High pressure

JJ

<p><span>We seem to be getting limited use of our columns due to high pressure after a limited number of injections. Even while pumping just mobile phase the pressure climbs from about 9000PSI at start to an increase of about 50 psi per hour. Once we shut off the pump it seems as if the pressure goes back down. Any hints?</span></p>

lizh

Seasons Greetings:

If the pressure rises even with mobile phase that says it probably is the mobile phase or something in it, that is causing the problem. However, with troubleshooting you must not jump to conclusions, but work through a process of elimination. Have you spoken to your local chemistry rep? They are a great resource. They can assist with the process of identifyig your issue. And or please contact your local technical support resource.

Firstly, you need to determine the location of the plugging, Head of column etc by replacing the inlet frit and if not then replace the outlet.

Secondly, what is casuing the plugging. It is always good to ask whether the method previously worked on HPLC columns. You will need to inject standards or blanks prepared in mobile phase, to see what is the column lifetime? This will help to rule out the solvents, system and method.

Assuming that long column lifetimes are enjoyed during this study in #2, prepare standards in water and subject these standards to the sample prep procedure. Inject these prepared standards. What is the column lifetime?

All that is left now is the samples. The system, solvents and lab practices have been eliminated. If UPLC column plugging or short column lifetimes are now encountered, the sample quality & preparation need to be investigated.

UPLC Columns act as a very fine filter. To realize the potential of UPLC, small procedural changes may need to be made - Analogous to past changes required to utilize 3 µm particle columns or microbore columns.

I hope this helps.

Liz

Outback

Sounds like >0.2um particles.

We have seen a few causes

1) Bacterial contamination... Solvent is filtered into containers that are re-used and contain a bio-film.

2) Wrong filter.. some types of filters leach binder and decompose at high organic composition. (check comparability)

3) Glassware.. we have seen cases where the glass containers were leaching silicates at high pH (NH4HCO3).

4) Sometimes a small amount of bacterial or other fouling has built up on surface of the inlet filter from an earlier run and during flushing is extruded into the filter causing much greater flow restriction.

5) If the system has been run in THF without the necessary precautions.

Contact someone at Waters to show you how to change the inlet end frit, if the backpressure is still there try the outlet as well. If this fails look at the history of what samples have been run through the column and choose a suitable solvent to best solubilize the compounds. (Protein contamination will require a different strategy)

dougm

Just a quick followup up to Ms. Hodgdon's and Outback's excellent replies. UPLC column plugging is rare. Because of this, troubleshooting UPLC column pressure issues is a fairly straightforward process since there is a very finite number of causes. In order to identify the cause of plugging quickly (without guessing) I also suggest you contact your local Waters Chemistry Operations representative who has been trained to troubleshoot LC column issues with customers. Waters can suggest a methodical, step-by-step troubleshooting process that will quickly determine the location (usually inlet frit) and then source (e.g., bacteria, sample particulates, etc) of UPLC column plugging. Please let us know if there is anything that we can do to help.

MikeT

I had that problem once because unknown to me my sample was not compatable with the mobile phase. Samples were filtered and absolutely clear but as soon as they were intruduced to the mobile phase stream something would precipitate out. How I determined this was to mix a sample with some mobile phase in a small test tube. It was then I could see something coming out of solution. Further investigation showed it was the ZnSO4 I was using to enhance precipitation of proteins that was not miscible in the ACN mobile phase. I had to change my extraction to a LLE rather than protein precipitation.

And not only the column got ruined. The stuff precipitated in the sample valve and it had to be replaced. Big lessen learned about sample vs mobile phase.

regards

Mike

ACQUITYEPM

Greetings,

There is one other thing to consider and investigate that I ran into in my lab. For a time, we (Waters) were supplying solvent reservoir filters (sinkers) made of Hastalloy. In general, Hastalloy is an excellent material that resists most types of corrosion and contains little or no iron to contaminate MS detection. This absence of iron was the primary reason for their introduction. However, over time, we discovered that Hastalloy did have one shortcoming. When exposed to acids (TFA, formic, etc.), Hastalloy will form insoluble oxides. When the oxide formation begins, the behavior is exactly as J&J describes. System backpressure will constantly, and inexorably, rise. This will continue as long as there is acid in the mobile phase reservoirs and Hastalloy solvent filters are in the solvent reservoirs. The Hastalloy filters are marked for identification. I believe it is an "X" imprinted on the bottom of the filter. If it is not an "X", it is some other letter. The standard 316 stainless steel filters have no marking. If this is what J&J is experiencing, the solution is simple. Replace the Hastalloy solvent filters with the standard stainless steel filters. I believe that the part number for a package of 5 is PSL613604, but this should be confirmed. The problem will almost immediately disappear. However, it is very difficult to recover any columns exposed to the oxides. Sometimes backflushing will remove some of the oxide particles, but the column almost never returns to the "original" backpressure. I am, absolutely, not saying that this is the issue that J&J is experiencing, but it is something that should be investigated as a possible cause.

Hope this helps,

pcb

aldij

all said before is true: especially about the basic trouble shoot pathway: eliminate possible origin of the problem one by one, and if you think you found the problem switch it back to the original to check if the problem comes back. we have had rising back pressure that was caused by the quality of the methanol used in our UPLC-method. when just pumping methanol/water the pressure raised after one night. when we switched to an other supplier of methanol the problem was gone.

rune

Hi

I believe that the correct par number for the Critical clean stainless steel sinkers is 700003616 which is a package of 7. The Hastelloy sinkers are marked with an XH at the bottom. The SS filters do not have any marking. We also have Titanium sinker that should work OK. These are marked with an XT.

best regards

Rune

ACQUITYEPM

Greetings Rune,

Thanks for the, accurate, corrections to my message. I got some of it right. Which means I got most of it wrong. I'll be more careful with the details in the future. However, I did want everyone to know about the issue since I ran into it on one of my own systems. It was rather mystifying until we determined the root cause. For those running acidic MS applications, the titanium is probably the best choice. Do you happen to know the P/N for the titanium solvent filters?

Regards,

pcb