Acquity pH buffer change switch methods

<p>Hello,</p><p></p><p>I have a query about switching methods.</p><p></p><p>We have an Open Architecture UPLC, with a column manager and a Binary solvent manager.  SCN 737.</p><p></p><p>We have 4 different columns in there, 3 dedicated to Neutral pH, 1 dedicated to Acid pH.   Line A1 = Neutral buffer.  Line A2 = Acid buffer.      Line B1 = ACN for both.</p><p>Starting conditions 98% A     2% B.    I normally run at 1ml/min.</p><p></p><p>When we switch from Neutral to Acid and back again, we use switching methods, either acid to neutral, or neutral to acid, which flushes the pumps with the next mobile to be used (to waste).
This is so that the correct solvent constituents are already in the pump for the next method.
The columns (& presumably the lines) themselves are always meant to be left in the correct mobile phase pH.</p><p></p><p>The analytical runs normally take 1.5 mins, but my switching method takes me 1 min.</p><p></p><p>(Please see attached pdf for switch method parameters).</p><p></p><p>Firstly I am wondering whether I can choose a better combination of flow rates and percentage A/B to reduce the time to do the switch method.
Perhaps somebody out there has already figured out the quickest & best way to do switch methods, and they maybe can share this info?</p><p></p><p>Secondly, I have noticed that the sequence of switching from acid column to neutral column and back again may possibly be allowing a little bit of acid getting into the neutral line, and vice versa.</p><p></p><p>E.g. The sequence of events at the end of a Neutral column run, whilst switching over to acid, as a result of the introduction of the switch method, is as follows:</p><p></p><p>1. The solvent A line switches from Neutral to Acid.</p><p></p><p>2. Within a second or two the flow stops.</p><p></p><p>3. The column valve remains set to the Neutral column for about 6 seconds, as the pressure continues to drop towards 0psi.</p><p></p><p>4. After the approx 6 seconds, only then does the Column selection valve change from the Neutral Column to waste.</p><p></p><p>In other words, because the mobile phase switches a number of seconds before the column changes over to waste, it might be that a Neutral column might get a slight slug of acid, when switching from neutral to the next (acid|) run, and vice versa.
(I do get a slight change in retention time when switching between pH buffers, as opposed to when not switching between buffers).</p><p></p><p>I can see that there is an “Equilibration Time” of 0.1 mins (6 secs) in the column manager window (see PDF).</p><p></p><p>I’m, not exactly sure what this time is meant to do, but it wont accept any value below 0.1 mins.</p><p></p><p>Regards.</p><p></p><p>A.</p>


  • Hello:

    Thank you for all the detail, this help, but I think we may still need some discusion on all your questions.

    The first thing is to clear up, is why you need 6 seconds and I think this will allay your concerns. The valve always vents the pressure before bringing a new column online. This is extremely helpful for column life as much as anything else. The vent valve on the BSM goes to waste and coordinates with the Column Manager. Any flow that is a reult of venting will end up in the waste.

    The control time line goes like this. The pressure is read at the end of the run, just prior to the run time clock stopping. For traditional reversed phase we would recommend setting a final line in the gradient table to high organic, flushing previous organic from column, before equilibration with initial conditions of analysis method. This also ensures the backpressure is low. This step promote RT reproducibility. In your case, perhaps an aqueous flush and then some organic and then the switch.

    I added a method example. Let me know if this makes sense. This is more MassLynx/MS typical example, with more application data I could suggest something more applicable to your work.



  • Could you please be a bit more specific regarding what your low & high pH buffers are? And, what columns & chemistries you are using? Also, are all the peaks moving or just some?


  • Hello Liz,

    thanks for the info.

    I will need to have a think about this for a while, to I see if I can understand it....

    I suppose the thing I couldn't quite understand on our system, is why the solvents would need to switch before the column is disconnected and the flow goes to waste.

    So, the Neutral column (for example) gets to the end of the run, and whilst its still on line, the acid line is activated, and only after some seconds the valve is diverted to waste.

    I would have thought it would be better to stick with the neutral line, depressure etc, then vent to waste, and then intoduce the acid line to waste.

    This is not a major problem by the way, and I don't have any overpressure issues, it just that I can see this happening, and I seem to get a very slight shift in RT for early eluting peaks on the neutral column, after a Neutral to acid switch has previously occurred.  

    I'm talking only a second or two.



  • Hello Doug,

    B is always ACN

    A1 = 0.1% Formic Acid in water.

    A2 = 10mM ammonium Formate in water.  pH = 6.4

    Column is BEH C8  1.7 um 50 x 2.1mm.

    There is a slight shift in RT (shorter) on early peaks on the neutral column after an acidic switch method has run.... only a second or two.

    Was wondering whether, after a neutral run, during the switch method, because A mobile phase goes from A2 to A1 a few seconds before column valve moves away from the neutral column to waste, whether maybe a slight slug of acid is entering into the neutral column.



  • I will wait to ee ehat Doug says based upon your input.

    The vent occurs first, before any switch in mobile phase, or viscosity as we are using the BSM vent valve, we are venting prior to the column and prior to change in MP, but as I mentioned you can affect that by putting a final line in your gradient table to do just that, and you can choose what makes bet ense to your chemisty, but you will ahve to allow for the system volume, to get to the column, and then check a 3 x system volume + x5 column volumes re-equilibration, based upon your method flow rate.