how to handle carbon load in separation

[smiley]

i am trying to increase the retention time of the eluted peak in reverse phase, so i got a higher carbon load C18 column(27%), the original column is phenomenex luna c18(2), carbon load is 19%.
but the side by side run on both columns turned out the peak eluted almost at exactly the same time(8.5 min.) from both columns, the general gradient is 90/10 water/ACN with 0.1%formic acid to 5/95 water/ACN in 15 min..
since the new column is almost 50% higher carbon load than luna, theoretically it should get more retention when all other conditions fixed. the vendor tech support can not explain this either, just suggest to run isocratically., saying that the gradient steepness may overcome the retention, that i doubted not true.
the compound is small drug discovery molecule(mw=450) with several secondary amine and amide structure, the peak shape is perfect on luna with acidic mobile phase.
i even tried another column claimed better for basic compounds from the same vendor, but it did not elute later than Luna C18(2) did.
so please suggest some highly retentive column for small molecule, i remember some vendors list charts of various columns comparison based on retention, polarity, and other criteria, but i can't find them now.
my purpose is to increase the organic content for signal sensitivity in LC/MS, since higher organic(less water) will help to increase ionization in MS.

[Uwe Neue]

To answer your last question first: since you have a compound with many amine groups and you want to increase retention, consider going to the alkaline pH with a suitable column. As a rule of thumb, you get a roughly 30-fold increase in retention, if the compound is converted from the ionic form to the non-ionic form.

Now to your first question: the carbon content on its own is a very misleading number. It is normalized by the weight of the silica, however, if you are dealing with silicas with a different specific pore volume, the carbon content by itself is meaningless. As a rough guess on which materials are more retentive, you can use the ratio of carbon content to specific pore volume. when you do this, you will realize that for a packing with a pore size of 10 nm the differences between different packings becomes small.

I also would not use the carbon content as a primary criterium for the selection of a column for your compound. Good peak shape for basic analytes is not easy to get.

[smiley]

thanks,
i will use xterra with 0.1%NH4OH to test your suggestion.
however, based from my past experiences with XTerra(i use it daily for screening) that the retention is not as great as to 30 folds as you said, we usually go to Xterra only if the compound got serious tailing on Luna, so can you also suggest several other good columns for basic compounds?
and what do you think about running basic compounds in HILIC mode? the web search on HILIC comes several c-grams all with very high organic content mobile phases like 90% methanol, which is very attractive.
do you know who or which vendor is the best to talk to about HILIC columns?
thanks.

[tom jupille]

Uwe works for Waters, so your question puts him in an awkward position . I'll stick my neck out and suggest:
- Waters
- PolyLC
- Sequant

others may suggest additional sources.

[Steve]

smiley

Poor surface coverage and high activity of residual free silanols often results in peak tailing with basic compounds. Uwe is correct to suggest working at an elevated pH to minimize or eliminate the tailing of the basic compounds. Macherey-Nagel has developed their GRAVITY columns made from ultra pure silica and a miltu step endcapping procedure to minimize free silanols to allow you to work at elevated pH for basic compounds. If you check the Announcement Board you will see a special offer for this column.

[smiley]

actually, i don't see much tailing problems from Luna C18(2) even for basic compounds., 95% of the time it is alright.
what i tried to improve is the sensitivity in LC/MS, i need to increase the organic solvent in the elution. so i am looking for highly retentive phase for RP HPLC for small molecules.

[Sallybeetle]

On such compounds, I have had better resolution using a acidic pH mobile phases, a gradient, and Waters YMC ODS-A or ODS-AQ columns. Can't remember exactly which column it was, but it was the column with the higher carbon load. I studied the theoretical pKa's, but I didn't get good chromatography with the higher pH mobile phase gradient.

p.s.: I don't work for Waters and I tried about 20 different columns on a few similar projects. In this case, with similar column and pore sizes, the carbon load appeared to make enough difference in retentions that I was able to calculate the resolution factor of the parent and the impurity.

[smiley]

YMC ODS-A is a little better than Luna in terms of hydrophobic retention, but only a little according to comparison chart(yep, i found it)

my molecule elutes in the middle of gradient so it's like around 50% of ACN, what i am looking for is to change it to elution at around 80% ACN. i don't believe YMC ODS-A or AQ will do it(AQ is probably for aqueous meaning higher % water?). i don't really care too much of resolution.

[Uwe Neue]

So the purpose is to get good retention in about 80% organic to get improved sensitivity. I don't think that there is any C18 around that can switch the retention from 50% organic to 80% organic without any drawbacks. It is possible with a packing that is full of silanols, but then you get tailing peaks for bases.

I suggest another avenue: use silica in HILIC mode. It retains bases nicely in a mobile phase with over 50% organic, does not create tailing peaks, and prefers to be used with mobile phases with small ionic strength, all ideal for LC/MS.