Chromatography Forum

Hi

I would like to know why SEC columns have fewer lifetimes compared to RP columns?

(Also most of the SEC column starts giving bad profile very suddenly and unpredictability ….we even have had SEC columns gone bad after 50 injections!!!)

Thank you

In order to understand this, I will need more information, since there can be many different reasons for a column "going bad". Would you please give me more details (what is the column that you use, what makes it "go bad", what are you injecting, what is the difference between a column that goes bad qucikly and another one that lasts, etc.). I am also curious myself, why this could be the case.

Do you try any cleaning procedures?

Hi

Sorry for the late reply….
Well went through all my column entry records for SEC and found that number of injection for an SEC column is between 200-300 ( there are variations, in some cases it it has worked well beyond 300, while in others the column has failed at 190 injections).. I even came up with a case were my column failed after 50 injection.
As we have multiple protein analysis, there is variation in type of buffer but most of them are phosphate buffer with pH on average around 7...
When I talk about “column going badâ€

Hi Rick,

(my protein peak starts getting merged with buffer peak…)

Do you experience shorter retention time for the buffer peak (the total permeation volume) which causes resolution deterioration? If so, this could mean blockage of the stationary phase pores.
In this case it is not a question of column stability but chromatographic conditions. Proteins are, as we all know, susceptible to aggregation followed by partial or total precipitation. One of the critical parameters is pH. Many proteins’ pI are around the neutral pH which means risk of potential aggregation at the pH you’re working. In order to secure “comfortableâ€

This seems like rather consistent results. Does the column typically go bad after it has been stored, i.e. with a new set of samples, or is it happening suddenly in the middle of a long run of samples?

Uwe Neue Posted: Wed Apr 02, 2008 12:58 pm Post subject:

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This seems like rather consistent results. Does the column typically go bad after it has been stored, i.e. with a new set of samples, or is it happening suddenly in the middle of a long run of samples?

yes this is what usually happen i have last set of samples giving me great/perfect sresults..bad witha new set of samples the column starts behaving werid...( are you suspecting column storage..but we use the usual recommented storage condition..and if the storage is problem why after so much injection??)
also do SEC columns have same life time as RP columns?( in our lab SEC columns are considered sarced)

I suspect the storage conditions. What are they?

Typical life time of a SEC column is in the order of 500 injections. I have not done any work myself to determine the failure mode of SEC columns.

Hi

We usually store our column in 0.05% sodium azid ? how should this create a problem??