Gradient HPLC - Massive peak at 65:35 Acetoonitrile:Water

[alterm]

We are running a starightforward gradient HPLC method which we have been using for several years. This is a gradient method using far UV HPLC grade acetonitrile (VWR) and purified water. Gradient starts off at 90% water and ends at 90% acetonitrile. Over the last few days we have suddenly started experiencing a massive peak at around 65% acetonitrile/35% water. This affects 5 HPLC systems that we have tried so far (Agilent 1100). We have tried 5 different batches of VWR acetonitrile (some of which we know have worked perfectly previously). We have tried water from 2 different purified water systems and purchased HPLC-grade water. We have tried old and new columns for 2 different (but very similar) methods - USP and EP. We have not yet scanned any of our materials on a UV/Vis to see what absorbance we get. We have flushed all of our HPLCs with IPA and acetonitrile to try and ensure that the flow paths are clean. This has got me puzzled - what is the root cause of the problem and why do we see this massive peak around 65:35 ratio (typically around 20 minutes into run)? We are even seeing this with a NO-VIAL injection (i.e. just the gradient being run with nothing injected into the flow path). Any ideas or suggestions would be greatly appreciated as this will soon impact our ability to release API for downstream processing.

[danko]

Hi Alterm,
The most obvious cause is typically some contamination (in the aqueous eluent) that is retained on the column, until an adequate amount of the organic arrives and elutes it. So, your focus should be directed to the water you use or the container you store the water in.
But to be more confident with this suggestion and to make sure that no equipment effect is causing the peak, you may start with removing the column (exchanging it with a connector) and running the gradient. If you see the peak then your efforts should be directed towards the equipment. If the peak is gone, then the eluents will need some more attention.

Best Regards

[juddc]

I'd do the following:

1. Run 3 gradients and vary your equilibration time between them - first 10 min, second 30 min, third 60 minutes. Does the peak get larger with increased equilibration time? If so, it's a MP contamination issue and those are most frequently caused something in the water.

2. Clean and rinse all of your MP reservoirs and lines. You might consider changing filter stones and frits if they've not been done recently.

In my experience, the HPLC is something of a "canary in the coal mine" with purified water systems. It's often the first sign that something is not right there. I've very rarely seen freshly opened solvent of appropriate grade cause problems, though I have seen bottles that have been opened for a while get funky.

[Bruce Hamilton]

Danko and Juddc have given the best advice, which should be investigated first, however one other much less possible cause may be volatile contamination in the laboratory.

Ensure that no volatile chemicals ( painting, restarted Air Con, solvent still, cleaning agents etc ) have recently occurred, or that a new glassware cleaning agent has appeared.

If possible, you should try to look at the UV spectrum of the peak- mainly to see if it relates to any compounds in the laboratory, whether samples or reagents.

Please keep having fun,

Bruce Hamilton

[Mach]

I have experienced the exact same kind of problem lately. From one day to the next a new peak apperared in the chromatogram. Gradient with water/acetonitrile added phosphoric acid and heptanesulphonic acid. The method has functioned smoothly for two years with a lot of samples. I have changed all chemicals and HPLC modules and column, and the peak is constant......

[tom jupille]

I've heard a number of recent reports about essentially the same problem, apparently as fallout from the ACN shortage. In at least a couple of cases, people have told me that they had to try ACN from several major suppliers to avoid the problem. The problem isn't new (see, for example, viewtopic.php?t=3951), but it does seem to have gotten a lot worse lately.

Bruce, juddc, and danko have all made good diagnostic suggestions. If you can confirm that the problem is coming from the "A" solvent (the "three gradient" test should tell you that) and you have a high-pressure mixing system, you can put a C18 guard cartridge in the line between the A pump and the mixer. If you have a low-pressure mixing system you can filter the A solvent through an "Empore" filter. Both of these are work-arounds until you can find suitable lots of ACN.

[juddc]

Amusingly, I walked into the lab this afternoon and was greeted with a very similar situation...diagnositcs in progress.

[juddc]

It was the ACN...but one of our analytes got into it, so not a vendor quality issue. Problem solved, source identified, and analysis continues.

[Consumer Products Guy]

It was the ACN...but one of our analytes got into it, so not a vendor quality issue.

How the $%&^**#@$%^*%##%&&*$#@ did that happen???

[alterm]

Thank you all so much for your replies to my question. I think we have solved the problem.

Firstly I was given some duff information by one of my lab chemists. It turns out that we had not tried purchased purified water as I previously stated since we did not have any in stores. However,you all pointed us in the right direction in terms of water quality.

The water comes from 2 separate purified water systems (but same feedwater) both of which are tested weekly to meet USP and EP requirements for purified water and have met spec., so we did not expect a problem.

We installed an analytical column (since we had no C18 guard columns) straight after one of the pumps in our binary system (the water channel). Lo and behold, the peak has disappeared and the baseline is the cleanest we have ever seen. Clearly the HPLC "guard" column (the same type as the analytical column) is removing some contamination from the water.

Our next step is to work out why we are seeing this contamination from both of these sources of purified water. Perhaps something from the feedwater is getting through but at the moment I don't see how as both systems appear to be operating well in terms of conductivity, TOC and micro-organisms. At least we have a path forward and the guard columns are on order.

Once again thanks to you all for the excellent advice.

[Bruce Hamilton]

Thanks for telling us the solution.

You have been very lucky to date. I would not rely on "purified water" as suitable for analytical chromatographic analyses- even when the specification is restricted to purified water produced by ion exchange, as suggested in some pharmacopoeia for "Water for chromatography".

There are minimal relevant controls in the specification, compared to dedicated laboratory instrument-quality water. Use this opportunity to push management for an appropriate laboratory water supply system.

Please keep having fun,

Bruce Hamilton

[juddc]

It was the ACN...but one of our analytes got into it, so not a vendor quality issue.

How the $%&^**#@$%^*%##%&&*$#@ did that happen???

Dunno...possible screw-up, but I'm not going to get torqued about it. It was in the last half-liter in the bottle, was identified and rectified within 4 hours, and the instrument was shown to be running well with data to be acquired overnight, so there was no data loss. The chemist noticed the problem in the first blank run and threw a flag right then and there. I consider it a training exercise because she learned how to ferret out a MP contamination issue.

[lmh]

In the sort of lab I've seen, a likely suspect would be someone making a dilution of a standard, who couldn't find the right solvent, and therefore took some from an hplc solvent reservoir, but forgot to change their contaminated pipette tip before sticking it in the bottle.

[juddc]

I'd doubt that...the LC reservoirs are inconveniently placed for that. I'd bet contamination in the bottle, which was nearly empty and was used to top up the reservoir shortly before the problem came to light. A bit of troubleshooting was learned by the chemist on-duty, who is excellent by the way; I'd say no harm was done.

[alterm]

Bruce,

In my haste to (a) pose the question and (b) describe the solution I neglected to state that we do actually polish the purified water through either Millipore or Elga water purification units so the original problem is all the more puzzling. What could possibly be getting through the purified water generating systems and the polishing units to affect the chromatography so badly? That is something we need to answer but in the meantime at least we have a solution that is giving us great chromatography