Chromatography Forum

I have been running polymer samples on a combined column set consisting of 2 Agilent PLgel Mixed C columns(5um) in line with 1 Waters Styragel HR-2 column (5um). We have been running on an old PerkinElmer system, and a new Agilent HPLC. Our problem persists on both instruments. The problem is, that we are required to use a polymer control sample for suitability. The Mw of the sample should be 110KDa +/_ 5%. Even though our plate counts are always above our 25000 criteria, the control sample often fails. It is typically too high. There are times when we put a brand new column set on the system and the control sample still fails. When it does work, we very rarely get a set that will last longer than 100 injections. Our mobile phase is chloroform (unfiltered, Fisher Optima), and we run at a flow rate of 1ml/min. We prepare samples in chloroform with a toluene flow rate marker. Has anyone else had an issue with a lack of column life similar to this?

Both column brands are excellent.
My first question is, at what columns temperature do you run the test.
Why you use unfiltered Chloroform?
First of all I would use always filtered clean solvents.
Also I would use a guard column.

Depending on the samples the number of injections is always relative.

Thank you for your response.
We are running our method at 40°C.
We filtered our chloroform at one point in time, but we stopped because it was not a very efficient process. We are using a brand that is already submicron filtered, and we performed comparison studies that showed no difference in results.
We do use a guard column.
We are 95% of the time running just polymer samples. A mix of 7525, 5050 or 8515 with various endgroups. We occassionally run microparticles or active ingredients.
Historically, we would get about a 2 month column life, running 2 runs a week. Now we are lucky if the column lasts for 2 runs. Sometimes we put brand new columns on the system and we don't even get a control check to pass, and they go straight into the trash.
One interesting thing is that when this first started happening on a regular basis, I had 2 sets that failed right away. I refused to throw them away because I just felt like it was impossible that they both failed like that. They sat in chloroform at room temperature for probably 2 months or maybe more. I put them back on a system out of desperation, because I had run out of new sets. And those 2 column sets lasted for about a year after that with no issues lol...I don't understand why that would happen, but maybe you may have some guesses? I have not repeated that process since then, because I couldn't remember exactly how long I had let them sit...Also, we are under a lot of pressure to produce test results, so I don't have a lot of time for experimentation. Limited by time as well as instrumentation.

Hi nsparks,

Could you describe a bit more about your "failure" or "trash" conditions? What is happening to the columns that makes them trash or failed, is it a difference in RT or like an overwhelming back-pressure issue, or is it more like all retention is being lost?

Thanks,
TS