Chromatography Forum

My gradient system developed a problem with pulsation at certain points of a run. It seems very stable when I pump either solvent, but when they start mixing, at certain ratios I get a nice sine function out of a UV detector and the pressure fluctuates quite a bit. And later in the run (close to pure acetonitrile) it disappears to come back when the mix goes back to 10% acetonitrile 90% water. The configuration includes a degasser, two Shimadzu LC-10ADVP pumps, a mixer, a RP column, and a VWD. What can it be?

Sounds like you might have problems with a too small mixer. What flow rate are you using and what size is your mixer?
Do you have the possibility to install and try a larger one?

When you start mixing you change also the viscosity.
So flow rate, volume of mixer and detector flow cell is of interest and what temperature you are running.

The system runs at ambient temperature (~22 - 25 C). The mixer has volume of 25 ul and the flow rate is 1 ml/min. I have a larger mixer, either 250 or 500 ul, but I was saving it for prep runs and thought that it would slow the system's response to gradient. I have to check the volume of the flow cell, but I know that it is a newer cell with volume smaller than usually used on these "high" flow systems.

The simplest thing I can do to test the idea of mixing issue causing pulsation would be to switch acetonitrile to methanol or isopropanol. How increase in viscosity would change the pulsation? At this point, back pressure with water is 3 times higher than with acetonitrile. Actually, the system is most stable when I pumped isopropanol through either pump isocratically. I did not run gradients with IPA/water.

To me it sounds like you have a buffered (A) eluent.
I’ve seen such cases with buffered A-eluent when the gradient goes to a critical organic modifier (too much B) the solubility of the buffer salt decreases and you get microscopic particles in the mobile phase. The result is too much light scattering it reads as if it was absorption and pressure rise.

Best Regards

No, there are no salts in the aqueous phase, just 0.1% TFA. Pulsation occurs in the high-aqueous part of the gradient and disappears as acetonitrile becomes the dominant component.

OK, do you have TFA in the organic (B-eluent)?
And which wavelength are you monitoring at?

Best Regards

You underestimate how much mixing you need here. Contact me off list and I'll send you some data.

Andy Alpert
PolyLC Inc.
aalpert@polylc.com

I agree with all statements above. Here are some additional information:
- To reduce mixing issues always use 80% of TFA from Eluent A in Eluent B (eg. A: H2O with 0.1% TFA, B: ACN with 0.08% TFA)
- The quality of TFA is absolut essential; prepare fresh mobile phases.
- 25µl mixing volume @ 1ml/min flow is definitely not enough. Increase mixing performance/volume to about 100 or 200µl

Here you can find some more and detailed information:http://www.thermoscientific.com/content ... 044-EN.pdf
and
http://www.dionex.com/en-us/webdocs/110 ... 851-01.pdf

I guess your wavelength is around 215nm.
Good luck!

Thank you so much for all suggestions. I tried several things and here is my take-home messages:
1) It seems that pulsation is indeed related to mixing issues, although it would not be visible without difference in absorption coefficients of the two components of the mobile phase. Thus, I take all pieces of advice for preparation of fresh mobile phases, using high quality TFA, and other pieces of advice related to reducing the observable component of pulsation. The easiest and most powerful change was to give up on 215 and move to 254 nm!
2) The pulsation did not go away completely even with a 500 ul mixer but it got better (amplitude went down ~5x). This makes me think that a part of the problem was related to the lines connecting the pumps to the mixer or other components of the system (see below). I guess I need to use a larger diameter lines if the problem persists. Initially, I connected two mixers serially and it had no effect on pulsation. Removing the smaller mixer helped.
3) Now, after the system stabilized, I re-installed the 25 ul mixer back to check what it would do. Interestingly, I had less pulsation than I had initially. Also, there was no pulsation in the pump pressure (which was more scary than the UV signal). I guess this may be related to some intermittent problem with a check valve or a piston seal or to degasser. The whole pulsation issue started after I introduced an online degasser. Before I degassed mobile phases off line.

I will keep playing with this system and let you know what I learn. Unfortunately, I cannot do extensive diagnostics because I have other projects to attend to and I have to run samples on this machine, but I am planning to put a larger mixer permanently on this system. I am also building a dedicated system for HIC, which, as I learned from Andy Alpert, is even more demanding in terms of mixing. It is good that this issue came to my attention early. Thank you, guys!