Chromatography Forum

Dear all:

My sample is a small amount of API in oleic acid in 20/80=1%TFA/Ethanol, samples were filtered via 0.45um GHP filter before injected. However, when I tried to use the new phenomenux Lusa C18(2) 150x4.6mm ,3um culomn, I only made one injection and my column collaped. It had a super high pressure and caused leak. I've been using agilent SB C18, 250x4.6, 3.5um, and this column started to show high pressure as well.

I tried to rince the column backward for around 2h with IPA, and then forward with IPA for overnight but didn't solve the problem. Does anyone have any suggestions?

Thanks in advance.

Aqueous/ethanol mobile phase typically produces high pressures.

Aqueous/ethanol mobile phase typically produces high pressures.

Oops, I actually used 1% TFA/ACH=85/15 not ethanol.

Thanks.

Aqueous/ethanol mobile phase typically produces high pressures.

hello,
im new in this site

my advise : use hot water 70C degree temperature ,make the flow start from 0.1ml/min then start increasing the flow rate every 30minutes to reach 1.0mL/min.then use methanol/De ionized water
1:1 at flow rate 1.0mL/min .finally store the column with 100%Acetonitrile.

Not sure about the exact value of your "high pressure", but since you are using 3/3.5 um particle columns, higher pressure are expected (compared with 5um columns).

I may be wrong, but I was a little curious about your zorbax-SB column as 250*4.6 was a little long (150mm being the standard for 3um columns). Not sure about 1% TFA either, in my opinion TFA is a very strong organic acid and is typically used in the 0.1% range. This high concentration of TFA can adversely affect your column lifetime, especially at higher temperatures.

Borbo, I am not sure about your cleaning/regeneration protocol. I think the OP is using RP columns and water being the weakest mobile phase will not wash out any hydrophobic gunk that may be trapped inside the column. 70 degree is very aggressive and the OP is definitely not removing precipitated salts in the column. I do agree though ACN is perhaps the best storage solvent for RP columns.

I will stand corrected if I am wrong

Actually, 1% TFA/EtOH=20/80 is my diluent and H2O/ACN=15/85 is my mobile phase. Sorry that I must be hafl asleep when sending out the message. 250x4.6 was the Zorbax-SB column referred by client.

I've tried clean with 100% ACN, Ethanol and IPA, bot none worked so far. Quite frustrated.

Thanks

Not sure about the exact value of your "high pressure", but since you are using 3/3.5 um particle columns, higher pressure are expected (compared with 5um columns).

I may be wrong, but I was a little curious about your zorbax-SB column as 250*4.6 was a little long (150mm being the standard for 3um columns). Not sure about 1% TFA either, in my opinion TFA is a very strong organic acid and is typically used in the 0.1% range. This high concentration of TFA can adversely affect your column lifetime, especially at higher temperatures.

Borbo, I am not sure about your cleaning/regeneration protocol. I think the OP is using RP columns and water being the weakest mobile phase will not wash out any hydrophobic gunk that may be trapped inside the column. 70 degree is very aggressive and the OP is definitely not removing precipitated salts in the column. I do agree though ACN is perhaps the best storage solvent for RP columns.

I will stand corrected if I am wrong

In addition to what you have done, I would try two more things:

-wash first with IPA 100%, then hexane 100%. After hexane, use again IPA 100% before using MeOH or ACN.

-change the filter at the column entry (if possible).

I've had issues with increasing pressure and decreasing N and peak symmetry from inject food residues. I've found solid particles weren't responsible as I use an .5um inline filter and the fits are 2um and I actually replaced the inlet frit on one column and it did not help at all.

What I do is run 3 30 min gradients at 80 deg (even if it is above the rated maximum) with 25 to 100% 33%CH3CN/66%IPA/1% TFA and A is just water .1%TFA. I also do 100ul injection of trifluoroethanol. I restored columns to perfect condition that I was ready to throw out using this method.

BTW I stopped using 100% water on any of the channels. I keep 10%CH3CN 90% H20 on A and find that solved a lot of problems stemming from microbial growth (even though none was visible I believe it was happening).

In addition to what you have done, I would try two more things:

-wash first with IPA 100%, then hexane 100%. After hexane, use again IPA 100% before using MeOH or ACN.

-change the filter at the column entry (if possible).

what do you mean by "Changing filer at the column entry"? Did you mean frit? Thanks.

I've had issues with increasing pressure and decreasing N and peak symmetry from inject food residues. I've found solid particles weren't responsible as I use an .5um inline filter and the fits are 2um and I actually replaced the inlet frit on one column and it did not help at all.

What I do is run 3 30 min gradients at 80 deg (even if it is above the rated maximum) with 25 to 100% 33%CH3CN/66%IPA/1% TFA and A is just water .1%TFA. I also do 100ul injection of trifluoroethanol. I restored columns to perfect condition that I was ready to throw out using this method.

BTW I stopped using 100% water on any of the channels. I keep 10%CH3CN 90% H20 on A and find that solved a lot of problems stemming from microbial growth (even though none was visible I believe it was happening).

So in your case food particle was not the root cause and it could be the oil in the food that cause the high pressure? And to clarify:

Here is your gradient for cleaning:

Time A%(0.1% TFA in H2O) B%(33%CH3CN/66%IPA/1% TFA)
0 25% 75%
3h 30min 100% 0%

Thanks.

Yes, you unscrew the part at the column head and change the frit/filter. It is not impossible that some particles clogged it.

Silly question: are you absolutely sure it's the column, and not a blockage elsewhere in your system?

Hello,

I actually had problems with my column as well, largely due to too much analytes injected.

What I tried to do to fix this was use a IPA/MilliQ gradient, reversing the column (and for about 4 hrs), then I reverse back the column, did 100% Mobile B, 100% Mobile A, 50/50, then 2% for re-equilibration.

After that I ran some standards and test samples and it seemed to work.

Hope that helps a bit!

Unless you have an axially compressible column, opening a column is one of the best ways to instantly kill it (void), even if you have extra stat. phase available. There are uncounted good examples of how to rejuvenate a column in this forum.