I am trying to quantify glycerol (CH2OH-CHOH-CH2OH; MW - 92.1 g/gmol), 3-Hydroxypropionic acid (3-HP; CH2OH-CH2-COOH; 90.08 g/gmol) and 2,4-Dihydroxybutyric acid (2,4-DHB; CH2OH-CH2-CHOH-COOH; 120.08 g/gmol) using HPLC. Glycerol can be detected by RID only whereas 3-HP, 2,4-DHB can be detected by both RID and PDA. I have used IEC and RPC with the following mobile phases:

• 0.5 mM H2SO4 - isocratic at varying temperatures (50-65 C for IEC and 40 C for RPC)
• 0 to 0.5 mM H2SO4 - gradient elution at varying temperatures (50-65 C for IEC and 40 C for RPC)
• 5 mM H2SO4 - isocratic at varying temperatures (50-65 C for IEC and 40 C for RPC)
• 0.01% formic acid - isocratic at varying temperatures (50-65 C for IEC).

All these compounds co-elute at the same time and I could not get three distinct peaks. Also, the peak of 2,4-DHB is broad in all these conditions. Can someone suggest me a suitable mobile phase or a suitable column to separate and quantify all these metabolites!