accumulates all sorts of junk from your samples, your mobile phase and anywhere else (such as any debris from seals). The smaller the particle size, the more "efficient" the columns are at collecting particles and therefore pressure increase will be much faster than with a 5µm column. This is even more pronounced due to the smaller frits used in UHPLC columns.
In order to increase column lifetime, just follow the usual HPLC hygiene rules, which are even more important for UHPLC.
- Filter your samples. Better use 0.2µm filters than 0.45µm. Filtering is better than centrifuging for UHPLC.
- Use an inline filter or a guard column to protect your analytical column. I'm not a big fan of guard columns if I'm not working with really "dirty" samples, but for UHPLC at least an inline filter is a must. Be sure to use some sort of inline filter dedicated for UHPLC with a small dead volume.
- It may be a good idea to filter your mobile phases, especially when using buffers

You may resurrect a blocked column by exchanging the inline frit. You can obtain new frits from Waters. Be warned, though, that by opening the column you may disturb the stationary phase and thus spoil the column beyond repair.

[HPLCaddict]

One further thing: BEH columns (as well as the identical XBridge ) are among the best I know concerning chemical stability. pH, buffer concentration, temperature - as long as the parameters are somewhat reasonable, you don't have to care much about it. There is a guy who uses these for high temperature LC at something like 130+°C.
I'm quite sure that your BEH column absolutely doesn't mind if you're using 10 or 20mM Ammonium acetate. I've abused that kind of column with much more disgusting things[:)].
Concerning physical damage, like the blockage you are describing, they are of course defenseless like any other column.

[kagliostro]

One further thing: BEH columns (as well as the identical XBridge ) are among the best I know concerning chemical stability. pH, buffer concentration, temperature - as long as the parameters are somewhat reasonable, you don't have to care much about it. There is a guy who uses these for high temperature LC at something like 130+°C.
I'm quite sure that your BEH column absolutely doesn't mind if you're using 10 or 20mM Ammonium acetate. I've abused that kind of column with much more disgusting things[:)].
Concerning physical damage, like the blockage you are describing, they are of course defenceless like any other column.

Thanks for your reply, may I ask you how many injections last your column under your conditions? just to have a sort of comparison.

[HPLCaddict]

I have a 1.7µm BEH C18 that is approaching its 3500th injection soon. On the other hand, there is an identical column that died a quick death after a mere ~200 injections.
What it boils down to is: Columns WILL die sooner or later. With a bit of chromatographic hygiene, it might be later. If you're lucky, much later. But even with ~500 injections, it will probably have earned its costs. If you break down the column costs to cost per analysis, you'll probably spend the same money on syringe filters.
With some nice analyses like biological fluids the analyst probably would perform a dance of joy if he'd get 500 injections out of one column .

[kagliostro]

I opened the column today and what I found was a dead frit (it was black in the middle). I changed it with a clean one and back pressure problem disappeared. I think that eludente filtration is a crucial thing.
The frit was almost completly blocked by black spots.