Analytes: phosphorylated carbohydrate (inositol phosphate)

These analytes having little pka difference and different number of negetive charge functional groups. ( we have only two standsrds, IP3 and IP6). These two are eluting almost at same retention time (chromatographic conditions: scherzo multimode c18 column, mobile phase A is water+methanol 80:20 and B is 50 mM ammoinum formate +50mM ammonium hydroxide + methanol 40:40:20,ph8.4, detector ELSD). With minimum flow rate (0.150 ), we got resolution about only 0.35 min only.

In literature, It was developed by using ion pairing agent such as tetra butyl ammonium hydroxide and C18 column. Here, column is modifing into SAX property by mobile phase layer.

Multi mode column is also works as similar without use of ion pairing agent. But we are not able to get the separation as achived earlier from the literature.

Please give me the suggestions for  the development of this method.

And alsothe appropriate retention mechanism and chromatographic conditions to separate these compounds.

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