Comparison of determination of sugar-PMP derivatives by two different stationary phases and two HPLC detectors: C18 vs. amide columns and DAD vs. ELSD

Reducing sugars, especially aldoses with their ring structures, have strong reducibility to react with 1-phenyl-3-methyl-5-pyrazolone (PMP) to form sugar-PMP derivatives detected by ultraviolet (UV) detector at 248 nm. In this study, several carbohydrates and some of their alcohols, including 1). seven main monosaccharides, 2). three common disaccharides, 3). two oligosaccharides, and 4). some sweeteners like sugar alcohols were investigated for their reactivities with PMP. Analytes were separated chromatographically by two common high performance liquid chromatography (HPLC) columns and subsequently detected by two tandem detectors. Results pointed out that C18 column had a stronger capacity to separate the reducing sugar-PMP derivatives rather than the sugar themselves. While amide column could only effectively separate the original sugars. These phenomena demonstrated that polarity of reducing sugars decreased after their PMP derivatization. Moreover, both diode array detector (DAD) and evaporative light scattering detector (ELSD) were able to detect the reducing sugar-PMP derivatives, including the PMP-derivative of the oligosaccharide hydrolysate of chitosan, though the DAD exhibits a higher sensitivity than ELSD. In conclusion, carbohydrates themselves are more likely to be efficiently separated by an amide column and detected by ELSD, while DAD combined with a C18 column has more power to determine carbohydrates derivatives.