Issues controlling Waters 2695 with Millennium 32 v 4.0

Basic questions from students; resources for projects and reports.

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Hi all, I am bringing an old waters 2695 with 996 PDA back to life. I have gotten it primed and connected to the computer with millennium 32 v 4.0 installed. When trying to make a single injection just to test the software, I am able to start the run using millennium 32 from the computer. However, the parameters of my method don't seem to be fully communicated to the HPLC. For example, I wanted to clean the system, so I set it to a high flow rate (7.5 ml/min) and ran it, however the display on the instrument only showed it raising to 1 ml/min and the diagnostics did not appear on the millennium 32 display. I also selected in the method to monitor system pressure, and there was no readout. To check that the system was capable of higher pressures etc, I ran it manually through the direct controller method and it responds nicely.

So my question is, does anyone have experience troubleshooting the connections/communication between millennium 32 and a waters 2695/996PDA ? Millennium 32 does not seem to be receiving information back from the instruments and is not sending the signal as I expected.

Thanks for the help!
Hi tmaxwell,

I'm not sure if this helps, but there may be a setting within either the 2695's firmware or within Millennium 32 v.4.0 called "Stroke Volume" that may have to be adjusted to allow for such a high flow rate--I may be in error here on this count.

Otherwise, it is possible to "scan the BusLAC/E" to determine if there is active connectivity between the PC and the Alliance--I think that would be in the Configuration menu option.

Best Wishes!
Thanks Mattmullaney, I have figured out how to properly control the flow rate now. Turns out it was a silly issue with me not creating the method correctly. I have now figured all of the issues with the 2695 and can nicely do gradient elutions. The remaining issue is erratic control of the 996 PDA. The first day I had things set up it seemed to respond nicely to millenium, but now when creating a method the PDA always reads at 254 nm regardless of what I specify. Do you have any tricks for such an issue? Any kind of step by step would be appreciated too. Strangely enough the first time I followed the instructions on this youtube video ( it worked great, yet doing the same set of clicks (I think) I now can't seem to control the PDA. Thanks again!

Hi tobi

The 996 will collect all wavelenghts in the range specified in the instrument method. The derivation has to be done afterwards in the method set.
by default, in run samples 254 nm will be displayed > right click in the trace window and select change channel (or similiar, bottom entry). There you can change and specifiy different wavelengths for "live view". To keep these settings for this project you then have to "save preferences" from file menu.

in general millenium/empower: right click is your friend. often gives you a selection of meaningful tasks at the actual position
Hollow, Thanks so much for the prompt and helpful response. I now see the difference between the real time view and the actual data collection and have found how to begin the processing in the 'review' part of millennium. For now I am working with waters guides, but if you have any other advice or resources for helping to process data, I'd love to know about it. Thanks again!

you're welcome

Millenium/Empower may be not that easy to begin but once you're into it gives you so many possibilities.

If not yet done, also use the help contents (F1). Sometimes it's a bit tricky to find the right infos but there's a lot of information built in. Normaly it will show you the topic related to the active window.

Just play a bit and try things out.
For this purpose, it's good to create something like a "training" project, where you can also import data from other "real" projects (right-cl>Copy to) and then just play around without messing up the real analysis.

Some known "beginners" mistakes (done myself...)
- when processing PDA data, you can't select a specific <processing method>. needs to be a <method set> (as from the previous post, the wavlength is defined there. otherwise you'd be trying to integrate a cube...)

- take some time to create a nice report with all the infos you want to have on your print out. Copy this to the other projects

- for referencing to your raw data, use the <result ID> or <injection ID>, these are unique numbers and with that you'll find your injection or sample set in a blink of an eye

- some nice feature to navigate is the <right click, view as...>

- hit <update> first, if you can't see what you've expected to see

- select the correct processing method type, when working with PDA data (needs to be <PDA> not <LC>), otherwise you won't see the spectral information for your peaks. And this type can't be changed later, so the processing method would have to be re-written

- use the <processing method wizard> until you're familiar where these parameter are written to. then wizard becomes obsolete for simple integration tasks (my opinion)

- many ways leads to "rome". find your own.

- for me, I normaly just use the "browse project" entry and do all my tasks from there (run samples, review, print etc.) and sometimes "configure system" for administration. Never used one of the bottom three entries.

- my way of processing (short, PDA data, no proc method yet)

-- load sample set into review
-- open the correct method set (see status bar which one is loaded)
-- go to "3d channels"-tab, select the PDA channel
-- in the toolbar, enter the wavelength needed, extract it
-- (use the "next injection" button to navigate trough the sample set)
-- manualy integrate the narrowest peak, click button for "peak width"
-- zoom in a region of baseline (as broad as possible and representative), click button for threshold
-- maybe set a miniumum hight (eg. 10x width of baseline ->LOQ)
-- right click, add integration events like "inhibit integration"*
-- toolbar integrate
-- select the peaks I want to label, right click, add name*
-- if everthing looks fine on all/most of the samples, save processing method
-- save method set
-- close review window
-- select sample set, right click, <process>
-- select method set of choice, ok
-> now there should be a result set with your chromatograms
-- if needed, load result set into review, do some manual re-integration, click quantitate (to re-lable the manual integrated ones), save <result>, close
-- print result set (should only include the newest (manualy intgr results)

*) don't remeber if this was already available in millenium or just came with Empower. otherwise go to processing meth window and fill in there.
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