Chromatography Forum

Hi,

I'm using Waters RI Detector 2414 and I run filtered culture supernatant as my samples to detect sugar utilization in E. coli. I hope someone could help me answer these problems I'm facing.

1. I often encountered LED intensity low error message after running the hplc for about a week and injecting about 70 samples. I asked the branch's Waters Engineer and he suspected the flow cell line was contaminated. Thus I tried cleaning with 6N nitric acid as recommended in the manual, it worked, but the problem surfaced again when I run the my samples again. The question is, how do I deal with this? I've been running this type of samples for over 3 years now without any problem, and this problem only surfaced recently. I prep my samples by centrifuging 13000 rpm for 10min and filtering through 0.2 micron nylon membrane.

2. Would frequent cleaning with nitric acid cause any damaging effect to our system?

3. In the procedure done for flow cell cleaning, it says to avoid getting the nitric acid into the reference flow cell (purge). However, I suspect that the reference cell line is also contaminated, because the pressure increase about 30 psi when I start purging. Is there any suggestion to what solvent I could use to clean this part too? I read some people use 10% phosporic acid for cleaning, but no information if it is safe for reference cell part too.

Please help me.
Thanks

Hello, Bintuabdullah,

1. The prep you're using seems adequate--and also, it seems not to be quite enough. I used to run fermentation broth samples under a similar prep routine. Only thing I can think of is to ask if at the end of every use of the 2414 RID, do you Purge both the Sample and Reference cells with water and then 90:10 (v/v) MeOH/water?

2. Nope. HNO3 is used to passivate the SS capillaries and is okay for the sample cell (quartz) as well. No fear of damage just expended time.

3. Remember that the line through to the reference cell is much longer than through to the sample cell, the difference in pressure drop could be due to length. On the other hand, I suppose there is the concern of damaging the pressure relief valve with cleaning solution.

Not sure about 10% phosphoric acid and that valve...I'd ask Waters.

Hi Matt,

Thanks a lot for the reply. I really appreciate it.

1. After each run, I usually purge the flow with water only, not methanol. I was not aware of the need to flush with methanol after every run like that and I'll make sure to make it our practice next time.

2. Thanks for the clarification.

3. I've asked the Waters Engineer about using phosphoric acid, but he's also trying to find the answer with his colleagues. I guess I need to wait a bit more.

Thanks again!

Hi Again, Bintuabdullah,

You're welcome. Best wishes going forward. The MeOH/water mix will hopefully help keep the quartz windows nice and clean after nitric acid treatment or an ordinary sample run.