C18 150x4.6mm 3um VS C18 250x4.6 5um

[ExLet]

Dear chromatographers

I am trying to select a new column for our company. I am happy with C18 columns and would normally use 150x4.6mm 5um column. For some applications BP advises using C18 250x4.6mm 5um columns which do give better resolution but increase analysis time and use much more mobile phase. I am thinking of C18 150x4.6mm 3um column instead of 250 5um, but I have never worked with 3um particle size columns. In theory they provide similar resolution compared to 250 5um columns and similar retention times compared to 150 5um columns, but increase pressure approx. threefold compared to 150 5um. Basically I need an advice/opinion from someone who has ever worked with 3um columns. If theory is anywhere near real life separations. Any problems/bad experiences? Any advantages/disadvantages?

Any advice will do, just share your experience

Thanks in Advance
Alek

[lmh]

3u should save some solvent and give better resolution for its length, so you're certainly thinking along sensible lines. However, there are some other things you could try too:

(1) If you're worried about solvent, have you considered reducing column diameter? A 2mm column has 1/5th of the cross-sectional area of a 4.6mm column, so you can reduce the flow-rate from, say, 1mL/min to 200uL/min, thereby saving huge amounts of solvent, but with minimal change to your method. The only considerations are that the dead volume of your system will delay things slightly more (very old HPLC systems may struggle, but anything vaguely modern will be fine), and that you may need to consider reducing injection volume if you're injecting in a solvent that is stronger than the running solvent, or if the sample is nearly overloading. In general, a narrow-bore column may allow you to increase sensitivity as many detectors (e.g. PDA/UV) measure concentration, not amount.

(2) Have you considered fused-core particles? My (limited) experience is that whatever your Waters rep tells you to the contrary, and however much theory he has to back it up, they actually do work. They give many of the advantages of very small particles, but with the back-pressure associated with larger particles. I don't want to say bad things about UPLC and tiny particles: they work too, but of course you need a UPLC to get the benefit.

There are quite a few ways to use solid-core. If you've got old HPLCs that can't handle high pressure, they're ideal. If you like 5u pressure but need a bit more resolution a 5u solid-core will give resolution similar to a 3u particle size on almost any LC system, at low pressure. You can also use, for example, 3u solid core to have a go at UPLC-like separation on a conventional pressure system, but UPLC-narrow peaks do depend on a short and dead-volume-free fluid path too, so you won't get really good chromatography from an old, large system just by putting a modern column on it.

[ExLet]

very old HPLC systems may struggle, but anything vaguely modern will be fine

I am working with Agilent 1050, so it might struggle

Have you considered fused-core particles?

You can also use, for example, 3u solid core to have a go at UPLC-like separation on a conventional pressure system, but UPLC-narrow peaks do depend on a short and dead-volume-free fluid path too, so you won't get really good chromatography from an old, large system just by putting a modern column on it.

Yes and yes, but not with the system I am using, as well as I dont want making it too complicated for now

Thanks for your reply Imh, really helpful

[HPLCaddict]

If you don't want to make it too complicated for now, a change from 250x4.6 5µm to 150x4.6 3µm is usually possible without problems.

However, consider that
- backpressure will be higher, but due to the shorter column it's only about 1.7-fold, not 3-fold
- the smaller the particle size, the more risk for clogging your columns. If you're dilute-and-shooting your samples, you'd be better off with staying at 5µm columns
- isocratic method transfer is usually straightforward: Change the column, leave everything else as it is. For gradients, a bit more work is involved as you will have to adjust the time points
- selectivity of your 150x4.6 3µm column will of course only be the same as the original 250mm column if you're using EXACTLY the same stationary phase (and even then there might be slight differences). Exchanging your 250mm C18 column to a different 150mm C18 column might or might not work...
- and finally, most of the methods run on 250x4.6 monsters, especially pharmacopoeial methods, have plenty of room for improvements without changing the column. Want to save time? Ramp up the flow-rate 50% (that's IMHO the adjustment allowed by USP/EP) et voila

[Consumer Products Guy]

Agilent 1050 may be old in "years" but its pumping system is mostly identical to the 1100 series.

Like above, I was going to suggest 3mm i.d. and 3µ particle 150mm long as a much more modern size column. Or shorter, if your separation allows for it and you don't need the additional resolution.

[ExLet]

Thank you for your suggestions guys. I've decided to go for 150x4.6mm 3um C18 for now. Ordered already, should get it in a couple of days