Chromatography Forum

Hello,

I am testing this column on a classical and old Hplc system, but optimized for high resolution . Unfortunately no QC reference came with the column, only via some software wich is installed on Waters equipment.
Could anybody share the maximum efficiency he could get on this column on a traditional Hplc system , or maybe transmit me the QC-report ?
Propably some appropriate connections are needed, because I could only obtain 6000-7000 theoretical plates, while I am used to work with 1.5 um wich gives me 12000-14000 TP. I tried this out on different flow rates 0.2-0.4-0.6 ml/min, wich gave no significant difference.
Any help is more than wellcome.

The Acquity UPLC column comes with an electronic tag that contains all QC information.

When running this column on a classical HPLC system, you will not get the performance that the column is capable of. The first reason is that you will not be able to reach the pressure needed to get at the plate count optimum. The second reason is that most likely and even with your best efforts, a rebuilt classical HPLC system is not getting anywhere close to the system bandspreading performance of a UPLC system.

Why do you think that Waters went through a few years of intensive R&D effort to develop UPLC systems, if somebody would have thought that you can get to the same spot on a classical HPLC system?

So it is the normal efficiency of the column itself, I assume?
I was just trying to understand why a 1.7 um gives nearly half of the efficiency of 1.5 um on the same system, because this is not possible. So do I understand well that in UPLC all performance is added by the UPLC system itself with the pressure, and not by the column ?
I was only looking if I could improve current methodology with other sub-2um technology, but it appears I better continue with the current methods and the 1.5 um column in the lab, without any investment.

Well, it is obvious that you are not telling me the complete story. I can not see how a 1.5 micron column gets you such a higher performance on a classical HPLC system compared to a 1.7 micron column unless you are comparing apples to oranges. Under normal circumstances, the true column performance for such small particles on an HPLC system is dominated by the diffusion term of the van Deemter equation. If you get a difference between different columns run at such slow velocities, the first suspicion is a significant difference in the retention factor. Considering the system that you are running, end-fitting issues and column diameter can be a factor. What is the i.d. of the 1.5 micron column, and what is the id. of the 1.7 micron column? What is the pressure at which you are using the columns? Is the UPLC column one that has been used extensively on a UPLC system and has been discarded (and is potentially half dead)?

The column performance is independent of the instrument, and it is not a fixed number, but varies with flow rate. This is why you need an instrument with the pressure capability to reach the minimum of the van Deemter curve for the particle size. To get all elements of the column performance, you also need an instrument with a very low bandspreading and fast detectors. I have not seen any refurbished HPLC system that is getting anywhere close to the bandspreading performance of a UPLC system. But admittedly, it is not impossible for a real good tinkerer to get there.

Sorry for all these questions, but you are making conclusions that are rather strange.

There is not much story to tell actually, tests were done in daily circumstances .
Ok, the only difference between the two columns is the ID. But as we ran 3 different flow rates without significant differences, ( we cannot obtain the optimal flow rate from the e-card), we didn't want to spend time on plotting, not for the 1.7 um colom, not for the 1.5 um. We used the standard flow rates.
It is obvious that peaks eluting early in an isocratic separation are even more susceptible to this broadening,because the volume of early eluting peaks [low k value] is considerably less than for later eluting peaks [larger k value].This observed peak broadening can result in a very significant loss in resolution. In fact,if the extra-column volume of a system is excessive, the performance of any analytical column can be compromised. But our HPLC system has a minimal extra-column volume gives nearly similar plates for both the 4.6 x 50 mm, and 2.1x 50 mm columns.
I can provide some hints on the optimized system for high resolution, it can help others as well without considerabel investment to improve the performances on traditional systems. Crucial point is to minimize the death volume of the system.
Sub 2um tests we usually run on a Shimadzu Hplc 10AT-series, equipped with a low dispersion manual Rheodyne injector 8125 , installed in the columnoven. Tubing from the injector to column 5 cm, ID 0.005 ", from the column to 5uml UV cell same tubing 15 cm. Soon we will have 0.001" tubings, we believe there is still a range for improvement.
Operational temperature 40 C.
Flow rate 0.2 ml/min for the acquity column ID 0.21 x 50 mm(no significant difference with 0.4 or 0.6 ml flowrate)
Flow rate 1 ml / min for the 1.5 um column ID 4.6 x 50 mm.
Software Class-Vp version 7, with the sensitivity: auxiliary range 1, range 1, response 1, aqcuisition frequency 20 ms.
Pressure for both columns is around 4000 PSI.
Acquity Column is a brandnew, wellwashed one.
And indeed, 1.5 um give some more retention, but longer runs with more hydrofobic samples on the Acquity give max. 7000 Plates/5cm.

It wasn't mentioned to bring strange conclusions, only to ask if 7000 TP is the maximum we can achieve on this column in Isocratic mode on conventional system.

You should get between 14000 and 15000 plates at a pressure between 4000 and 5000 psi for the 1.7 micron column. The fact that you are not getting there tells me that you would still have to work out a lot of extra-column bandspreading issues. The same is true for your observation that the plate count remains low when you increase the flow rate, which should improve the plate count to the target number above.
To get to the performance that you want, you will be better off with a 7.5 cm 4.6 mm 2.5 micron XBridge column or with a 10 cm 4.6 mm 3.5 micron XBridge column. You get the same performance 2 to 4 times slower but within your backpressure and bandspreading range.

This all seams very clear, but doesn't explain the different results of the two columns,same conditions and same peak.
Because the same rules are applicable and even more applicable for the 1.5 um.
Thanks very much for the reaction anyway.

I don't know what is unclear. Your home-made system has an intolerable extra-column bandspreading and not enough pressure capability and is therefore not suitable for running a 2.1 mm UPLC column. I said this very clearly...

same conditions and same peak.

That's the point, you are not really running the "same" conditions!

To expand a bit on what Uwe was saying: you are comparing a 4.6mm id column to a 2.1 mm id column. That represents a factor of 5 difference in the cross-sectional area (cross section area is, of course, a function of the diameter squared. That means that your linear velocity is five times higher on the 2.1 mm id column if you run at the same flow rate. And it means that you really need a system with five times less extra-column volume.