Chromatography Forum

Hi

May I know what are the cause of column-to-column (column from same brand/manufacturer) peak profile variability in reverese phase column (for example:- variability in closely eluting impurities… one close eluting impurities in some RP-columns merges with the main peak and in others RP-columns are well separated)?
What factors may contribute to it..(is it packing differences, is it particle size distribution difference, is it difference due to the surface coverage or difference in bonded phase..etc)???

The most likely causes are column age and batch-to-batch variability of the packing material ("batch" means different preparations of the powder that goes into the tube). Differences in retention between columns made from the same powder are negligibly small, unless of course the columns have a different history. If it can be demonstrated that the observed differences come from different batches, the more likely source is variability in the preparation of the packing, especially of the surface, rather than particle size differences.

We have comparatively evaluated columns from two different batches, (1)Batch no:-5267-32 (2) Batch no:- 5267-33, three columns from each batch and their were marked difference in profile between the two batches, especially in terms of resolution of closely eluting impurities with this impurities completely non-resolved in Batch 1 and well resolved in all three columns of batch 2 .

I was just curious what factor could be leading to such a difference???
these are C-4,4.6 X250mm 300A columns
mobile phase is Water with 0.1% and ACN with 0.1%TFA...

It could be almost anything:
- the pore size distribution might be slightly different
- the acidity of the silica support might be slightly different
- the surface coverage of bonded phase might be slightly different

It doesn't take much of a difference in surface chemistry to make a big difference in selectivity.

If you really want to get into it, I can recommend Uwe's book on HPLC column technology. Here's a link to it on Amazon: http://tinyurl.com/2fjnrz2

Maybe Uwe will remember that story, many years ago.
A customer called me and complaint that a new purchased column showed a better resolution and 2 more peaks, baseline separated, than all the other columns (same brand, same specification and modification) before. In the lab of the customer I first saw the chromatogramms and I was impressed. Than I asked when they used this new column and when was the last time they prepared the buffer solution. Because I saw a bottle, named buffer stock solution, take out 100ml and dilute with Water to 1 Liter and this will give the buffer pH6,0! I asked if they messured pH and I was told that this is not necessary because it is a validated method. After calibration of the pH Meter the used buffer had pH5,2 and not 6,0! After the buffer was adjusted to pH6,0 also the new column had the same performance than all the other columns before, and the 2 new peaks dissapeared. It`s magic? No, just pH!
And pH is also linked to temperature. So there are 2 more parameters.
Perhaps that give you another idea. Good luck. Gerhard