Buffer salts and pressure problems in reptile blood assay

[kernowsam]

Hi,

I've just discovered this forum and it seems like there is a lot of good advice circulating, so I was hoping someone could help me with a problem I've run into.... I've just started running a new assay to measure malondialdehyde in reptile blood. The protocol uses a mobile phase of 60% 50mM KH2PO4 (pH6.8 ) and 40% MeOH with a 5um ODS guard column and analytical column.

Initially the method was working fine but over time the pressure started steadily increasing from around 50bar to 180 bar! Eventually the pressure started fluctuating dramatically aswell. We are using a Dionex P680 pump and have changed pump heads several times, which works for a while but then the new pump head starts fluctuating too.

I should mention that I'm an ecologist not a chemist by trade so I'm a little out of my depth in the technical side of HPLC..... however, I have done some detective work and it seems like the source of the rising pressure is the guard column. Pressure with just the guard attcahed can be around 120 bar and unscrwewing reduces pressure to about 3 bar. I have replaced the column and this temporarily solves the problem but then pressure starts increasing again. The old guard column had powdery residue over the surface.

From reading other posts on this forum it seems to me the problem mught be buffer salts precipitating in the mobile phase and blocking the guard over time. Does this sound right? Could the same problem also be causing the fluctuating pressure in the pumps? I am only using 50mM buffer and 40% organic so I thought this would avoid precipitation? MOST IMPORTANTLY, what is there I can do to stop it happening?

Would be really grateful for any help from people more schooled in HPLC than I am.

Cheers

Sam

[Uwe Neue]

You are probably accumulating components of the reptile blood onto the guard column. What kind of sample pretreatment are you doing?

[Bruce Hamilton]

I'd want to insult your intelligence, but I'm going to list a few possible causes, and you can review your procedures...

Mobile phase components - all grades of reagents ( including buffer components ) should be HPLC grade.
Mobile Phase preparation. When you make up the buffer, you should take a small sample and test the pH, then dump it. Don't put the electrode into the bulk buffer solution..

I'm assuming you are using a single pump and pre-mixing the mobile phase, rather than having the buffer in one channel and the methanol in another. If you are using the two channel system, trying switching to asingle channel premixed buffer system - because it avodis issues arising from the mixing of water an methanol in the pump mixer.

So, mix your two mobile phase components, ensure that you have a visibly-clear solution that remains clear on standing for at least an hour. Any trace of cloudiness would suggest your KH2PO4 is not suitable.

Then filter through a fresh 0.5 or 0.2 um filter. Be aware that a slow vacuum filter can cause evaporation of the methanol, so one litre should filter in a few minutes, and the filter should be visually pristine.

Ensure any solvent reservoir and pump filters are free flowing ( fill with distilled water and it should drip out within a few minutes. If not, clean with warm water in an ultrasonic bath, or solvent - if water does not work.

Ensure that your complete HPLC system ( including injection loop - by performing solvent large volume injection of the mobile phase ) has been well flushed with a 90:10 water/methanol mixture ( 30 minutes or stable baseline and pressure ), and check/record the pressure at your normal flow rate and operating conditions.

If the column is temperature controlled, but is at ambient, set the temperature to 5-10 C above ambient, it shouldn't greatly affect separation and may reduce future precipitation.

Substitute your buffer for the flushing mxture, flush the solvent, degasser, and pump channel, and connect your mobile phase, pump for 30 minutes, and record pressure.

I can't review your sample preparation, but the last stage should be a technique to remove any particulates, preferably a 0.2 or 0.5 um filtration, or a high speed centrifuge. The sample should be in a solvent that closely matches your mobile phase composition, including the pH.

Take some filtered sample and mix with about twice the volume of mobile phase and allow to stand for 30 minutes. If there is cloudiness, you have to review your sample preparation further, as you will if your samples form insoluble rubbish on the guard column.

First of all, inject about 4 - 5 mobile phase or sample solution blanks, and cleck that the pressure remains constant. If it doesn't, you have to review your mobile phase and sample solvents.

Then inject samples, and watch the pressure after every 10 or so samples, if it increases, review you sample preparation. Especially the final filtration, or sample solvent. Filters are cheap. Try to record pressures every now and then to ascertain trends as batches are processed.

It may not be particulates, it may be rubbish from your samples that deposits on the guard - however that should not have affected your pump, so that's why i focussed on the mobile phase.

Also, revert to the 90:10 flushing solvent whenver pressure is increasing significantly, increase the column temperature and see if you can dissolve up the precipitated junk. If you can, review you sample solvent preparation and composition to see if you can remove the junk before injection.

When you have finished a sequence, flush the system with the 90:10 flushing solvent for about 30 minutes. Do not leave buffer in the pump or system. Record the pressure, as this helps identify unusual trends, such as sudden pressure increase next time you come to use the system, as well as being good routine monitoring of column back pressure.

It's highly probable that your pump problems are caused by buffer components depositing in the pump check valves, and flushing and storing with a higher-water-content solvent mitigates the problem.

Your column pressure problems could be either the samples or the mobile phase, and the guard column is doing it's job in catching the particles, so ensure you have plenty of spares, and replace at defined pressures, or numbers of samples.

The best investigation is to watch the back preesure, and identify how, when, and why it increases. Then address the cause.

I could go on, but you're probably already falling asleep from boredom, so I hope this give you some points to consider..

Please keep having fun,

Bruce Hamilton

[kernowsam]

Hi,

Thanks for the rapid replies guys.

