Thermo Hypercarb column high back pressure

[HaruYap]

Hi all. I am experiencing high back pressure for my HPLC column. I'm looking at pressure of more than 1400psi.

My column: Thermo Hypercarb 150mm X 1mm; 5um

I have the technical data sheet attached together with the column that I've purchased and the column back pressure was noted at 1100psi. What does that mean? Wasn't it suppose to be the maximun allowable pressure for the column?

What does back wash means? Is it using the column on the reverse side?

When I tried to recondition the column by flowing the solvents through the column with the HPLC system, I cant get the pressure to get lower; in fact, the pressure keeps on going up even though the end of the column was not attached onto the LCMS-MS when I try to increase the flow rate through the column.

Does that mean the column has choked or something or the frit has clogged? Do advise recommended actions to be taken.

Solvents tried for flushing the column: ACN:iPA (1:3) and THF (100%) (I did not try to flush the column in reverse order)

If you have the technical data sheet for me to note the maximum allowable pressure for the column as well as back washing or running the column backwards; that would be really helpful for me. Do attach to me too.

P/S: BTW, just curious; since reverse use of the column can be done, why there is an indication note that stated which side the flow should be?

Do let me know ASAP. Thanks a lot!!!

[Uwe Neue]

There is no reason to panic. The backpressure is just a bit higher than what the manufacturer has measured, and you are not stating the conditions for either your test nor the manufacturer's test. The difference could simply be the solvent composition.

The packing is not a soft polymer packing. It is hard. Maybe not as hard as silica, but unless I get a warning from the care and use manual not to go beyond a certain pressure, I would not worry about it.

If the column was tested at 1100 psi, I am fairly sure that you can use the column at 1400 psi. If it were a silica-based column, would you panick? Most silica-based columns of the same dimensions and particle size are probably tested at pressures of 1000 psi or less, and used without difficulties to pressures in excess of 4000 psi.

In order to judge, if the column is clogging, one needs to know more about your conditions, and the column test conditions. Unless I know for sure that the column is clogging I would not even dream about inverting the column. If it is clogging, I would first exchange the column inlet frit before inverting the column. This is the reason why they put an arrow on the column. Of course, if everything else fails, one could potentially invert the column, but it is NOT the first thing to to.

[Kostas Petritis]

I cant get the pressure to get lower; in fact, the pressure keeps on going up even though the end of the column was not attached onto the LCMS-MS when I try to increase the flow rate through the column.

I would expect a minimal increase of the back pressure when you connect your column in the MS. Also if you increase your flow rate the pressure will go up so I do not quite understand your statement.

From what you described it seems like the column is fine, especially if you do not experience continous back pressure increases during several analysis.

I would recommend you to be carefull if you try to open the Hypercarb column in order to change the frit. If you do not keep your stationary phase wetted at all times it starts to fly...

[Victor]

I agree with everything that has been said and that you have nothing to worry about.

You could try disconnecting your column and recording the pressure using the same flow rate of the same solvents. It is possible that you will see an appreciable residual pressure due to the tubing , mobile phase filters and injector in your system. Of course, the pressure will vary with the exact nature of all of these- for example small ID tubing generates a much higher pressure.

[HW Mueller]

Uwe, your suggestion is quite a surprise to me. From previous discussions here, and talks with some representatives of manufacturars for the last ~30 years I was under the impression that especially more modern columns are not suffering from reversed flow, unless the inlet frit is too coarse to hold stat. phase. Now one always looses some material when opening a column, and stat phases are hard to get at, recently, etc. Thus when there was flow interference I have tried (since I can remember) solvent washes first. If necessary I did similar washes in reverse flow next. Replacing a frit has been dead last.
So my question: Is the stat phase packing on very new columns more sensitive, for some reason, to flow change, or is the practice of using unsymmetric frits spreading?

[HaruYap]

Thank you for the reply.

According to Thermo, it is advisable not to change the frit urself. Also, they have given the assurance that the column will be able to take pressure higher than 1100psi; which means that the pressure of 1400psi that I'm experiencing is ok.

