In an article is described an HPLC analysis of sulfonated metabolites of chloroacetanilide herbicides; separartion is obtained by reversed phase chromatography with C18 column and mobile phase containing 0.1% acetic acid.
The chromatograms shows peak splitting due to rotational isomers but, what are rotational isomers?
(note that a chiral carbon atom is present in only two of four analyte)
Thanks for any suggest

For completeness double bonds are not present in the analytes (except for carbonyl and phenyl group)
Thanks

Dear Anonymus;

In some compounds the position of groups is such that free rotation is obstructed by stereochemical blocking, in other words, groups block each other.

The energy to surpass the blockage and yield two or more distinct isomers from the same molecule can be such that at room temperature or close to it, the isomers are in equilibrium. If the transition between the isomers is slow enough to be in the same order of magnitude as the LC separation (seconds or minutes), then isomers of this type can be separated. This is very temperature dependent.

I have seen this type of separation in peptides containing proline, and there many other examples in the literature.

I hope this helps.

Benjamin

Metolachlor is such a compound. It has restricted rotation about the bond between a phenyl carbon and the nitrogen. The temperature required to interconvert between the two forms is well above room temperature, and the rotamers can be separated by HPLC.

Thanks to Benjamin and Anonymous for your suggestions. The argument is very interesting!
I use a RP-HPLC-MS method for herbicides residues and metolachlor is among analytes. I had never been observed a splitting of metolachlor peak at room temperature with C18 column.
With this method metolachlor and alachlor are partially resolved; metolachlor show a simmetrical peak while alachlor peak show a prominent heading; the broad peak for alachlor is perhaps due to unresolved peak of rotational isomers?
I have tried a graphitized porous carbon column for this separation and I have obtained a splitted peak for dimethenamid and metolachlor (identified by respective ions). I have attributed this phenomenon to the GPC column capability to separate chiral isomer: is this correct?
thanks in advance

A graphite column will separate the rotamers of metolachlor, but not the chiral isomers. If you were to run metolachlor on the proper chiral column (in normal phase), you would get four peaks: the two optical isomers of each rotamer.

Hi, Anonymous #2
(I'm Anonymous #1) I have revised the property of Porous Graphitic Carbon column (Hypercarb) and, in effect a presence of a chiral selector in mobile phase is required to obtain an enantiomeric separations.
I have tried C18 column and PGC column with the same mobile phase (a gradient acetonitrile-water acidified with formic acid)at room temperature; with PGC column I have separated rotamers only for dimethenamid and metolachlor but not for alachlor. Have you an explanation for this behavior?
With C18 column I had never been observed the separation of rotational isomers for the three analytes dimethenamid, metolachlor and alachlor (for alachlor I have previously reported an ipotesis).
Thanks in advance for any ideas.

Anonymous #1,

I would suggest that you look at the structure of these three compounds. Locate the ring-to-nitrogen bond which has restricted rotation. Next, look at the symmetry of the ring on either side of the C-N bond. If the two sides of the ring are different, you will have rotamers. With metolachlor, for example, you have a methyl on one side and an ethyl on the other.

Anonymous #2

Anonymous #2,

with your useful suggestions I have fixed some points:

a-> metolachlor and dimethenamid present asymmetry respectively on phenyl and thiophene ring (as you already noted for metolachlor) therefore rotational isomers are present
b-> alachlor present two ethyl group in symmetrical position (2 and 6 on phenyl ring) and so no matter of rotational isomers
c-> with my chromatographic conditions (RP on C18 column, gradient acetonitrile-water acidified with formic acid, room temperature) the rotational isomers of dimethenamid and metolachlor are not resolved
d-> with practically the same chromatographic conditions but with graphite column instead of C18 column, rotational isomers of dimethenamid and metolachlor are baseline resolved
e-> in both situations (c and d) alachlor show a unique, headed peak
f-> with chromatographic conditions as in (c) I have tentatively separated sulfonated metabolites of alachlor and metolachlor (SO3H instead of Cl); the chromatogram show double peaks probably for both analytes. Probably because identification is performed on a real sample on the basis of ions reported in literature and not with pure standards.
Sulfo group can change stereochemistry? Acidic mobile phase have influence on rotational isomers in this case?

Thanks in advance
Best regards

Anonymous #1

Anonymous #1,

About your points:

a. Correct.
b. Although the ethyl groups on alachlor make the ring symmetric, they can be turned in opposite directions and make the ring appear that it is not symmetric. This interconversion occurs at a fairly slow rate at room temperature, so you see a broadened peak even though there are not true rotamers.
c. Your results are consistent with what I've seen in my work. Several metabolites of these compounds, however, have rotamers that can be separated on a C-18 column.
d. Again, this is consistent with what I've seen.
e. See b.
f. The sulfonated metabolites are difficult to deal with. I've had some luck using an acidic ammonium dihydrogen phosphate buffer and acetonitrile on an AquaSep C8 column. I have also used the ion pairing reagent TBAP with these compounds, but the peaks are broad.

The addition of the sulfo group does not change the stereochemistry. I would not expect the acidic mobile phase to influence the rotamers in any way.

Anonymous #2

Anonymous #2

thanks for your very useful suggestions.
You have worked with sulfonated metabolites of chloroacetanilide herbicides; this metabolites are commercially available?

Thanks
Anonymous #1

As far as I know, they are not commercially available. They can be custom made by some contract labs.

Thanks,
Anonymous #1