Uwe - Sample prep involves liquid-liquid extraction of the fluorescent marker I am detecting from blood plasma into n-butanol. An aliquot of the n-butanol phase containing the sample is then injected. Hopefully this step should remove blood particulates? Plus pressure increases even when just equlibrating with mobile phase which made me think this was the source of my probs.

Bruce - That is very comprehensive, thanks! Some of those things I am doing and some i'm not. All reagents are HPLC grade and mob phase is premixed. The column is incubated at 37 C presumably to reduce precipitation (should mention this is not my method, it is taken from published work). I will try washing with flushing solution and filtering all solutions and see if that helps. Is it adviseable to flush/store the analytical column with the flushing solution (change properties?) or disconnect it before flushing the rest?

As I mentioned above samples are in 100% n-butanol and it occured to me when I was handed this protocol that this might not be very miscible with the mob phase, so maybe this is causing problems?

Anyway, thanks again....looks like I'm spending my sunday playing with the HPLC (again!).

Cheers

Sam

[Uwe Neue]

The simple advice is to just replace the guard column regularly. Guard columns are less expensive than analytical columns, they protect the analytical column, and when they have a problem, you just throw them out.

The source of the problem can be carry-over of plasma components through your sample preparation, or the wear and tear of new pump seals, or other reasons. As you have seen from the giant outline by Bruce, you can spend a lot of time on the troubeshooting. Simply repalcing the guard may be the best approach.

[Bruce Hamilton]

Leave the column in for flushing, and you can even flush with a higher methanol content solution as well, if you think that will remove the junk ( IMO, unlikely ).

You can easily check butanol miscibility, by mixing some of your mobile phase with butanol. I'd expect it to be miscible..

Uwe is correct, if you can live with changing guards as required, that's the easiest solution.

Bruce Hamilton

[HW Mueller]

It could be that a better workup would improve things. I doubt that butanol gets rid of as many proteins as ACN, for instance. This assumes that the pressure increase without injecting sample occurred after the guard was already cruddy from earlier runs.

[Mark Tracy]

Unlike mammalian blood, avian and reptile red blood cells have nuclei. I suspect you may be picking up a little DNA in your extraction and that precipitates on the guard.

You can try disposable pre-column filters from any of the usual vendors of HPLC accessories. They are an order of magnitude cheaper that guard cartridges.

[kernowsam]

Hey, well thanks for all the comments on this everyone. I think I have solved the problem now so to close this here is what I have done and what worked...

Flushing with 90:10 H20/MeOH solved the fluctuating pressure problems so seems this was a problem with salt precipitation on check valves. I now wash through with 60:40 H20/MeOH at the end of every run (substitute H20 for buffer) to flush all buffer out and this seems to have stopped the prob coming back.

I have started mixing the buffer and MeOH and leaving for an hour and then filtering through a 0.45um filter to remove particulates that precipitate. Sometimes there is a white resiude on the filter which seems to be impurities from the KOH used to adjust buffer pH as we wern't using high grade (only 90% purity). Doing this has stopped problems of rapidly rising pressures during runs.

Pressure still ticks up very slowly over long runs which is due to some particulates in the butanol samples injected - suggestions that this is DNA or protein - filtering should solve this I guess but samples are very tiny (c. 30ul) so don't know if filters small enough are available??? At the moment I just swap guard columns when back pressure reaches double its starting level and this sorts it.

Many many thanks to all who replied.

Cheers

Sam

[Bruce Hamilton]

Thanks very much for reporting back.

Incidently, there was a "not"missing at the beginning of my long post, as I didn't want to insult your intelligence .

Maybe you could consider microcentrifugation ( or cheap in-line filters as suggested by Mark), if the particle problem becomes an issue, as low-volume filters can be quite expensive.

Bruce Hamilton

[Mark Tracy]

When we were developing a QA test based around a phosphate buffer, we found that in order to prevent precipitation, we needed to reduce the concentration to 30mM. Also, the price of good quality reagents is minor compared to the lost time and data that crummy ingredients cost you.

[DR]

Lots of good advise... just from my read of the initial post, I'm thinking

1) replace the guard column every time you see the pressure increase by a predetermined amount (10 or 20BAR, for example).

2) investigate SPE cartridges as a final cleanup (check recovery or add internal standard before 1st extraction step).

[AICMM]

Kernowsam,

If I am not mistaken, there is an ELISA method to assay malondialdehyde. I have requested more information from a collegue of mine and hope to get a method or reference from her in the next day or two. They were doing this by GC/MS when I got involved in repairing their GC/MS. I think they would like to switch to the ELISA method if it works for them. Are you interested?

Best regards.

[kernowsam]

Hi,

AICMM yeah if you have a reference for a method it would be good to take a look as the current HPLC assay does have problems.

DR I am just getting to grips with SPE for another assay I'm doing so I had thought briefly about using it to clean up samples....I guess you just use a similar sorbent as the solid phase in the column?? This would also mean I could potentially get the malondialdehyde out of butanol and into something more compatible with mobile phase I guess, as injecting in butanol is giving crazy peak tailing (I assume its this anyway)....worth a look.

Mark/Bruce....Haven't come across these filters before, do they just screw into the solvent line in the same way as a guard column?? Need to do something like this as my budget holder isn't happy about going through a guard column a week!

Thanks

Sam

[Mark Tracy]

For the filters, look in Upchurch www.upchurch.com or Isolation Technologies www.iso-tech.com or Valco www.vici.com or Optimize Technologies www.optimizetech.com The selection is bewildering.