Waters HPLC system newbie

[FragranceChem]

My company just recently requisitioned a complete Waters HPLC system w/ Empower software from our parent company because they no longer use it. The setup is a 2695 Separations Module, 2414 RI detector, and 2996 PDA detector.

It hasn't been used much the past couple years except to send out old reports to customers, and the person who originally ran it is no longer here. I've got the machines set up and connected to a workstation, but I'm struggling to figure out the Empower software. Admittedly, I'm pretty fresh out of school and chromatography was never an academic focus. I worked a lot more with NMR, IR, and MS.

The work I'm using the HPLC for isn't anything unique, and I'm pretty comfortable with the theoretical setup/methods for my runs, my issue is mastering the software/hardware aspect of the system. Any help in pointing me towards some specific topics on this forum or other resources would really be appreciated.

Thanks in advance.

[LC_labrat]

Waters software has a steep learning curve. They have well written manuals on their website which go into great detail so read those throughly. Use the wizard guide to help you through until you get the hang of it. Otherwise feel free to ask specific questions.

[FragranceChem]

Thanks for the response. I've been able to set up a few runs just for the sake of running them with a couple of sample sets, and I'm starting to get a hang of the "hierarchy" of samples set methods/instrument methods/method sets etc. The two that are confusing me are the processing and results methods. I'm leaving analyzing the data for later which seems the most complicated.

I imported an old processing method from a previous project because the default processing method wasn't doing anything. I would make a run, and all I would get was the 3D absorbance spectrum. When I've tried to make a new processing method in my recently created project I'm missing a purity tab, and a PDA library search tab. Aside from just copying the old processing method into my new project is there another function I need to add to my current project? The software recognizes all of the hardware as far as I can tell.

My easy question is does the empower software support remote changing of columns? I have two columns connected to a waters controller that does support manual and remote control, but when I've tried to make a new instrument method it usually doesn't allow me to change the column selection box, and if it does nothing ever gets changed (even when the LED on the column switch is set to "remote control").

I've managed to find some support manuals on waters.com that don't require a support plan to look at so I'll keep pouring over those. Thanks again.

[rwang]

If you are using PDA, you also have to define a derived channel in your method set window:

[benW]

Hello FragranceChem!
The reason that you do not see the purity and library tabs in the processing method editor is because the processing method was created for LC data and does not have the PDA features enabled.

To create a processing method for PDA data, right click anywhere in the main project window and select new method > new processing method. Then when prompted, select PDA and not LC. If PDA is not available, then you may need to add the Photo Diode Array option to that project (Configuration manager>right click the project>select properties... > and view the Enabled Options selected in the general tab.

If you want to use the processing method wizard, take some data into review (right click a channel in the project window and select review) then click the processing method wizard button at the top left.

Couple of extra tips,
right click is your friend in Empower! these reveal context sensitive menus based on the object and window you are working....
and the Empower (build 1154) help is very good.

[FragranceChem]

Thanks for the help. Suggestions on looking through the Waters instruction manuals has definitely paid off, and so has right clicking. Right clicking definitely gives me a lot more context specific options which is what I've been looking for as I'm starting from scratch.

One thing I haven't come across is a distinction between "Data Channels" and "Derived Channels". When I have an instrument method that includes the 2414 RI detector it will be automatically added to the Data Channel as W410, but the 2996 PDA detector can only be added as a derived channel for MAXPLOT or single wavelength absorbance. How come the PDA detector is never seen as a raw Data Channel? In what situation would I need to set up a derived channel for maxplot or single absorbance? As far as I can tell the PDA doesn't need to be defined in order to collect and process data.

Again, thanks for the suggestions. It's definitely making my job a lot easier.

[benW]

The the reason you are seeing channels for the derived channels is because you have specified these in your method set and probably checked the boxes for save extracted channels and delete 3 D channels. If you uncheck these boxes you will find that the 3D 996 channel is saved. The extraction will then occur when you take the 3D channel into review and open and apply the method set there (or extract a channel directly in review if you like).

[FragranceChem]

So i'm starting to get the hang of Empower, and I'm gaining headway in what has quickly become my part time job at my full time job.

I have a data channel for the 2695 psi levels, and under review it comes up as a 2D channel that the processing method attempts to process. Is there an easy way to keep that as a data channel, but exclude it from being processed?

Nevermind; I assumed when I went to review it would automatically apply my method set, but I forgot I turned that setting off. I opened up my method set, and it then correctly applied the processing method to my injection, and not the pressure data.

Quick aside: My processing method found the peaks it was supposed to, and integrated them, but the amount was not quantitated. I'll look into that today and next week, but any ideas why the processing method didn't use the calibration curve would be appreciated.

[ChromatographyKid1]

Did you quantitate the calibration curve right? In channels, highlight all your standard injections, right click > Alter Sample, then I think it's edit > amounts, then File > Copy component from processing method. Choose your processing method, the components should show up, enter the amounts. In units, my work only uses ppb, so we type "<ppb" in units without the quotes. Save everything, then open your standards in review, open your processing method, and quantitate each. Then check the curve, make sure it looks good and do File > Save > Calibration. Then open that processing method to analyze samples.

I'm not writing this from a computer with Empower so some things may be a bit off, but that should work I'd think.

[cody84]

Go to injections tab of Browse Project
-highlight injections you want to use, right click go to alter sample
-make sure you add your weights and dilutions and purity in the sample set methods
-you also need a line for clear calibration, calibrate and quantitate and labels and if you have any custom calculations you'll need a 'sumamrize custom fields' at the end of your sample set method

Keep in mind any time you alter the original samples you will have to reprocess to see new results.

The Help menu is awesome, use it a lot. Search for things with "" around your search terms for better results.