2.5um particle size in HPLC & UPLC

[upamaniah]

Hi Chromatographers,
waters has introduced some chemistries with 2.5um particles, has any one used this, any feed back.
or any information about other brands with this particle size.

[HPLCaddict]

Actually, Waters has been around with 2.5µm particles for some years (at least I'm sure of XBridge and Sunfire columns with that particle size). The "new" addition you're referring to are the "XP" columns, I suppose? These are now in the Acquity (UHPLC) column hardware, whereas the "old" 2.5µm used normal column hardware. These XP columns are officially good for pressures up to 600bars.
AFAIK XBridge and XSelect columns are available as 2.5µm XP columns. We're using a XBridge 2.5µm XP and are quite fond of it. Gives you pretty high efficiencies at considerably lower backpressure than sub-2-µm columns. Be sure that you need an optimized system (aka UHPLC) to fully exploit these columns.
If you need maximum peak efficiencies - go for sub-2-µm. If you can live with a little less, these 2.5µm columns are a good compromise between efficiency and backpressure.
BTW I've seen little difference in efficiency between the "old" XBridge 2.5µm and the new XBidge XP

[lmh]

I'm becoming increasingly convinced by solid core particles. I've been trying out Kinetix 2.6u, which is solid core and therefore comparable to smaller particles while keeping the reasonable back-pressure. Phenomenex aren't lying: it really is as good as the 1.7u particles from the point of view of resolution, and yet I can run my 50mm*2mm easily at 400 uL/min on a conventional LC system.

[HPLCaddict]

I'm becoming increasingly convinced by solid core particles. I've been trying out Kinetix 2.6u, which is solid core and therefore comparable to smaller particles while keeping the reasonable back-pressure. Phenomenex aren't lying: it really is as good as the 1.7u particles from the point of view of resolution, and yet I can run my 50mm*2mm easily at 400 uL/min on a conventional LC system.

You're right, I'm also quite happy with the Kinetex. Astonishingly high efficiencies at a reasonably low backpressure. Nevertheless I'm not so happy with Phenomenex' marketing claim "UHPLC perfomance on any HPLC system". According to my experience, you won't get those high efficiencies by just putting a Kinetex in a conventional HPLC system. You MUST optimize the system (smaller volume flow-cell, optimized capillary connections) in order to exploit these columns. Even then, it's better to stick with 3.0 or even 4.6mm ID columns, as the extra-column band-spreading with 2mm columns will be to high on these systems.
I'm using the 2.6µm Kinetex only on UHPLC-systems, so I don't have to worry too much about system volume. Here, I usually don't see significant difference between 2.6µm core-shell or sub-2µm fully porous.

[danko]

Do you downscale the injection volume or/and the loaded amount when shifting from 3 to 2 mm diameter column?

Best Regards

[juddc]

+1 on the Kinetix columns and HPLCaddict's comments regarding column diameter. I've had excellent luck with these on a reasonably well optimized 2695 with unmodified detector (std flow cell).

Peak widths of 5-6 seconds are achievable without running rapid gradients.

They're pretty rugged, too.

To danko's question: For me, it depends upon what the solvent is and what I want the assay to do. If it's mobile phase or very similar to it and I need to optimize for sensitivity, then I would try to avoid downscaling. For a situation where I was simply trying to optimize for time or solvent consumption and the downscaled injection volume is easily repeatable for the instrument in question, then I would do so.

[danko]

I wasn’t thinking of solvent effects.
The thing is, if you load a certain sample amount on 3 mm column - for instance – and if it’s the optimum load for that column, then switching to a column with narrower diameter – 2 mm for instance – will necessitate sample amount reduction in order to avoid sample overload. Sample overload can often be confused (looking at the chromatogram) with extra column effects.
Injection volume reduction may, or may not be necessary. Depends on several factors and is needed mostly when sample solvent is a stronger solvent than the mobile phase, while the opposite (weaker solvent) is often beneficial.

Best Regards

[HPLCaddict]

I wasn’t thinking of solvent effects.
The thing is, if you load a certain sample amount on 3 mm column - for instance – and if it’s the optimum load for that column, then switching to a column with narrower diameter – 2 mm for instance – will necessitate sample amount reduction in order to avoid sample overload. Sample overload can often be confused (looking at the chromatogram) with extra column effects.
Injection volume reduction may, or may not be necessary. Depends on several factors and is needed mostly when sample solvent is a stronger solvent than the mobile phase, while the opposite (weaker solvent) is often beneficial.

Full ACK. I usually scale the injection volume down according to the changed column diameter for a first try. Depending on the result I'd further optimize injection volume, solvent and sample concentration.

[juddc]

In my experience, column overloading in an analytical scenario is rarely a problem compared to solvent effects. I'd be more concerned about going beyond the linear range of the detector than overloading the column in most situations.

As I said, it depends upon the nature of the assay. If you're doing trace analysis, mass overloading should not be an issue. If you're assaying a drug for purity, then perhaps you should scale the injection volume.

[HPLCaddict]

In my experience, column overloading in an analytical scenario is rarely a problem compared to solvent effects. I'd be more concerned about going beyond the linear range of the detector than overloading the column in most situations.

When talking about 250x4,6mm monsters, ACK . With smaller columns, like 50x3 or especially 50x2.1, column overloading can become a serious problem according to my experience. And unfortunately, the core-shell-columns like Kinetex seem to be especially sensitive, probably because of the lower surface area compared to fully porous columns...

[lmh]

I'm with judd: it really comes down to what sort of chromatographer you are. If you generally work on concentrated samples and are verging on semi-prep, then you'd need to scale down your injections, but if you're like me, and very often scraping to see the peak at all, in a minute amount of sample, narrow-bore columns make a lot of sense because I can improve my sensitivity by injecting more provided it's in a solvent as weak as, or weaker than, the running solvent. By definition, if you've got a sensitivity problem, you (almost certainly) don't have an overloading problem.

I'm rather happy that Kinetix is now available in big particle sizes for prep applications, because it's embarrassing to admit to someone that I've got them a good separation at analytical scale, but if they wish to go semi-prep (for example to get enough material for NMR of an unknown analyte), they need to change column material.

I'd agree that "UHPLC perfomance on any HPLC system" is a silly exaggeration (silly because it's unnecessary). If you put a narrow-bore Kinetix column on a 20-year-old system with tubing so wide you can use it to hold propelling pencil lead, and a 1mL volume flow-cell, it will obviously be disastrous. Speaking to a Phenomenex rep recently about their new Kinetix that combines solid core with stupidly small particles, she reckoned the improvement the solid core was giving at that level compared to fully porous stupidly small particles was quite small compared to the (huge) improvement you get when comparing solid core and fully porous 3um or 5um particles. She attributed this to the fact that very few HPLC systems are really suitable for that sort of performance; extra-column effects dominate. I'm sure Phenomenex are well aware that even their larger particle sizes do make demands on the HPLC system too. A column is just one bit of the system, and the system is as weak as its weakest part.