Help! Settling in manually-packed columns! Weird!

[dkreller]

Hi,

Before reading on you might first want to look at the chromatograms in this figure:
http://chemistry1.che.georgiasouthern.e ... quartz.jpg

The data is from an experiment in which I made a succession of identical 10 microliter injections of 0.01M acetone onto a column that I (dry) packed with quartz sand. The flow rate was 0.5 mL/min. There is essentially no interaction of the acetone with the quartz; it is a conservative tracer that is completely unretained.

The changes in peak shape are presumably due to changes in the (dry-packed) column that occured over the time-scale of the experiment. Some of you here may vaguely recall having a similar discussion with me about this in the past, but this time I am seeing slightly different changes in peak shape.

Do you have any insight into what is going on in the column? I learned from previous discussions on this forum that the total void volume is a constant, but there may be, over the course of the experiment, setting in the column and the formation of a larger void space, probably at the head of the column, as the particles in the rest of the column settle and pack more efficiently.

My million-dollar question: How could the retention time of most of the tracer slowly increase, while a shorter-retention time shoulder appears? I have some thoughts, but I won't share them here yet. I would love to hear from the expert HPLC community.

BTW, it is abundantly clear that our current column packing protocol is failing us (sucks, to be blunt) and that we need to change it. I humbly admit that! I am open to suggestions on manual packing procedures. We are next going to experiment with slurry packing, first at atmospheric pressure, and then at higher pressure if problems persist.

Thank you in advance for your consideration and thoughtful/constructive/informative responses.

David

[Ron]

I suspect that the column packing is shifting, and there is some channelling occuring. When you develop open channels the flow is faster in the channels than in the bulk of the packing, and the retention time is shorter for the fraction that passes through the channels.

I am surprised that you are packing the columns dry, it isn't much more work to pack a slurry, and columns tend to be more uniform.

[dkreller]

Ron,

The 'channeling' hypothesis is what I was thinking. I understand that the formation of one or more channels, through which some of the acetone moves at a higher than average speed, would explain the shorter-retention shoulder. I don't as yet understand what might cause the peak retention time of the main peak to systematically increase. We are now attempting slurry packing - I have two students in the lab setting this up as I am typing this.

Based on your experience, would you suggest doing the slurry-packing under pressure, with some kind of packing device, or do you feel that slurry-packing at atmospheric pressure would be enough.

Thanks for your input!

David

[JGK]

David,

If you take the column apart and inpect the high pressue end (the one nearest the injector), do you see a void (deadspace) caused by the bedding down of the column packing under pressure?

If a suspect you will, this is the cause of your observed chromatoraphy.

Initial injections will pass directly into the packing giving you the initial peak with the expected shape.

However, as the void forms and grows the sample injected is partially held within the void and does not enter the packing material as a single "slug" of analyte but diffuses in over time. This broad band of material causes the lower responding shouldered peak effect you are seeing.

If you repack the void with additional staionary phase you could see a return to the initial chromatography.

[Ron]

If you can slurry pack under pressure that is the best way of packing, and the way all commercial columns are made. Packing to a consistent elevated pressure gives the most reproducible results from column to column. Slurry packing at atmospheric pressure is better than dry packing, but may still have void formation.