Am I going to wreck my HPLC or autosampler by doing this?

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

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Hi,

Several days ago, I posted about contamination issues in a Waters 717 autosampler. A suggestion that surfaced multiple times was to change my seal pack. I just finished up that job today. That was probably a good thing to do, because Waters says it should be changed once a hear and I have had my A/S 4 years without ever changing it. So I don't regret the expense.

However, this hasn't corrected the contamination issues. When I do zero volume injections of mobile phase, there is extremely little contamination coming off. But, when I do 100 microliter injections of mobile phase (which at this time is HPLC grade mobile phase right out of the 4L amber bottle), I see the contamination. It's not exactly a small amount, reaching absorbances of about 0.01. When I make 100 microliter injections of 2M NaOH, about 4-5 x the amount of contamination comes out, as quantified by peak area. BTW, all injections I speak of are made w/ a union in place of the column. I then did a little test in which I made 10 consecutive 100 microliter injections of 2M NaOH and the contamination peak area slowly decreased. This made me think that perhaps this contamination stuff can be washed out of the system. I just need to do enough injections of NaOH. So, I have set up my system to make 100 100 microliter injections of 6M NaOH.

My question is, is injecting 10mL of 6M NaOH in 0.1 mL increments going to damage either the 717 or the system (also Waters)?

Thanks,

Dave
hm, I'm not familiar with injections of conc. NaOH on LC systems at all, so not sure about doing so.


But some more thoughts:
- are the peaks that you see really your contaminants or just fluctuations of the pressure/refractive index?

- the 717 has a flow through needle, where 95% of your mobile phase is flowing through your loop and needle all the time. Therefore I wonder how things could get stucked there?
Nevertheless, if so, you maybe can set up an ammonia based mobile phase e.g. NH4OH 0.1-0.5M and just let it flow through the system for some time. This sounds less harsh than the 100 injections of NaOH 6M, and you're able to use a even bigger volume to purge your system.

- another way to passivate/clean your system could be the use of nitric acid. If I remember it right you can use HNO3 6M for this.
!!!But before doing so, make sure that every solvent line, syringe etc. is well purged with pure water and no organic solvent is left in the system! For sure, don't collect the HNO3 in your normal waste container!!!

- if nothing's going to help, consider to replace the needle and loop as well (if not already done with the seal pak replacement)

- think also on contaminants in your vials, your NaOH (you don't see real peaks with low volume of fresh solvent...)

- another source could also be the Needle Wash line and or solvent. Maybe you're able to clean/replace these line as well.
As in the last post, is the needle wash working properly?


-->
I just found out in the waters manual, that the Alliance can tolerate up to 10M NaOH and NH4OH 3M, nitric acid up to 6M.
So I think the 717 should also be able to handle this.
When you excluded contaminations of your chemicals, it sounds to me that you have contaminations in your AS valves (syringe or / and loop). You should passivate your system as Hollow described (don't forget to inject the cleaning solvents!!!) and inject again your mobile phase. You should also think about the replacement of the rotor seals.
Hi
Before you do all the above did you change the small needle frit when you changed the seal pak?
If one has a problem of this type a systematic search of the cause is in order, not replacing the apparatus, one piece at the time.
Hi Folks,

Thanks for your insightful and stimulating comments regarding my issue of 717-related contamination. I have continued to work on this issue. Rather than try to describe it all here, I have prepared a presentation that will walk you through the tests I have done, and their results.

https://docs.google.com/a/georgiasouthe ... 24c3t4kt2m

Although I am not confident that I am personally any farther ahead in knowing exactly where in the 717 the contamination lies, you may see on the second last slide of the presentation that I may have found a practical strategy for leaching/flushing the contamination out of the system. After 0.5 M NH4OH sat statically in the system overnight, quite a significant amount of contamination came out when I started the system flow (with monitoring) the next day. Perhaps repeated cycles of leaving the basic solution in the system for overnight periods (the LC should be able to handle this solution, according to manual) and then running the pump for a while the next day will wash much of this contamination out of the system...

Still open to your comments.

Thanks again,

Dave
dkreller wrote:
I have prepared a presentation that will walk you through the tests I have done, and their results.
https://docs.google.com/a/georgiasouthe ... 24c3t4kt2m


->login with password needed

Could you please provide these or upload somewhere else?
Thanks
Sorry about that, I didn't realize it wouldn't be a publicly available website.

Try this:

http://chemistry1.che.georgiasouthern.e ... l_pack.pdf

Dave
:?

