Waters Acquity

[gregg]

After a couple of months trying to seperate a couple impurties from the main peak (small peptide) on a standard HPLC (Alliance) w/o sucess, we borrowed a Acquity (UPLC) from a different group and achieved baseline resolution. Since this method will be transferred into QC, my question is how robust is the Water's Acquity UPLC? Specifically, I would like to hear from anyone who has used these systems analyzing biomolecules in a GMP environment.
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Thanks in advance for you input.

[tom jupille]

The Acquity system has been out for a couple of years now. I would ask your Waters sales rep to provide you with a couple of local references.

[DR]

Wow - 12.5 hours of deafening silence

Kidding aside, our labs have had some success with transfer of Acquity based methods. We have also had some miserable experiences but they are generally due to a developer's failure to realize that the capacities with respect to µg of analyte and injection volume are considerably lower with an Acquity column's dimensions than with a regular analytical column.

Given that the increased sensitivity is mated with a reduced (on column) sample capacity, the dynamic range you can get from an Acquity system is a modest improvement over conventional HPLC.

As advertised, you do get better resolving power as a function of the smaller particle sizes of the packings and higher pressures employed. This also leads to reduced run times. As long as you look at Acquity as a means of doing the same old things faster, with better resolution - you won't be dissapointed. If you're looking for a magical instrument that will give you another order of magnitude of sensitivity while you run the same 1mg/100µL samples you're already doing via HPLC, you will be disappointed.

That said, have you tried some of the smaller particle sized packings that are HPLC friendly? One of these may get you there with the Alliance...

[Victor]

DR- you say that you are regularly injecting 1mg/100ul samples ..that's 10,000 mg/l. Is this really correct? Are you working in the pharma industry-is this what people usually do? Is it a big problem to dilute the samples, or does this add unnecessary complexity or time to the method?

I guess you mean that the reduced capacity of the Acquity system is due to the dimensions of the column say 10cm x 2.1mm ID. A similar capacity would be expected on a 10cm x 2.1mm HPLC column packed with 5um particles-but I guess people are using longer, wider bore HPLC columns.

[DR]

Not really - I just pulled a number out of the air...
1mg is doable on most conventional LC columns, as is 100µL injection volume. My point was that Acquity columns require lower volumes and analyte concentrations.

I do work in pharma. Ideal methods around here have linearity out to fairly high concentrations and down to ~0.05%, by weight, of whatever it is we're assaying. That's a fairly stiff dynamic range criterion especially considering that the main analyte of interest might be <<1% of a dosage unit's weight.

[Newman768]

@gregg:
Have you tried a xbridge-column with the alliance system?
Most differences came from different column materials, in rare cases the uplc system (the high pressure that leads to temperature effects on the column head and the system dead volume) itself is responsible for the "extra kick".
With the same column material and (nearly) the same column temperature you may be able to transfer the method from uplc to hplc by scaling up.


@dr:
Lets say that the acquity requires a lower amount of analyte injected. I normally use the same sample conc. as with hplc, but I inject only 0.5 ul instead of 5, 10 or 20 ul.
The biggest problem with any ultrafast lc system is isolating the matrix of the injection from the compounds of interest within a few seconds compared to the "ultra"short run time. If one manage this problem it is possible to inject 20 ul or more with uplc.

[DR]

Agreed - the lower column volume of UPLC columns means that extra column volume effects, strong solvent effects (anything associated with potential hazards of large injections) are a lot more likely to foul up a method than with regular LC.

[jvj]

What do you think about the new ultra fat systems that lately had appear in the market specially th Agilent 1200 RRLC system that let you work in normal HPLC and UPLC mode in the same equipment. I just buy two of this systems


Javier[/img]