Hello,

I am trying to analyse pectin ( polygalacturonic acid ) by saponifying with NaOH and then HPLC using C18 4.6x15mm column and water : H2SO4.
Since this is the only column in my possession, does anybody have any other methods for analysis as the peak elutes very rapidly.

Thanks for any suggestions!

I'm not sure it can be done on C18 without derivitization. It may be simply too polar, especially in the saponified form.
Two observations: 1)you may be hydrolyzing the bonded phase of your column with the sulfuric acid in the mobile phase; 2)C18 ligands need a certain amount of organic modifier to solvate them. 100% aqueous mobile phases cause them to collapse like greasy hair in the shower.

You can try ion pairing method to increase the retention, 5 mM tetrabutylammonium phosphate, 20 mM phosphate buffer at pH 6, 10% acetonitrile can be a good starting point.

Warning: The column equilibration can be lenghty (overnight) and the column will never be the same.

Do not forget neutralize your samples before injection, as a few ul of concentrated NaOH solution injected will destroy your column

I have also heard that columns are sometimes "never the same" after the aliphatic chain collapse that can be induced by pure aqueous phases. Keep this in mind if you do not find a successful method (i.e., a new column might work if cared for properly) and if you do develop a method and purchase a new column (it will not have been subjected to this collapse and may behave differently). I generally don't invest large amounts of time developing methods on columns which are not new.