Resolution problems on Agilent A1100 system

[Techie 33]

Hi there. I've been having some difficulty acheiving resolution using Agilent A1100 systems. The plate count of the peaks is dropping and the peaks are not as tall as they used to be. If I take the mobile phase, columns nad solutions and setup on a Waters Alliance 2695, these issues dissappear, and I can acheive resolution. The problem is we have mostly Agilent systems. We have very little room to change our methods, so if anyone has come across this problem before I'd appreciate some input. I'd rather "fix" the Agilent system than have to go out and purchase more Waters systems.
Also, I just use these systems for analysis. They are maintained externally, and the methods are written and validated off site - i.e. I don't know too much about LCs other than what I need to run them and stop them from breaking down.
Also, I can't supply too much detail other than the exact same method, setup etc. provides different results on the different models. Any help would be really appreciated.

[danko]

Hi Techie 33,

The plate count is lower because the peak width is larger (I assume that the retention time is constant). You can convince yourself by comparing the areas. My guess is that the peak areas are roughly the same regardless of the system. Here I’m assuming that the detector types and flow cell dimensions are comparable. If all this is true, you need to focus your efforts on the plumbing: Make sure that the same diameter tubes are used on both systems – especially the one that connects the column to the detector. Also, check the connections/fittings etc. And finally make sure the column temperature is roughly the same (plus/minus 1 – 2 degrees) on both systems.

So, no need for method changes

Best Regards

[mbicking]

Could you provide more detail on how the peaks are changing?

Did this happen suddenly, or has it been happening slowly over days/weeks/months?

Are the "bad" peaks tailing more than the good peak?

Is this an isocratic or gradient separation?

Please specify your Agilent system (pump, detector, autosampler) and mobile phase components.

[Techie 33]

Could you provide more detail on how the peaks are changing?

Did this happen suddenly, or has it been happening slowly over days/weeks/months?

Suddenly.

Are the "bad" peaks tailing more than the good peak?

The "bad peaks" have a tailing factor of about 0.64. The good peaks have tailing at about 1.0 to 1.2. The bad peaks are fronting very badly, but the column is o.k.

Is this an isocratic or gradient separation?

Isocratic

Please specify your Agilent system (pump, detector, autosampler) and mobile phase components.

Agilent system A1100:
G1322A Degasser
G1311A Quaternary pump
G1313A Autosampler
G1316A Column Compartment
G1314A UV Detector

Mobile phase 0.05M KH2PO4 pH=3.0:Methanol (60:40). Flow rate 1.0ml/min, column temp 40 deg C. Leeway of +/- 2% for methanol, +/- 0.1 pH units for buffer, 0.1ml/min flow, =/- 5deg C column temp.

I've attached jpegs of the chromatograms:

The black trace is from the Agilent system.





This isn't the only setup that gives problems with resolution. Some of my colleagues have mentioned this may be due to the fact that the Alliance systems have a larger dead volume, so I will investigate that further, but in the meantime any input would be appreciated.

[mbicking]

When I look at the chromatogram, I see two common explanations:
1. An injection solvent problem. If your sample is in methanol (or other strong solvent), and your mobile phase is only 40% methanol, the solvent is causing the fronting/distortion that you see. (For the first few mm of the column, the sample is in a "stronger" mobile phase because of the injection solvent, and some of the sample elutes a little faster than the rest of the sample, until the sample solvent is diluted out by mobile phase.) This problem would be most common for either a relatively large injection volume, or a narrow bore column. The Agilent system does have relatively little tubing between the injector and column, which means not much mixing occurs before the column. If the other LC has larger tubing and/or longer tubing length, more mixing would occur before the sample reached the column.
2. Your column has a void, partially blocked frit, and/or damaged stationary phase. Try reversing the column and repeating the injection. If the problem goes away or is less severe, this might be the problem.

The only problem with explanation #1 is the fact that this problem occurred suddenly. Typically, injection solvent affects appear immediately; they don't just appear at some later time. For explanation #2 to be true, you need to investigate what happened to the column just before the problem appeared. Was the column in storage? Was a new sample type analyzed? Has the system pressure increased?

I am not completely happy with either suggestion, but you may be able to figure it out with the answers to the above questions.

[unmgvar]

Techie 33

from what i am reading and seeing if you say that you took everything from the agilent to the waters without changing anything else other then that then the problem seems for me to be mainly in a connection.
you say the peak shape is not like it use to be, so it means that the method use to work fine when run also on a 1100.
primary suspect would be the connection to the column.
in 1100 there are mainly peak tubing with peak connector. change them.
start with the column outlet connection then move to the column inlet.
if not there then you will need to either go toward the autosampler or the detector flow cell and check connections. but it is most probably the outlet or inlet connection of to the column.
it could be like mbicking suggested that the tubing to the inlet of the column has been shorten for some reason. this can also give you your problem for 2 reasons:
1. like mbicking says the sample solvent is MeOH and the mobile phase too different and there is no more time for mixing.
2. your column temp is 40 degrees. your lab temp. is probably less. the difference in temp. shows now as a split peak. it can happen even with the pre-eluent heater; or maybe you have not connected to it.

most probabl thou it is the connections

the chance that the problem is due to the difference in dwell volumes here is very close to zero since this is an isocratic method.

[danko]

The longer retention time seen on the Agilent system tells me that there is a leak on this particular system.
Try to compare the backpressure in the Agilent to that of the Alliance. I would guess the latter generates a higher backpressure.
The most probable place to look for is the connection to the column inlet.

Best Regards