LC contamination

Discussions about HPLC, CE, TLC, SFC, and other "liquid phase" separation techniques.

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Hello all. I've got a Shimadzu Prominence LC-20AT/SIL-20A HT/SPD-M20A (an HPLC-PDA system with autosampler), and I've got a contaminant coming up in every sample (lambda max 222nm). My routine pre-batch start-up includes purging the pump, 3x's autosampler rinse, and an autosampler purge with both aqueous and organic mobile phase.

I've since tried preparing fresh mobile phase, back-flushing the column with 95% CH3CN/5% water for 20 column volumes, prepared a new blank vial - nothing seems to work.

I've also tried disconnecting the column and letting it flow directly to the PDA with a female-to-female adapter, but I don't think I can see it distinct from the solvent front (looking for the lambda max, it doesn't come up).

What else should I be looking for/checking? I've never taken apart an autosampler before, so I haven't tried to replace the needle or loop. Many thanks in advance for your help!
Isocratic or gradient method?
-- Tom Jupille
LC Resources / Separation Science Associates
tjupille@lcresources.com
+ 1 (925) 297-5374
tom jupille wrote:
Isocratic or gradient method?


My analysis method is gradient (40% CH3CN/60% water to 100% CH3CN, then 10 minute hold at 100% CH3CN before reconditioning back at 40%). The contaminant elutes late, around when the gradient reaches 100% CH3CN.

Since my method runtime is almost 30 minutes and I know it's a late eluter, I've started troubleshooting using a 10 minute isocratic run using 95% CH3CN/5% water. Using this method, the contaminant elutes about 1 minute after the solvent front.

Retention times are consistent, but the peak area increases with each subsequent injection (10 uL injection volume). Started at ~5000, currently sitting around ~14000. Decreasing the injection volume (tried 1 uL) doesn't affect anything.

Today, I wiped down the rinse port covers (including the little septas) and flushed the needle seat with MeOH and water, then flushed the needle with 95% CH3CN for about 5 min @2mL/min. Alas, the bugger is still there!
Where I was going with that question is checking out the possibility that there was some low-level contamination in the "A" solvent reservoir that accumulates on the column during the pre-equilibration. The standard test for that is to run a series of blank gradients with significantly different equilibration time and see if the size of the contaminant peak increases with increasing equilibration time. That hypothesis doesn't seem to fit the results you have just given, but it might be worth doing anyway just to confirm.

It doesn't depend on the injection volume, which argues against contamination in the sample. When you rinsed the injection port, did the area come down from that 14000 value? For that matter, does the area *ever* come down? And if it does, how long does it take (and what do you have to do)?

Sorry that I have more questions than answers!
-- Tom Jupille
LC Resources / Separation Science Associates
tjupille@lcresources.com
+ 1 (925) 297-5374
No worries at all, I'm grateful for any suggestions and your questions helped trigger an idea!

I tried your suggestion of extending the pre-equilibration time from 3 min to 7 min and 11 min, and I didn't see an increase in height/area in either. I was never able to get a decrease in peak size with any remedial steps or cleaning (although I got rid of some itty bitty ghost peaks that had always been there and were never a problem).

Solved it!
Your query about mobile phase A made me rethink mobile phase B. I had just opened a new bottle of acetonitrile that was a new lot when these problems started (I also replaced mobile phase A, milliQ water, at the same time). Today, I opened another bottle of CH3CN of yet a different lot. I just topped off the mobile phase (~200 mL to ~1000 mL) and the contaminant peak is now exactly 1/5th of what it used to be! It was mobile phase B, as in "Bane of my life!". There must be an impurity in that bottle/lot, and as any evaporation occurred through the filtered vent in the reservoir or as I was topping off the solvent, the impurity was probably increasing in concentration.

I will probably continue to use that bottle just to not let it go to waste, as I know it is eluting off the column and it doesn't actually interfere with my peaks of interest. I was mainly worried about mucking up the column with some contamination in the autosampler.

Thanks again for your advice, as re-looking at the mobile phase was the right path!!
Hi cmf09,

First of all, congratulations on finding the source of your contamination! I'd like to give you a tip on how to, potentially, avoid a problem like this in the future. From your previous comment, I noticed that you like to "top-off" or save as much solvent as possible, however I would advise starkly against this. Topping off bottles, or opening and reopening them, exposes your mobile phases to the atmosphere which can introduce impurities and contaminants sporadically. The issue with some of these impurities is that they will NOT pass through the column, but get stuck somewhere along the way. If you clean your frits, things like dust and small particulates will have a tougher time getting through, but it is not unheard of when a tiny particle gunks up a whole instrument. The best practices would be to ditch that entire lot of acetonitrile, call the manufacturer and get a refund! They will be happy that you called and reported this error in quality control (if they're a decent company) and should reimburse you for the mistake that THEY made. There is no waste in this scenario-- except if you are wasting your column on their bunk acetonitrile. I'd suggest using that newer lot and to mark/waste the tainted lot of acetonitrile. I used to do something similar with topping off my reservoir bottles, but have see far superior, and cleaner, chromatographic results ever since I switched. Please do not wait until a hidden contaminant gunks up your machine-- trust me!

Anyway, congratulations again on solving your problems-- I hope that your chromatography is as smooth as a baby's bottom.

Have a nice day,
Tyler Smith
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