Alternative Columns to C18 for EPA TO-11A Analytes

[mattmullaney]

Hello,

I've explored five different columns--with various types of polar-embedded phases--in an effort to separate the 15 DNPH derivatives comprising the EPA TO-11A set of analytes. Below is a link to a .pdf of the EPA TO-11A method.

www.epa.gov/ttnamti1/files/ambient/airtox/to-11ar.pdf

Has anyone looked at using HILIC, porous graphitic carbon, porous graphitic carbon-clad zirconia or normal-phase HPLC to separate the E- and Z-isomers created in the formation of the 15 DNPH derivatives as they are collected on-cartridge?

My thanks for your help, in advance.

[danko]

Then there is this possibility of another issue than the column.
Equipment, mobile phase and other conditions discrepancy.

Best regards

[mattmullaney]

My thanks--your point is well-taken. Please, though, consider the following article:

"Isomerization of aldehyde-2,4-dinitrophenylhydrazone derivatives and validation of high-performance liquid chromatographic analysis"

Shigehisa Uchiyama, Masanori Ando, Shohei Aoyagi; Journal of Chromatography A, Volume 996, Issues 1–2, 9 May 2003, Pages 95–102.

Here is a link to the Abstract of the reference noted above from Elseveir ScienceDirect:

http://www.sciencedirect.com/science/ar ... 7303005429

The authors discuss the formation of E/Z isomers of DNPH-carbonyl derivatives (in particular where the carbonyls compounds are short-chain aliphatic aldehydes) in acid solution. It is possible for this isomerism to occur in samples gathered on DNPH-saturated silica cartridges, impinger solutions or in standard solutions. This E/Z isomerism seems to be effected by trace amounts of acid (perhaps phosphoric or perchloric) used to catalyze the formation of the DNPH adducts.

Uchiyama describes isomerism of ketones (such as methyl ethyl ketone) and unsaturated carbonyls (such as crotonaldehyde, benzaldehyde, acrolein...) in other papers he has co-authored.

While the polar-embedded phases provide partial to complete resolution of the E/Z isomers from themselves (for example, E- and Z-DNPH-hexaldehyde), I have not found any polar-embedded phases that completely resolve All of the E/Z isomers from other derivatized analytes in the set of the 15 listed in EPA TO-11A.

Application Note 347 from Supelco (t005347, document G003341) shows a good example of what I am talking about--without meaning to "pick on" the RP-Amide column in the least. Note in particular the co-elutions of E-DNPH-benzaldehyde with Z-DNPH-isovaleraldehyde and E-DNPH-isovaleraldehye with Z-DNPH-valeraldehyde. These co-elutions to harm to method specificity...

As an aside, the majority of C18 columns provide little or no separation between E- and Z-DNPH carbonyl isomers from themselves...for example, E- and Z-DNPH acetaldehyde(s)...thus, if there are differences in the ratio(s) of the E- to Z- forms of DNPH-acetaldehyde, DNPH-propionaldehyde, DNPH-butyraldehyde, DNPH-isovaleraldehye, DNPH-valeraldehyde and/or DNPH-hexaldehyde, there is the probability of analytical error due to differences in the molar absorptivities (extinction coefficients) of the E- and Z-isomers. There is no easy way that I can see to determine the molar absorptivities for Z-isomers here as these species are not stable in the solid state (possibly they could be inferred from study of the formation of the Z- isomers from their respective E-isomers, but this kind of thing would take a while to execute).

My thanks!