Seek suggestion on LC-MS configuration

Basic questions from students; resources for projects and reports.

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Hi,

We have a Bruker QTOF coupled to a Dionex 3000 ultimate. The maximum pressure the LC pump can handle is 400 bars. The column we have right now is a C18 RP, 5 um, 4.6 x 150 mm.

The problem is, it takes a huge lot of time to run each sample. Since, I cannot allow more than 200 uL/min flow in to the MS.

I seek your suggestion on which column/extra equipment will be beneficial for proteomics and metabolomics applications. We process targeted and un-targeted profile studies.

Looking forward to your responses.
The good news is you can improve this enormously at relatively low cost, because you couldn't really have chosen a worse dimension of column for the job!

The column you are using, with a 4.6mm diameter, has a huge cross-sectional area for a 200uL/min flow. I would normally expect to use at least 1mL/min down a column of that size, and wouldn't expect a back-pressure anything like your 400bar maximum. If you can only pump 200uL/min without causing trouble to the mass spec, then you are running at 1/5th of the linear velocity of solvent that I've got, so viewed crudely, your method is likely to be 5 times longer than mine would have been.

The first, obvious, change is to go to a 2mm column, with 1/5th of the cross-sectional area, which means that 200uL/min is now a sensible flow-rate.

The next thing to do is to think about your back-pressure. At the moment, you probably have a tiny back-pressure (if you don't, then a lot of it is probably tubing rather than the column). Assuming you have a very low back-pressure, and most of it is caused by the column, you can estimate that your new pressure will be about 5 times the old (because you're now using a column with 1/5th of the cross-sectional area). If this value is still much less than 400bar, then you could consider using a smaller-particle column. 5 micron is quite old-school. 3 micron will improve the resolution of your method. Improved resolution means that you can either get better separation of compounds, or, if you don't need better separation, you can run gradients a bit steeper, sacrificing some of the improved resolution to get shorter analysis times.

But since you are buying a new column, you could also consider solid-core columns. These give resolutions similar to that of a smaller-particle column, but without increased back-pressure. Thus, if you decide you can get away with a 3micron particle instead of 5micron, if you get a solid-core 3micron particle, you will probably get the performance of a 1.7micron particle (normally the territory of UHPLC).

Of course this performance depends also on extra-column issues with your system. If it is plumbed in extremely wide tubing, with long lengths of tubing, and a bad, old-fashioned autosampler, then you won't get much benefit in going solid-core.

A final thought: if, after you've got the new column and checked your plumbing, you find yourself limited by the 200uL flow to the MS, but the pressure is still below 400bar, and you think your method would be shorter and the chromatography good with a higher flow-rate, then you can invest in a simple tee-piece. This is much cheaper than a flow-splitter, but will do the same job. Direct your flow post-column to the tee-piece, put some narrow tubing to waste on one side (narrow tube creates an appropriate resistance) and attach the other side to the mass spec, and adjust the length of the waste-tube until you get the desired split ratio. In this way you can run a method at 300 or 400uL/min, while still preserving 200uL/min to the mass spec, and with little or no change in signal because ESI depends mostly on concentration, not total amount of material.
Awesome lmh! Thanks a million for the inputs.

I've got 107 bars at 0.2 mL/min, 5% ACN This comes down to about 70 bars at higher ACN %. So, I guess it will be risky to go for the change you suggested if we are staring at 5 times more back pressure?

Should I carry out a column and tubing wash with overnight ACN flow or something?

One doubt! You mentioned that if we go for a 2 mm column, it will be 1/5th of the one we are using now. But, we are using a 4.6 mm now. Isn't that 1/3rd?

- Arnab
To deal with the simple bit first:
Cross-sectional area calculated as pi * r-squared
For 2mm diameter, radius = 1, cross sectional area = pi
(because 1 squared is 1)
For 4.6mm diameter, radius = 2.3mm, and 2.3 squared = 5.29, so the cross-sectional area is 5.29*pi. This is where my estimate of roughly 5-fold came from.

The difficult bit is the pressure. For a 4.6mm diameter, 150mm long, 5 micron column, I wouldn't expect anything like 100 bar pressure, but it's not a size of column that I use myself, so perhaps someone else here can advise. I worry that either you have some unnecessarily long lengths of unreasonably thin tubing on your system, or something has got a bit blocked. It'd be good to check what the pressure is if you substitute a simple union for the column (find out how much of that pressure was caused by the column, and how much is the system). I would expect to be able to run a 100*2mm 3 micron column at 200uL/min without a problem on a conventional, 400-bar maximum system.
Thank you lmh.

I removed my column from the system and checked the pressure. By the way, I have a guard column attached to the system just before my column. Here are the values. Buffer A is water, B is ACN.

Flow ACN Connections Pressure (bar)
1000 2% -Column 18.6
1000 50% -Column 14.7
1000 90% -Column 5.2

200 2% -Column 4.3
200 50% -Column 3
200 90% -Column 0.9

1000 2% -Column-Guard 1.8
1000 50% -Column-Guard 1.5
1000 90% -Column-Guard 0.8

So I tried washing the guard reversing it. But couldn't see any improvement in the back pressure.

But since I can see only maximum of 4.3 bar pressure contribution from the guard at 200 uL/min flow rate. So, I guess, either I have to change the guard cartridge or live with it.

I can check the back pressure once by using a demo 2 mm ID column.
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