Chromatography Forum

Greetings. I'm trying to analyze an urea solution using an amino column in the reverse phase mode. We've been running into problems converting the column from the normal phase solution it was shipped in to a 80/20 ACN/Water.
The column manufacturer says this should be no problem and recommends purging the column 1st with IPA (isopropyl alcohol) and then running the reverse phase mobile phase (ACN/Water).

We've tried characterizing the column utilizing chromatograms (and the associated parameters) from the manufacturers' literature (sugars or toluene/pyridine/phenol mixtures) which have resulted in poor chromatography or very little separation respectively.

Has any one had similar experience using these columns in this mode or would have any suggestions.

We've had the same thing happen on 2 new columns supplied from the manufacturer and they are at a loss to explain

Regards,

DTM

Some Amino columns can hydrolyse fairly easily, losing the stationary phase. I've managed to kill a couple after reasonable numbers of yucky, hydrophobic samples, but it took a few months. Some amino columns are compatible with 100% water, others aren't as robust.

When you say poor separation, do you mean the peak retention time has decreased, or the peaks have broadened or become mishapened?.
Have you seen a diifference in the column backpressure ( with the same solvent ) over time?.

Did you actually get to feed urea into the columns, or did they die first?. Are you confident the mobile phases and sample solutions that you are using are suitable to maintain Urea solubility at your sample concentrations?.

Not trying to insult you, but were all the solvents, especially the IPA, of good quality ( HPLC grade, and kept sealed ). Did you lower the IPA flow rate rate to keep pressures similar and ensure you flushed the injector as well?. When you flushed the system, did you ensure there were no cross leaks through the pump mixing valve?

Did you run the manufacturers Certificate test mix when you got the columns?. What happns if you cycle the solumn through reversed phase and normal phase and retest the column, keeping an eye out for pressure changes.

I'd cycle the columns through a couple reverse/normal phase transitions and retest to ensure that the transitions.are OK before investigating other issues.

Please keep having fun,

Bruce Hamilton

if you want to convert the Amino columns from normal phase to reverse phase,you would better follow steps as listed below
1.flush the system with hexane-acetonitrile(99:1) for 10 colum volumes.
2.flush the column with chloroform ,isopropanol,methanol-H2O(50:50) at the same flow rate.
when it come to the poor resolution. since,you directly flush the column with 80/20 ACN/Water which is immiscrible with normal phase solvent such as hexane chloroform and so on. bring air bubbles in column and hard to remove them. i strongly recommend you to flush the column with IPA for a long time with relatively high flow rate.

Bruce: In answer to your questions:

Some amino columns are compatible with 100% water, others aren't as robust.

I'd didn't want to specify the manufacturer, but their claim for this amino column states that it is 100 % water compatible as well as extended pH range

When you say poor separation, do you mean the peak retention time has decreased, or the peaks have broadened or become mishapened?.

Retention times did not match with their application data as well as peak shape was broad and mishapened

Have you seen a diifference in the column backpressure ( with the same solvent ) over time?.

No backpressure once established with new solvent was stabile

Did you actually get to feed urea into the columns, or did they die first?. Are you confident the mobile phases and sample solutions that you are using are suitable to maintain Urea solubility at your sample concentrations?.

No we never got as far as actually running the urea solution as the test applications were so poor. Recommended mobile phase was 80/20 ACN/Water for the urea test

Not trying to insult you, but were all the solvents, especially the IPA, of good quality ( HPLC grade, and kept sealed ). Did you lower the IPA flow rate rate to keep pressures similar and ensure you flushed the injector as well?. When you flushed the system, did you ensure there were no cross leaks through the pump mixing valve?

We didn't use HPLC grade IPA we only had spectrograde available and because we weren't actually running any samples with the IPA I thought this would be adequate. We kept the flow rate the same at all times (1 mL/min) but backpressure for the IPA was significantly higher for the IPA (2700 psi) vs the ACN/water (570 psi). These were all below the recommended max for the column and we flushed the injector from start to finish.

Did you run the manufacturers Certificate test mix when you got the columns?. What happns if you cycle the solumn through reversed phase and normal phase and retest the column, keeping an eye out for pressure changes.

The column is shipped in 1% ACN/hexane. I was reluctant to run the test chromatogram that came with the column (with this mobile phase) as I run only reverse phase separation (and changing over is a pain)

I'd cycle the columns through a couple reverse/normal phase transitions and retest to ensure that the transitions.are OK before investigating other issues.

