flow splitter für HPLC-PDA/MS

Discussions about GC-MS, LC-MS, LC-FTIR, and other "coupled" analytical techniques.

8 posts Page 1 of 1
Dear all,

we are looking for a flow splitter that would allow us to combine our Ultimate 3000 HPLC with PDA with a Q Exactive MSD. At the moment, we cannot use both detectors at the same time, because if we want to see peaks in the PDA we would load too much on the MSD....

We use core-shell columns and a typical flow rate of 0.6 ml/min. I have tried some simple splitters, but the problem is that I have observed severe peak broadening in the MSD - if we split e.g. 1:100, we have a flow rate of a few µl/min going to the MSD. This should be OK for our HESI-II source, but I think the distance/volume from the splitter to the tip of the ESI needle is too large and allows for too much diffusion...

So... does anyone have experiences with flow spiltters to combine DAD and MSD, and can give some recommendations (manufacturer, type of splitter, split rate, etc.)? In the future, we even might want to add an additional ELSD to the set-up, so a 3-way split might be great. But this is not mandatory right now...

Thanks says The Alchemist
If you are afraid of diffusion due to the low flow rate, mount the splitter as close to the MS source as possible (maybe use a column coupler to connect directly to the MS) and accept the longer path to the UV detector. Difference in retention time between MS and UV should be negligible.
A simple split won't work anyway. ESI gives a signal related to concentration, not to total amount of stuff reaching the source (that's why nano-flow is so good for proteomic work), so if you merely split 100:1 you'll still have the same issue that the signal at the MS will be enormous if you couple it with a much less sensitive detector.
If you want to make this work, you need to split the flow 100:1 and then add a make-up flow on the MS side of the split, to dilute the tiny flow of concentrated peak and make it a rapid flow of diluted peak. This overcomes the problem of diffusion/peak dispersion in the tubing, and also balances the signals.
This is the typical approach used in mass-directed fractionation, which has a similar problem (desire to inject a tremendously large amount of stuff, preferably to recover most of it in the fraction, but not overload the MS detector).
But this is a world of pain. You need an extra pump. You now have two t-pieces, one splitting off the PDA, and the other adding in the make-up flow. If you get the balance of resistances wrong, the pressure at the second tee will slightly exceed that at the first, and the make-up flow will go the wrong way, not only supplying the entire flow to the MS (which will detect nothing) but putting a little excess out the other way to the PDA. Your only saving grace is that you can at least measure the flow to the PDA, but working out whether it is 594uL/min (good) or 610uL/min (disastrous) is not going to be easy. People who make this work, tend to set it up once, and never touch the plumbing! you can buy, off the shelf, splitters that do the job, with an input for the make-up pump, but remember that they are, in the end, just resistances, and everything you add to the outputs of the splitter (tubing to detectors, the detectors themselves) will affect the split, so the world of pain doesn't disappear. Also, the moment anything gets clogged up, you have a horrible diagnosis problem to work out why it's not working any more.
100:1 is quite an extreme split. If you can get away with a less drastic split-ratio, then the world will be decidedly less painful. It may still need a make-up flow, but it will be easier to get it right. Can you sacrifice quality in one of the two detectors in order to get enough quality in the other? For example, if you're doing MRM's or single ion monitoring, can you work with an isotope peak rather than the main peak in order to reduce MS sensitivity? If you must use PDA with a very good MS, it helps to get the very best PDA you can.
Thank you, lmh, for the answer. In fact, I had not thought about detector overload before: I had more been focused about avoiding contaminating my poor MSD with too much substance...

Thanks also for making me aware of the problems that might arise - this sounds as if you have been through a horrible time... ;-)

Following your adviceI had a look into make-up flow splitters. The extra pump is not an issue, we already have one sitting around. However, I think it would be a pain to asseble one myself with t-pieces and tubing... :-/ So I had a look around for commercial sources and found the QuickSplit from ASI. Do you know any other commercial suppliers? They seem to be the only ones I can find...

Of course, the 1:100 was just an estimation - no idea if 1:25 or whatever will also work. I simply have too few experiences with the sensitivity differences between PDA and MSD...
Hi,

I fully agree with lmh, and also had a hard time in the past with flow splitters at the preparative scale. For an analytical job, my bet is that you might save time by injecting a diluted sample in the MS followed by a concentrated sample in the UV, rather than trying to optimize a split system. High resolution MS instruments have become so sensitive in recent years that the working concentration ranges between UV and MS are probably too different to properly combine both detections.
Might be worth asking Thermo if you're on good terms with them! How bad your contamination problem will be might depend on the exact samples you're using - if your analyte absorbs really strongly and ionizes not so well, it might be OK, particularly if you can tolerate the instrument getting a bit dirty (if you're also using it for work at the very highest levels of sensitivity then this may not be an option). Also if the main contamination concerns of your sample are hydrophilic sugars, salts and buffers that elute very early, you can help your MS by diverting to waste at the start of reverse phase runs.
I'm not sure who are the best manufacturers of pre-made splitters with make-up flow input; we have one, from a company imaginatively named "Analytical Scientific Instruments" (in the USA), but it was supplied to us as part of an LC-MS solution by one of the major MS manufacturers, so I never did a market comparison on who makes what.
I know you mentioned splitting the effluent to go to both the UV and the MS, but have you thought of running the flow through the UV, then split after the UV at the MS? This would allow for a higher flow rate through the UV for better peak shapes there, then reduce the flow as close to the MS as possible to prevent peak broadening.
The past is there to guide us into the future, not to dwell in.
James_Ball wrote:
I know you mentioned splitting the effluent to go to both the UV and the MS, but have you thought of running the flow through the UV, then split after the UV at the MS? This would allow for a higher flow rate through the UV for better peak shapes there, then reduce the flow as close to the MS as possible to prevent peak broadening.


That is precisely how we split ours. We still have an older PDA (Agilent 1100) that works better with higher flow rates, and we split post PDA. If we're running in tandem (we can, but normally just run one or the other) we tend to de-tune the MS so as to not swamp the detector with the concentrations we have to use for the PDA, rather than re-diluting the stream as lmh suggests. We switch to less sensitive ions, turn down the EM (PM in our case) and run well off of optimum ionization settings for the ESI
Mark Krause
Laboratory Director
Krause Analytical
Austin, TX USA
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