UPLC - can tubing length pre-mixer affect retention time?

[EmpowersBane]

I run a gradient method UPLC using a 1.7micron 100mm C18 column pH 3.00 for impurities and because of ghost peaks, I add a small column between B1 line and mixer inlet. This has always worked well so far until the tubing was replaced recently. I guessed about 6cm length of tubing from column outlet to mixer inlet and attached it. Now my critical pairing of peaks are on top of each other co-eluting badly (was previously resolution of at least 2.5)and im not sure is this something to do with the tubing being extra long, maybe it should be less kinked (its sticking out a bit because the space wasn't designed for an extra column) or....the machine hasn't been used for 3 weeks so maybe a longer prime and then several condition columns before starting a run.
Any ideas on what i can do to restore the best resolution?

[Multidimensional]

When using tiny columns with tiny internal column volumes, even small changes in tubing or anything in the flow path can change the chromatography. This takes time to learn and something that is so very important when you use an HPLC system with some of the low volume columns.

Here is a link which will help you understand the importance of HPLC tubing ID when changing connections. The ID has a major effect on the overall volume, which of course effects both delay volume and diffusion (which can lead to loss of resolution). http://www.hplctools.com/capvolume.htm

Here is another one with Column Volume info too: http://www.hplctools.com/columnvolume.htm

These are fundamental to learning about liquid chromatography and developing good methods.

[Multidimensional]

< because of ghost peaks I add a small column between B1 line and mixer inlet >

Please do not do that. Toss that extra column in the drawer and find the cause of the ghost peaks. Adding delay to the method with a saturation column may just cover up real problems. Good methods do not need such cheats.

[EmpowersBane]

Thanks for the information, im stuck with the extra column as its in the method but I also wonder would kinking in the tubing cause peaks to be co-eluting as well? Because the method was fine beforehand but the tubing had become worn so was replaced and now I cant get resolution, is it possible that a bend somewhere between mixer and column inlet could cause a void and contribute to this issue? I know pH could it too so im not ruling out that either..

[Multidimensional]

A "kink" in the HPLC tubing (not "UPLC") can only result in: a leak (from cracking) OR an obstruction (partial or full blockage). Most SS tubing is far stronger and resistant to internal kinking than people realize (esp the high quality thin SS tubing sold by Agilent from Germany). Plastic Tubing (i.e. PEEK) is easily damaged by kink'ing and should be disposed of (replace the line). When in doubt, toss it.

Poor quality connections (far more common) may result in voids between connections causing changes to chromatography.

HPLC Tubing and Fittings; An introduction to Nuts, Ferrules and Tubing Choices
https://hplctips.blogspot.com/2018/07/hplc-tubing-and-fittings-introduction.html

[Rndirk]

Can you elaborate: in the first post you describe a change in the system between the "ghost" column and the mixer, and in the second between the mixer and the analytical column? The second situation, in my opinion, is more prone to cause problems (since here your compounds are in the flow path). A pH change, definitely, but this is a parameter that did not change between now and when the method was still running fine (?)

Does the B line provides the aqueous or the organic phase?

A chromatogram before/after would be helpful here.

[lmh]

If you are using a high-pressure binary system with a column fitted between the pump and the mixer, to remove a contaminant from the solvent, then once the clean-up column has become fully equilibrated with solvent, it will not affect the retention times.

Although I have personally never found it necessary, I know of many reputable people who have used a clean-up column in this way, once they've traced the cause of a ghost peak to a contaminant in their solvent.

Extra volume before the mixer should not affect the gradient. That is the thinking behind binary systems compared to quaternary.

If the tubing becomes so kinked that it develops a leak, then yes, there will be drastic differences in the elution, because you'll then have a lower flow, and the wrong percentage, and possibly even weird dead-volume issues from the mixer itself if, at any point during the gradient, the pump on the leaky side is running at such a low flow that there is a net flow from mixer to leak.

I am also assuming the mixer is the right one for the job, and that the system now is in its 'bad' state... the most difficult situation is if the system now is in its 'good' state, and unfortunately your method isn't working, while the method used to work, for some obscure reason connected with a fault (such as a small leak on the clean-up column that no one had noticed) that you've now corrected.

I like your suggestions of a bit more priming and a few conditioning runs - certainly can't do any harm, and might sort out a lot of the more trivial causes of unreproducible retention times. Check the solvent really is what you think it is, and any buffers have been made correctly. What does the system pressure look like now, compared to when the method worked? From resolution of 2.5 to coelution is quite a drastic loss of quality - something moderately drastic must have happened to cause it!

[EmpowersBane]

Thanks Imh, funnily enough when I primed out the lines and system for a lot longer today (20 mins per line) then tightened up the fittings along the flow path and also ran 5 condition columns, the resolution went up to almost 2 which is what I had been getting before so I presume the lack of equilibration caused this issue. Or perhaps a bit too much organic in my sample diluent pushing peaks closer together.
Kinks in the tube when you are trying to accommodate that extra column are inevitable but im glad to hear they don't normally cause RT issues.