OK I will try, thanks for your input


Regards,

Dan





Please keep having fun,

Yaoguocan: Greetings. In answer to your questions:

If you want to convert the Amino columns from normal phase to reverse phase,you would better follow steps as listed below
1.flush the system with hexane-acetonitrile(99:1) for 10 colum volumes.
2.flush the column with chloroform ,isopropanol,methanol-H2O(50:50) at the same flow rate.
when it come to the poor resolution. since,you directly flush the column with 80/20 ACN/Water which is immiscrible with normal phase solvent such as hexane chloroform and so on. bring air bubbles in column and hard to remove them. i strongly recommend you to flush the column with IPA for a long time with relatively high flow rate.

We ran with IPA for 2 1/2 hrs @ 1 ml/min and then 1 1/2 hrs with ACN/Water (same flow rate). we did notice a difference in pressure ( see above reply) so I was reluctant to use a high flow rate so I wouldn't go above Pmax

Perhaps longer?

Thanks for your input.

Regards,

Dan

What is in your urea solution and what are you trying to analyze? Are you injecting sample dissolved in water? Water is the strongly eluting solvent in HILIC, and can be the cause of peak distortion (same as injecting a reversed-phase sample dissolved in DMSO).

Unison UK-Amino is shipped in ACN / H20 eluent. No need to flush
it with organic solvents.

Rereading your responses told me that you never got around to test the urea sample. Apparently you tested some sugars, and the toluene pyridine phenol sample.

I dont know what to expect from the last sample, probably all nearly unretained peaks. Not sure that this means anything.

You did not specify the sugar sample. I expect sugars to be well retained on an amino column in 80% acetonitrile. I would run 70% acetonitrile to test with sugars.

On the other hand, all of these test just complicate things, and all of these tests can suffer from the problem that the sample is not dissolved in the mobile phase. I would think that this applies especially to sugars, since it is a PITA to dissolve sugars in 80% acetonitrile.

Why don't you just inject your urea?

Thanks for the comprehensive responses. Very helpful.

I'd say that 2.5 hours of IPA is far more than needed, 30 minutes should do, so that's not the problem.

Check the specification for your IPA, the only difference is likely to be the HPLC grade would be 0.2um filtered, so that's not the issue.

Based on that, I'd suggest you have performed the changeover OK, and the columns may be OK. If you have performed a couple of cycles, you should find no change in behaviour.

I would just check your tubing connections etc to ensure that you don't have any holdup dead volumes,, and if you have a guard column, try removing it to check peak shape.

It almost seems as though the test mixes - Sugars, etc, aren't relevant to your proposed analysis, but you are trying to ensure the column is OK. That implies that your method hasn't been performed by you before, and you are trying to ensure all items are OK before starting?.

When you say the peaks did not match their application data, did you use similar concentrations to their data, and the same solvent for test mixture?. Is their solvent compatible with the mobile phase to maintain test sample solubility and peak shape. Were there any trends, and did both columns behave the same way?.

You haven't done anything that would kill the columns, and so I'd want to confirm the column is OK for your analysis by injecting one of the standards for the analytes you were planning on measuring. You might be pleasantly surprised.

Please keep having fun,

Bruce Hamilton

Bruce: Greetings. Thanks for the reply. In answer to your questions:

It almost seems as though the test mixes - Sugars, etc, aren't relevant to your proposed analysis, but you are trying to ensure the column is OK. That implies that your method hasn't been performed by you before, and you are trying to ensure all items are OK before starting?.

Yes, correct. I wanted to confirm that the column was going to work in the RP. mode


When you say the peaks did not match their application data, did you use similar concentrations to their data, and the same solvent for test mixture?. Is their solvent compatible with the mobile phase to maintain test sample solubility and peak shape. Were there any trends, and did both columns behave the same way?.


HPLC parameters matched the application data completely (flow, mobile phase, loop and column oven temp) No use comparing apples with oranges. Both columns behaved in the same way.

Uwe:
I dont know what to expect from the last sample, probably all nearly unretained peaks. Not sure that this means anything.

Yes, correct very little retention. nearly all the same.

You did not specify the sugar sample. I expect sugars to be well retained on an amino column in 80% acetonitrile. I would run 70% acetonitrile to test with sugars.



I think I going to take both your advice and just try the urea and see what happens.

Thanks for you input.

Regards,

Dan

Greetings. Just to keep those who are interested updated. Urea analysis worked just fine. Nice retention (~ 4 minutes) peak shape good.

Live and learn

Regards to all,


Dan