help: high pressure of safe guard

[wxmwqr]

I used the waters HPLC system, Phenomenex column and safe guard.
Mobile phase is Acetonitril and 0.025M phosphate buffer (pH=7.0 phosphate acid adjust)
Sample is the microsome incubation system, after extraction and reconstituted with initial mobile phase, all the samples were filtered by 0.45um membrane unit.
But the cartridge of the safe guard got high pressure (more than 3500psi, only use the pure acetonitile) make the pump stopped automatically.
I change 3 of them and got the same problem. First one used less then 20 days, second on used 1week and last on used only 3days and analyzed only 12 sample.
Pleas help me to solve the problem! Thanks a lot!

[peptidemetdev]

Maybe your mobile phase is salting out on you?

Have you tried using pure water (or, at least, unbuffered water with a small amount of organic in it if the column isn't good for 100% aqueous)? See if that will reduce the pressure, you may have to reverse the column and do a slow flow for awhile to remove the blockage. If that helps, be sure to wash your column with unbuffered water/ACN at the end of each day to clear out all of the salt.

[wxmwqr]

I usually use the water : methanol (50:50) to wash the column and safe guard after the analysis procedure for about 30 min and used the pure acetonitrile to wash for about 30 min.

I test the system. Use the pure acetonitrile at 1mL/min flow rate.
no column and safe guard, pressure is 160 psi
only column, pressure is 824 psi
only the safe guard and cartridge, pressure is more than 3500 psi
only the safe guard one end is empty, pressure is 188 psi

that means the cartridge cause the high pressure. and may be something is wrong in it. But I can not find the reason of the problem.

[peptidemetdev]

It may be that the sample is not very soluble in the mobile phase? Do you see fluctuations in response when you do multiple injections from the same sample preparation?

You could try cleaning the guard with low or high (if you are using a high pH stable column like the Gemini or some of their mixed mode columns) pH eluents. I'm not sure what exactly it is that you are dissolving, so I wouldn't have a suggestion. My experience with the Gemini columns is that they handle changes in pH pretty well after an initial "conditioning" at both high and low pH.

[Uwe Neue]

What do you do for extraction from the microsome incubation system except for filtration?

[wxmwqr]

4mL acetonitrile added to 1 mL microsome incubation system (pH=3-4) for precipitation of protein, and centrifuge it at 6000rpm, take the aqueous layer to other tube, and dry under air stream at 40 centigrade. Then, added 150uL initional mobile phase(acetonitrile:phosphate buffer=30:70) for redissolved, and used 0.45 um syring filtered unit to filtered sample, and 50 uL injected in the system. used the gradient elution, the end mobile phase ratio is acetonitril:buffer=80:20.

all the chromatograms is good. but the cartridge is blocked. today I used the acetonitrile:water=5:95 to wash, little changed of pressure!

[Kelly Johnson]

We once had a customer with a similar issue and it turned out to be his pump seals. You may want to double check to be sure it isn't a degradation of a part causing the issue.

[Uwe Neue]

OK, you did a reasonable job with the sample prep. Residual stuff is expected to hand up on the guard cartridge.

Is the guard cartridge made with the same packing as the analytical column? If not, then it may not have retained residual junk that is now retained on your main column and has clogged it.

Alternatively, your guard column has bled out junk (maybe fine particles) and blocked the inlet of your analytical column.

Just some thoughts...

[HW Mueller]

Yes, it is a reasonable workup, but from your description not good enough. There will certainly be some proteins left, sometimes these do something (clogging), sometimes not. An additional SEC or ultrafiltration might do the trick.
(On this "lifetime" of the guards: Here it has happened that as a workup step becomes routine it is done less carefully. If this happens in wxmwqr´s example it could mean that later extractions had more protein left).

[HW Mueller]

Ugh... my SEC should read SPE (had solid phase extraction in mind).
Actually, size exclusion is not completely out of place either, I have used it to get rid of proteins.

[wxmwqr]

Thanks all friends!

I had used the precipitation method before on the Agilent ZOBAX SB column and safe guard. All the things are ok.

And I had tried the SPE method also. Because I want to analyze 6 compounds at the same gram, so the SPE method has bad repeatability of all 6 compounds.

It is really has many protein left in my extraction method, but after I used the 0.45 um syringe filtered unit to filtered, the sample looks clean and good.

I suspected that the syringe filtered unit that was made by nylon can be dissolve by acetonitrile. I will change it to PFTE.

Maybe “peptidemetdevâ€

[HW Mueller]

A 0.45µm filter will not get rid of any dissolved proteins. There won´t be enough proteins left to cause turbidity, you will not see them, your guard frits most likely will.
A salt precipitation should very easily and quickly be removed by injecting enough water or changing the mobile phase to water for a short time (as long as there is no total flow stoppage). Anyway, where should so much salt come from? Didn´t you extract with a large ACN excess? If you think it comes from the buffer you should get an increase in flow resistance by just running your gradient without any injections. Also, don´t you people check your mobile phases for solvation compatibility?