someone can help me?packing unrepeatable

[moon704520]

I met with a strange phenomenon!I am packing our spring columns ,all the conditions were the same ,the same packing system,the same HPLC system,almost the same amount silica (C4,300A,10-15u,214TPB1015),about 280g,the same column hardware!this time ,with higher packing pressure about 1000psi,last time the packing pressure is about 750psi. and the result column pressure drop is higher than the former one,this time is 3.3MPa,last time is 2.0MPa,but when I test the column effiency,I found that the testing probe's(Naphthalene and 1.54mg/ml phenanthrene) retention time decrease from the former 25min to the now 20min,finally,when I press the silica out of the column ,I find that the length of column bed is almost the same!I do not know why is so ? do you have some advices ?will the piston speed affect the column efficiency a lot when packing the column?
the lot of the silica was different ?and the ACN used as mobile phase maybe from different company,I forget it!
testing conditions as follows:
mobile phase:50%ACN/50water
flowrate:50ml/min
wavelength:254nm

[Mark Tracy]

I don't know much about axial-compression packing, but I do know that when slurry packing, the compression rate is critical to good efficiency. Also, higher packing pressure tends to give higher operating pressure, but the relation is not linear.

The difference in retention times may be related to the lot difference. Calculate the k' and see if they are also different; if so, blame the silica. See if the vendor can supply a 4.6x250 mm column packed with the same lot of silica, and pre-qualify the lot before buying in bulk.

[moon704520]

Dear Tracy
is there any relationshipm between the compression rate with the column effiiency? Does higher compression rate means higher column efficiency?
if the K' is the same ,do that mean no silica lot difference?thank you very much!

[tom jupille]

For starters, you need to be concerned with two aspects of your stationary phase:
- chemistry (surface characteristics)
- physics (particle size distribution)

The first can be verified by packing a small column and running a test probe (a known compound) under isocratic conditions and measuring the k' as suggested by Mark Tracy.

The second is more difficult. It is possible to have to lots of packing meeting the same specification (10-15 μm) with very different particle size distributions. Aside from a particle size analyzer, the best way to tell is to pack two columns under identical conditions and measure the efficiency (plate number) and the pressure.

Having said all of that, if I were your supervisor, I would be concerned about the apparent lack of consistency in your procedures.

all the conditions were the same ,the same packing system,the same HPLC system,almost the same amount silica (C4,300A,10-15u,214TPB1015), about 280g,the same column hardware this time ,with higher packing pressure about 1000psi,last time the packing pressure is about 750psi.

If you packed at 1000 psi this time and 750 psi last time, then all the conditions were not the same. And what does "about" 280g mean? Was that ± 1g? ±10g? There is an old saying that "the devil is in the details", and that certainly applies to chromatography columns.

[Mark Tracy]

The relation between compression rate and efficiency is empirical. There is an optimum rate for each combination of packing material and column geometry. Manufacturers keep the exact knowledge a trade secret. As Tom suggests, you need to set your qualified packing conditions and their allowable tolerances, and stick to them.