Selectivity changes with YMC ODS-AQ 3µm 4.6x250mm column

[Mike H.]

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.

[HW Mueller]

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

[Mike H.]

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.

[HW Mueller]

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.

[pharmaguy]

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.

[Mike H.]

Thx man. This makes a lot of sense.