Chromatography Forum

Hi all! For the past few months I have been having trouble with the an RP HPLC method on one instrument specifically. I am running the same method on 3 other instruments with no issues. I am running on Waters 2695. A mystery blob peak has been appearing with varying retention time of between 5 and about 40minutes. I have tried just running zero volume injections and after doing a bunch of wet priming and purge injectors it may go away for a few injections and then come back. It does usually not show up in the same exact retention time injection to injection. Peak shape is usually blob like and stretches about 7-10 minutes. Peak height is about 10mAU.

Column- XBridge C18 5 μm x 150 mm x 4.6 mm (tried three different gel lots) Columns all additionally work fine on other systems.
Mobile Phase A 90% HPLC Grade Water/ 10% LC/MS grade ACN + 0.1% TFA
Mobile Phase B 100% ACN + 0.08% TFA
Gradient- 1 min hold 100% ACN. 1-36 minutes 80%A/20%B to 5%A/ 95% B-hold at B and then return to starting condition.
Water and TFA are JT Baker and ACN is Fisher.

Flow rate 0.3 ml/min
Runtime 60 min
Injection volume -25uL
Detection UV at 220 nm

I have tried cleaning on the instrument using water, ACN, 30% Phosphoric Acid ( no column on), 50%/50% ACN/Water. Nothing seems to work. I have taken off the sinkers cleaned them and even run it with no sinkers on just to see what I could see and the blob was still there.

I ran 12 zero volumes with the gradient going through lines A and B yesterday and 12 zero volumes with the gradient going through lines C and D just to see if the contaimination is in one of the lines. I saw the blob regardless of what lines it is going through.

I have HPLC specific glass containers I use to ensure that there is no contamination coming from the bottles. I have run the same mobile phase, in the same bottles, on a different instrument and see no blob.

PDA spectra is seeing peaks in the 320-360nm range.

I could really use some advice! I appreciate your help in advance.


Kelly

Wow! You have *really* been thorough!

You have pretty much exonerated the columns, the solvents, and the glassware, so it has to be *something* in the system, and you have already looked at the most likely culprits there.

About the only other source of junk that I can think of would be your autosampler rinse/needle wash solution. If that isn't it, the only other thing I can think of (and its *tedious*) is to start swapping modules with another system (autosampler / injector first, then pump; you have already done the columns) and see which one the problem follows.

Is there stronger absorbance near 220 nm than at 320 - 360 nm ? Maybe some aromatics involved?

Have you tried extending your run time dramatically to see if the mystery peak comes off at a much later but repeatable time? It could may well be a contaminant in the sample or even part of the sample itself eluting much later if the RT is all over the place from a previous injection working its way through the column. Way to check is do you see it in your first sample active sample? If you don't but start to see it after in subsequent injections its possibly this especially if its "walking through" your chromatograms as it were.

As you're not seeing it on other machines/detectors - is this system more accurate/newer? We had a similar issue with our TSP systems where a sample was running fine then when we switched it to an agilent 1200 we got a small carry over peak randomly in the chromatograms much later than our run time "cut off point".

What do you use for your needlewash also as this could help if you use e.g. 100% MeCN to help shift it if this is in fact the issue.

lynz x

Thanks I try to be as thorough as possible. This peak is giving me nightmares!

Ok Tom Ill give that a shot. Right now, my needle wash is 20% MeOH I could probably bump that up.

I have tried extending the gradient but Im sure that I can extend it more. The peak does appear to be "walking" up the gradient so there may be something to that. The system is the same age as two other systems in the lab. Waters performed a PM around the same on each of them. I would say that the only difference is that this instrument has a 2998 PDA detector and the other instruments in this lab have a UV 2489 detector.

I appreciate your thoughts! Thanks so much!

do you have a chromatogram of the pressure as well when running this instrument?
i suggest you make a few runs collecting the runs as well.

have you tried to rule out the degasser?
i am less inclined to it being the problem since the peak seems to move around so much