I am trying to determine the mw of my polysaccharide sample. the hplc system is agilent 1260 equipped with tskgel pw5000. the mobile phase is de-ion water. the problem is my pectin control (mw ~160K ) RT is 4.5 min, while my 410K dextran standard RT is 6 min. why this happen? is anyother factor contribute to RT other than MW in this case? i am really new in this area, expect experienced person to help. thanks!
Chao: pw5000 column basically follows gel filtration principles, and that standard molecules should be ideally homogeneous, globular in shape and neutrally in charge to show their linear RTs. However, the choice of standard molecules are very limited in meeting the two standards, e.g., pectin are rich in galacturonic acid (negative charges) while dextran is a polysaccharide derived from the condensation of glucose (neutral charge), let alone their shapes and heterogeneities. So, HPLC-pw5000 is only a rough estimate of your polysaccharide sample, and a different method shall be used (e.g., thin-layer chromatography, polysaccharide-gel electrophoresis....). Good luck.
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