Chromatography Forum

I'm new to chromatography and need a little help coming up with a mechanism for the following.

We are trying to develop a separation for 3 very similar compounds using a YMC ODS-AQ 3µm 4.6x250mm Column 120Å [AQ12S032546WT].

The exact structures are proprietary but the only difference between the three is the substituent groups. Compound #1 can defined as [X], Compound 2 is [X-CH2-OH] and compound 3 is [X-CH2-NH2].

At pH 2.5 the elution order is [X], [X-CH2-OH], [X-CH2-NH2].

At pH 3.2 the elution order changes to [X-CH2-OH], [X], [X-CH2-NH2]

The retention time for the [X-CH2-NH2] compound is constant at both pHs, but the retention times for [X-CH2-OH] and [X] increase but at different rates over the 2 pHs.

Can anyone think of a mechanism to explain why the elution order of the [X-CH2-OH], [X] compounds flipped? Why is the X-CH2-OH compound affected less that the X compound from pH 2.5 to pH 3.2?
thanks for any help.

How many times did you repeat the prep. of mobile phase and these results?

How many times did you repeat the prep. of mobile phase and these results?

I prepped the the KH2PO4 twice at each pH. The elution order of the 3 compounds are the same when using a 2year old column and a new column at pH 2.5.

You didn´t try both pH with both columns?
It doesn´t make sense that the XCH2NH2 doesn´t move, but generally this sort of thing is explained via changes in charge state.

The differences between NH2 and OH can be quite dramatic especially when talking about electron density and pKa - look at these two groups and see how they may be affecting the pKa of other heteroatoms on X, especially if nearby on the structure. Also, Aq-type columns generally rely on silanol activity to aid in polar retention, by raising pH you may be affecting not just the compounds but also the silica surface.

Thx man. This makes a lot of sense.