Chromatography Forum

I work with Waters instruments excusively. In particular, all current and past QC analysis was run using Waters Alliance HPLCs. I am advocating going to UPLC, however, there are two types of UPLC instruments. The Acquity UPLC and the Acquity H class UPLC. I am more inclined to go with the UPLC over the H class instrument due to the improved fluid path. This is potentially risky since the UPLC only instruments are more prone to problems that arise from less than ideal operator methodology. Does anyone have experience making the switch, and how much trouble occurs from teaching old operators new tricks? It seems that the transition might be less from HPLC to H class than from HPLC to UPLC. We would continue to have access to our current HPLC instruments during the method validation process.

The type of operator who listens to missives about cleaning out columns without precipitating salt in them and takes the time to make sure that the lamps get shut off at the end of a run tend not to have problems with either type of UPLC. Careless operators will have more land mines to stomp on.

IF you are high volume or have a large number of users, you will experience issues with the UPLC classic. They are very picky and need to be maticuously cleaned, and shut down properly after each run. They will not take any where near the abuse of and HPLC. No experience with the H-Class... yet.

We have a lot of Alliance separation modules, and also a lot of UPLCs (BSMs).

A while ago we tested the H-class, and it looked very promising.
The only bad side is the fact that we already had regular UPLCs, and this would mean a lot of revalidation work.
If we could turn back times, we would go for the H-class instead of the UPLCs.

The injection system of the H-Class is almost similar (FT needle) to the Alliance.
With the UPLCs, we sometimes run into recovery issues, just due to the injector design.

HTH

Ace

There are half-way-houses that are worth considering. Despite the best efforts of some Waters sales staff to prove otherwise, the solid core particle columns that are widely available now will allow you to benefit from the ability of a UPLC system to work efficiently with very narrow peaks, but will spare you the loss of robustness that comes with very tiny particles, and the extra wear on a system of having to use very high pressures. Honestly, they do work.

If you have to buy exclusively from Waters because of deals your company has struck, or because of software/training issues, then fine, but it's worth looking round if you can. There are a lot of very good high-pressure systems out there now, many with very good efficiency, very low carry-over, very flexible hardware, and easy use and maintenance. We, the end users, can benefit a lot from competition between a number of extremely capable companies.

It has been my personal experience that solid core technologies work very well as well. Where solid core seems to fall short is when the separation of a critical pair is involved. In all instances that I have encountered this, the solid core column produced unacceptable resolution where the UPLC column actually produced increased resolution.

As far as an H-Class goes.... I never got a HPLC method to successfully run on an H-Class. I think the fluid path needs to be re-plumbed with larger diameter tubing. Column pressures will be significantly higher than what you experience on a HPLC. I also had baseline quality issues, which I assumed because solvent flow was moving fast through narrow tubing and then it expands into this massive column.

As for operator teething problems; you are going to have them. I can't speak for a classic Acquity system because I have never used one. However, an H-Class can not simply be started or shutdown either. A H-Class system must be cared for as well.

Because one can not simply start it and forget it, the analysts at the company I worked for where I switched from HPLC to UPLC did not like the UPLC instruments. The UPLCs were rarely used by the QC group. I used mine all the time in development ... then I took theirs.

I thought the users would eventually catch on and would be able to handle UPLCs. They did not and they could not. Retrospectively, I should have purchased newer HPLCs and left the UPLCs to R&D only.

As a foreword, I don't have any pratical experience with Waters UPLCs, neither the "classic" UPLC nor the newer H-class or I-class. My experience with UHPLC is based on hardware from other companies. Nevertheless, I'd like to share some thoughts.
- Concerning the transition HPLC -> UHPLC, I've also made the experience that routine QC staff often seems to shy away from UHPLC. It's new, it's unknown, it's expensive, the pressure is so high, the column is so tiny, whatever. They rather use their wellknown 250x4.6 monster with a sequence that runs three days than having the same work done with a UHPLC method in three hours. R&D staff, on the other side, tends to embrace UHPLCs and the possibilities they offer. I know, generalizations like this are always problematic, but I think there's some truth in this one .
- If you want to use classic HPLC methods on UHPLC, no matter which manufacturer, be warned that you might run into problems. Lower dwell volumes lead to different gradient shapes, lower system volumes may lead to peak shape problems when using "weird" solvent/mobile phase combinations, detectors can be less sensitive because of lower volumes, ... UHPLCs are not worse than HPLCs for running those methods, they are just different. What it boils down to is that you will see how robust your HPLC method really is .
- Generally, what I really dislike about the whole UHPLC hype is that everyone behaves as if the wheel has been reinvented. It's still HPLC, even if you put an "U" at the beginning . The same old-school rules about chromatographic hygiene apply and some common sense is still needed. It's nothing new that buffers might precipitate if you're not flushing your columns and your system before shutting down. If you properly learned what you're doing and follow the rules, I don't think that you will have much more problems with UHPLCs than with HPLCs. We don't use "UHPLC-grade special filtered Acetonitrile" or similar stuff, we usually don't even filter our buffers, and we don't have any problems with blocked capillaries. An inline filter still is your best friend .
- Concerning solid-core vs. sub-2-micron, one should be very careful not to mix up efficiency and selectivity. I think it's rather common sense now that 2.6-2.7µm solid-core particles can deliver about the same efficiency as sub-2-micron fully porous particles. This does NOT mean that any solid-core column will give you the same separation as a sub-2-micron column. I'd say that most examples in column manufacturer brochures that are meant to prove that fully pourous is better than solid-core or vice-versa (depending on what the manufacturer is selling ) actually don't show better general performance of the respective column in terms of efficiency but just a slightly different selectivity. Which means that column A is not better than column B but column A is only better suited for THIS particular analysis.

It is strange that well-trained chromatographers think that there is a cliff between HPLC and UPLC, not to mention the difference between "LC", "HPLC" and "UPLC": It is a gradient: you may use tubing from 1 mm (or larger) in preparative LC, down to around 10 µm in nano-LC. The dwell volume of “ordinary” “HPLC” may differ by a factor four, depending on model. After all trimming I have done on LC-equipment during 30 years to improve resolution and speed, the conclusion is: if you want a robust LC system do not use inner diameter below 100 µm. The original Acquity systems in use where I work presently (not H-class) do require a lot more service and support than other LC equipment I have worked with, mostly all kinds of Agilent 1100, Waters Alliance and Dionex Ultimate. So for a non-critical analysis where you don’t need the best resolution in shortest time, it is better to use the other LC systems with capillaries down to 100 µm. But when you need a rapid analysis because the sample is not stable and if the Acquity system is down, your sample is wasted. That is a high cost. And if you do GMP analysis and have to write reports why the samples are re-run, it is a pain, not an efficiency gain. I had no possibility to work on the H-class chromatographs at my previous employee but I have heard from there that the H-class chromatographs now in use are a lot more robust than the I-class ones.