HPLC columns: 2.1 mm vs. 4.6 mm

[parkc23]

Hi,
Does anyone have an experience on comparing the performance between 2.1 mm and 4.6 mm columns?
Thanks.

[Gerhard Kratz]

YES
If you reduce inner diameter you will increase the mass sensitivity.
What are you specifically asking?

[HPLCaddict]

With 2.1mm columns (of exactly the same stationary phase, of course) you usually get lower efficiencies than with 4.6mm columns. Because of the low column volume of 2.1mm columns, leading to very low peak volumes, extra column effects are much more pronounced. Even with the most advanced and optimized UHPLC systems you'll lose some efficiency due to extra-column band broadening when using 2.1mm columns.

[parkc23]

Thanks for the reply. I need to choose a column size for LC-MS among 2.1, 3.0, and 4.6 mm sizes. Given that a low flow rate is preferred for MS, I was inclined to get 2.1 mm column. On the other hand, 2.1 mm columns are more sensitive to other factors such as lower column efficiency and extra column effects. Therefore, I am not sure which one would be better at the end. Any suggestions?

[Hollow]

if you're able to go directly to the MS and therefore don't need a splitter, this may be a plus and balancing some other effects out. but you should also check and reduce the ID of the tubing from column to detector to the smallest possible.

Also check the gradient dwell volume of the system. Due to the lower flow rate (ca. 1/4 of that of 4.6) the gradient performance may be compromised. also injection volume should be adapted.

[M Farooq]

It is well known that 2.1 mm always show lower efficiency than 4.6 mm i.d. columns. A good compromise is 3 mm i.d. column when low flow rates are required.

[mattmullaney]

Hi parkc23,

I agree with almost all of what is said above...all of that said, if you're running gradients, the efficiency of the stationary phase doesn't matter as much (though Hollow is quite correct also, which does countermand what I've just said), if you're running isocratic methods, efficiency does suffer much more, particularly for later eluting peaks.

What LC system are you using...do you know or can you obtain the gradient dwell and system volumes? This link can help provide some insight into the so-called extra-column volume...may give some insight also into setting your data rate for acquiring your spectra: (that isn't too terribly different at all from UV detection, you want to have a certain minimum number of "data points" across your peaks)

http://www.mac-mod.com/pdf/technical-re ... uceECV.pdf

In the very little bit of LC-MS I've done with an Agilent 1260 plumbed with the 0.17 mm capillaries--and I'll be honest I am just starting out with this (I'm using a standard DAD for the 1260 and a 6130 MSD in series), I've had pleasing results with 3.0 mm ID columns and 3.5 um stationary phases...so I rather agree as well with M. Farooq.

[lmh]

I'm in favour of keeping columns small. Yes, the efficiency of a narrow-bore column of the same length will be slightly lower than the wider bore column, but depending on your system, this may be irrelevant, and there are things you can do:

In real life, if you're coupling to an MS, if you use a 4.6mm column less than 400mm long(!) you will probably find that its optimum flow-rate vastly exceeds the optimum flow-rate of the MS, and if you start doing flow-splitting, you will probably find you've reduced the efficiency of your system. You are right that if you use a 2mm column, the naturally optimum flow-rate of the column is far more likely to be directly compatible with electrospray MS.

If you have lost too much efficiency, you can always use a slightly longer column to recover it. In theory, this would make your method slower, but in practice nowadays it's rarely the case. Most 4.6mm methods are running at rather less than the ideal flow rate of the column, so with a 2mm column it is likely you will run the method at a faster linear flow anyway - potentially speeding things up. Particularly, if you're doing a gradient method, you have to consider re-equilibration, and a combination of a good, modern, UPLC pump and a pressure-tolerant column is a sure-fire winner here: you can re-equilibrate much faster than you'd consider with a big old column. Of course, if you're stuck with an old system with a huge extra-column volume, you may be obliged to stick to matching, 4.6mm columns.

Columns are tools (different ones are right for different applications): the proteomics crowd clearly feel that sensitivity is more important than anything else, and despite wanting high resolution for complex peptide mixtures, they still regularly choose nano-flow. I like 2mm because I don't like throwing away vast bottles of very expensive and environmentally unfriendly solvents. My feeling is that if the loss of resolution between 4.6 and 2mm is the deciding factor for a method, then the method is sitting on a knife-edge anyway, and just waiting to go wrong...