Sample Degradation in Column

[AY]

I am running my samples at 80 deg C with a C18 column and a mobile phase containing acetonitrile and pH3 phosphate buffer. Run time is 1 hour. How do I test whether my sample has been degraded in the column or not. Thank you in advance for any suggestion.

[amaryl]

I am running my samples at 80 deg C with a C18 column and a mobile phase containing acetonitrile and pH3 phosphate buffer. Run time is 1 hour. How do I test whether my sample has been degraded in the column or not. Thank you in advance for any suggestion.

you want to degrade sample or your column?

Working at 80 deg C won't get you anything. The column will be dead, no more in workable position.

Regards,

Amaryl.

[Koen Hollebekkers]

I do not fully agree. The work at evaluated temperature does indeed requires somewhat more advanced columns. So your first concern should be: What is the stability of my column.

To investigate whether my analyte is stable or not under these conditions, inject your sample at different column temperatures, if your peak area decreases, your analyte degraded. If it stays the same you have a reliable method.

[AY]

Thanks, Koen and Amaryl;

Our sample is an early stage API. Due to its sophisticated sample matrix, it contains quite a large amount of impurities. we tried many columns and mobile phases, and found that a gradient run at 70 or 80 degree with a C18 column is the most optimum condition. We also tried a prolong injection at 80 degree(kept injecting for 48 hours). The column life seems OK. Now we go to Koen's second question, whether my analyte is stable under these conditions. I will probably test my sample at 2 other lower temperatures and compare the peak area.

Does anyone has any other suggestion?

Thanks

AY

[syx]

I am so worried to your column lifetime. You may try gradient run instead of isocratic, with lower temperature.

[Bill Tindall]

1. Put the pure material in mobile phase and heat at 80 degrees in an oven and sample every XXminutes. Analyze to see if it goes away and how fast.

2. The column can have some catalytic effect. If the stuff degrades in the column it will degrade TO something. The degredation product will front or tail from the main peak depending on whether it moves faster or slower than the parent component. If degredation is significant this front or tail will be obvious.

[Kostas Petritis]

I like Koen's and Bill's suggestion.

As for the column lifetime I wouldn't worry that much. For sure the column lifetime is decreasing with higher temperatures but some compounds give much better peak shapes in higher temperatures.

Things to consider: The degredation compound might not have a chromophore so you might not see it, so the decrease of the peak area is a good test.

If you care to characterize your impurities, those might degrade as well and might be harder to investigate their stability at higher temperatures...

[AY]

Thanks for all suggestions. I finally get some point to start. Thanks again for all your opinions.

AY

[sjmarin]

Hi AY,

There are several things to consider when operating at elevated temperatures. One of the most important things is the column. Most traditional silica columns will break down at temps above about 60C, but there are several silica-based packings that are stable at higher temps. I can recommend some for you if would like.

Another thing to be concerned about is thermal mismatch band broadening. You need to make sure the mobile phase is properly preheated. Since you say you are getting sharper peaks at higher temp, your system may have a preheater. What HPLC system are you using? What flow rate are you running your analysis at?

I have also done some work on degradation of API's and other samples at high temperature and can provide that info to you if you email me. You may see a baseline rise due to on-column degradation, or the appearance of extra peaks. I disagree that simply putting the sample in an oven and checking it will mimic what is happening on column. It will tell you if your sample degrades at the temperature over time, but the time the sample is exposed to high temperature on column is often very short, and my experience has been that most samples are stable. I did some work with a compound that was known to thermally decompose to study degradation at high temp, and discovered that it was not as pronounced as you might think.

I am glad to see you are using temperature in you HPLC method development. It is an important, but often unutilized, parameter.

[KarenJ]

Hi,

If you are using extreme temperatures because of a difficult matrix, why don't you try to clean up your samples before the chromatography run? Maybe if you used some type of solid phase extraction cartridge (C18?) prior to the HPLC run, you could get rid of some of your matrix and then you wouldn't have to work so hard to resolve your analyte peak.

KarenJ