- do you have PDA data as well? How does the UV-Spectra looks like?
- do you also have the pressure chanel logged? If not yet, mayb you could turn it on. Do you see any correlation of the pressure and the Fluor/UV signal?

- maybe you can repeat the 100µL NaOH 6M experiments (e.g. the first 10 inj)
-> if you get the same curve with same intensities like the first time, I doubt that this is realy a contamination effect that we see (after all the flushing you did)

- when you do the overnight "soaking", will there be something left in a flask after you evaporate the solvent? If you would reinject it in two concentrations, is there a correlation with the signal or not (>is there a real contamination to flush out?)

- did you check your system for proper work?
-- leak/pressure test (are all of the valves working (sealing)?)
-- injection volume accuracy?
inject 6 x 50 µL of water, out of a weighted vial, weigh back and calculate the volume used (>would also point to some valve problem or to the injector draw speed (generating "bubbles" if two fast or vial is sealing to thightly)

- was the 717 once used with "incompatible" settings of loop and syringe (e.g. 100µL loop and 2500µL syringe), so as to contaminate a part which normaly never sees any sample (like valve #3/4 or the syringe?)

Interestingly the "retention" times varies with NH4OH and in the end also with air? very suspect... :? there is nothing in the flow path that can retain...
Hello,

No I do not have PDA data. Sure wish I did though! It would be useful in my research.

On the weekend I set the IM to display the pressure and I did a few mobile phase injections. In brief, the appearance of the curves in the detector channels did not coincide with pressure fluctuations.

My LC is working well, overall. We keep careful track of the performance of the various system seals and check valves. I periodically test the injection volume calibration by doing the very sort of test you suggest, by weighing a vial before and after a series of injections. My A/S is very precise.

That is an interesting suggestion about collecting effluent when contamination is being flushed out, and then evaporating solvent. However, at this point there are a lot of ionic species in my system again and I would be concerned that the ions left behind would mask any organic species.

Hollow, feel free to contact me directly by email: dkreller@georgiasouthern.edu

Thanks again for your interest and the discussion.
Hi Dave

thanks for your e-mail adress. I will use it if I have more suggestions or something to attach. But unfortunately, my ideas of what's next also start to decrease...
If no PDA is available, do you then have access to a normal UV/VIS spectrometer?
Then I would try to collect a spectra from the flushed effluent against the mobile phase used (NH4OH).
Due to the cut-off of the solvent (what has NH4OH?) you probalby won't see too much in the low UV region, but if there is really something, acc. to the UV trace, it has also some absorption at 300 nm (and acc. to the Fluor it absorps also at 350nm). Maybe this could help.
After evaporation/concentration, maybe try also to collect an IR-spectra if possible.

By using NH4OH there shouldn't be much ions-left, other than the contaminants after evap at reduced pressure.
If concerned about ions, maybe SPE on a C18 cartridge could help to get rid of them, and change the solvent to something organic ->mass spec?

Deppending on your suspected contamination, also keep the HNO3 cleaning/passivation in mind.

Recheck all of your chemicals for purity, and what about absortption effects from air (how does CO2 behave in the fluorescence det?)

Regards Sam
Hi Dave

I wonder if there are any news on your contamination problem?
Did figure out what caused the signals?

regard

sam
Hi Sam,

I never really understood either the exact nature of the contamination or where in my A/S it was originating. However, some of the work I did in trying to flush out the injection system must have helped. When I switched back to a normal mobile phase (back from those caustic solutions I was flushing with and making injections of) that contamination/ carryover was greatly decreased. A new set of samples has been sent to the lab that performs the intensive fluorescence analyses. I am waiting to hear from them before I conclude that everthing is OK, but I am feeling better about the situation now.

Thanks for the discussion and being interested.

What kind of chromatographic work do you do? Are you a professor, or do you work for one of the big pharma or agrochemical companies? Waaaaaaay back in time, after I finished my college degree, I worked for a summer for Sandoz in Basel. Of course Sandoz n'exist plus... I had the nicest time that summer.

Dave
I am experiencing UNIQUE issue in Waters Alliance 2695 ver 2.04 . It works fine but during the injection the Needle Wash Pump do not asprirates or noting comes out from Yellow waste line. It happens in PURGE mode too. The valve opens and waste come out from CLEAR waste line but Needle Wash Pump doesnt activates. Not the Needle Wash Valve Green Tubing consumes washing bottle solution. We have checked both Needle Wash Pump and Needle Wash Valve in Waters 717 Autosampler both operates and functions well.
Plz input with guideline
Thanks
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