Chromatography Forum

hi all:

I am packing spring column 25mmID*250mmL,with 5u particle size,C18,but I find that the peak of the testing probe always tailing ,but I do not know why?can anybody help explain ?

What is the test probe? There can be two reasons for tailing: 1) the stationary phase is damaged and a basic probe will be sensitive to that, or 2) the packing protocol is not optimized and a neutral probe will be sensitive to that. Have you talked to the manufacturer of the column hardware?

Dear tracy:

the testing probe is phenol and toluene !the tailing factor is 1.3?I do not know why?is there any relationship bwtween the tailing factor and the flow rate?or there are something wrong with my packing protocol?do you have some valuable experience on the packing ?

Actually, a tailing factor of 1.3 is not bad (I'm assuming the USP tailing factor measured at 5% of peak height). What have you achieved previously?

Actually, for a neutral probe, a tailing factor of 1.3 is borderline, not terrible. First establish that the packing material is not damaged by looking a some basic probes. Second, budget yourself some time to empirically optimize the packing conditions: temperature, type of solvent, volume of solvent, weight of silica, compression rate and final pressure. Keep a notebook. When you have the optimized conditions, write an SOP. You will get better with practice.

Column manufacturers spend a lot of time developing this knowledge, and don't give it away.

Dear tracy and Tom:


would you please recommend me some common used acid and basic probes?thank you very much!

For basic probes, amitriptyline is a popular choice. We use it to test our columns. (80:20 MeOH:buffer pH 6.0 or 7.0) You should get an analytical-size column of the same type as your prep column to determine the expected behavior. Another popular basic probe is N,N-dimethylaniline.

For acid probes, 4-butylbenzoic acid is good.

You should also discuss this with the vendor of the silica you use. They will have QA tests; perhaps they will share them with you.

Mark's suggestions are good.

However, if you are evaluating how well you have packed the column, a neutral probe is a better choice. Acidic or basic probes typically respond more to the chemistry of the column packing; tailing of neutral probes is usually related more to the quality of the packed bed.