Chromatography Forum

i have the same normal phase columns that are obtained from two vendors.
i believe they are very similiar for a simple 2-compound test mixture elute the same pattern from both columns under same isocratic conditions. only on column 2 they come out a little earlier.

the issue is by the calculations of the same plate counts, the sharper peak 1 has higher counts on column2 than on column 1, but the later eluting peak2 that is relatively broader is the other way around, the plate counts for it is higher on colum1 than column2.

i am wondering what this means for the column efficiency, are they supposed to follow the same trend? i can see the peak 1 is narrower on column2, while the peak 2 is less so. two peaks is about a minute apart. so peak broadening shall not affect that much.

does this mean the packing quality is different? i actually like the colum2 although peaks elute a littler earlier but the resolution is actually better. since this is normal phase column, does this mean the silanol activity is less on column2?

thanks for the help.

How far off are the plate numbers? Unless you have exactly symmetrical peaks, plate counts are subject to lots of variability.

the plate counts for peak 2 is 20% lower on column2 than on column1, while for peak 1 it is the other way around. the peak shape are similar so many variability shall be cancelled off?
i can't think of reason for this difference between peak 1 and peak 2.

Perhaps one of the columns had a little more moisture than the other.

How do the plate counts align with those shown on the certificate?. If you haven't, you could try using a certificate column test mixture on both columns - that way you would confirm one column, and also the difference between columns - if they use different test mixtures.

Bruce Hamilton

Bruce beat me to it!

Normal-phase columns of the "liquid-solid adsorption" type (e.g., silica gel) are very touchy about the trace water content in the mobile phase (to the point where, many years ago, I could see variations that correlated with the relative humidity of the air when working with bare silica columns). Also variations in pore size distribution, surface area, acidity of the silanols, etc. can all play a role, so that you may well have multiple retention "mechanisms" going on.

I'd be more concerned if you were seeing that kind of variation on two "same" columns from the same vendor.

i don't really trust the certificate for good reasons. also i can't use the same mixture since they are very toxic.

if it is the moisture, shouldn't it affect peak 1 in the same fashion? the peak 2 has an extra ketone in it so it may be the reason it behaves differently with water?

thank you.

In my experience, the water will affect the chromatography in various ways that are not predictable.

You could attempt to dry the columns using anhydrous mobile phase and perhaps higher oven temp. but, if you have consistent chromatography, I would just accept that the difference exists, and use the columns.

I wouldn't try to diagnose the chromatograms without a lot more evidence that the columns are not performing as certified by the manufacturer.

If I didn't trust a supplier's certificate, I'd be very unlikely to purchase a column from them. Also, most normal phase test mixes I've seen use
compounds like m-xylene, nitrobenzene, cinnamyl alcohol, nitrobenzyl alcohol, which should be safely handled in most labs.

Bruce Hamilton