I am having some doubts because I've tried to run my standards with the columns and after a few injections; the peak for the standard will not be detected ie from what I see, it must have been retained in the column? Am I right? So, that is why I'm concerned about the column being clogged.

FYI, I've filtered my samples using a 0.20uM syringe filter; however, it seems that the column will gets clogged.

My gradient system consists of 0.5% ammonia in water and my organic will be Methanol:ACN at 40:60 with 0.5% ammonia as well.

Any advise that you guys can give me to verify whether the column has clogged or not? FYI, this is a new column that I've just purchased and I've not used it since the day when I saw the backpressure being higher than 1100psi. I've kept the column in the organic solution that I've used for my run.

Thanks for the attention.

[Uwe Neue]

Column clogging is not very likely to be the cause of your problem. Plus, you should be able to measure the flow rate that comes out of the column with a graduate and compare it to what it should be.

[HaruYap]

If clogging is not the problem, then can anyone let me know what happened to the standards that I've injected into the column??

I've got the standard peaks from initial runs before it 'disappeared' from my chromatogram.

Uwe, do you mean that by checking the flowrates of the column with a graduated, this will showed that the column is not clogged rite? However, I am experiencing higher backpressure than my initial usage of the column. Initially, the column is being used at a backpressure of less than 1000psi. Now, as I've mentioned, I'm experiencing backpressure of about 1400psi. I didn't change the solvent systems nor any other settings on my system; which is why I'm having concerns about the column. I dont want to further 'damage' the column; if I can termed it that way because this column is rather expensive (approximately USD1K). So, any help would be greatly appreciated.

Thanks.

[rhaefe]

Since we do not know what you are trying to separate and how it is hard to respond to your problem.

cheers

[Bruce Hamilton]

If you're worried about material blocking the column, purchase and install a short guard column ahead of the main column.

Make certain that all of you mobile phase components are HPLC grade and suitably filtered after mixing. Ensure that you are flushing and storing the column in accordance with the manufacturer's suggestions. I would normally wash ammonia, acids, and salts out of columns before storage.

Bruce Hamilton

[HaruYap]

Sample would be: human serum

Testing on: Isoprostane level

I did installed a guard column initially but the guard column leaked only after a few injections? I have filtered my samples prior to running; so am I not sure what exactly caused the pressure to go up.

Do advise where necessary. Thanks.

[Uwe Neue]

The sample would be human serum ????

If you inject a sample of human serum onto any column without sample preparation, you can say "good bye" to the column. Depending how much you inject, the column might die after one injection or after 50. Thus the cost per injection is somewhere between 1000 US$ and 20 US$.

If you have injected such a sample already, and the pressure has increased, you will know already how quickly the pressure increases for each injection. The worst case in my life was an injection of milk onto a reversed-phase column. The backpressure immediately went through the roof, and I learned that this was not a good idea.

Try to save yourself from my experience and do some sample preparation and use a guard column! If you contact me, I send you some references about how to do that.

Otherwise, give me your address, and I volunteer to become your personal column supplier

[HaruYap]

I didnt inject a direct human serum sample into the system, Uwe. No worry about it. I have done the necessary sample prep before the injection. I have also installed a guard column but to no avail.

[Bruce Hamilton]

Would you like to share the details of your sample prep with the group?. It could very cathartic.

Bruce Hamilton

[HW Mueller]

If you did the necessary sample prep you wouldn´t have a flow restriction. While a flow restriction is compensated by the pump (flowrate doesn´t change unless the restriction exceeds the pumps capabilities), and therefore should not influence rt, it is an indicator of "dirt" reaching the column. Some is causing the restriction, some may change the stat. phase characteristics. I suspect that something could have changed the pH, so that the negative isoprostane is attached to the plus charges on the stat. phase (or dirt?).
I have worked with leukotrienes, from that experience I would tend to suspect that the stability of the compound(s) could be a cause to count on as well.