The standard method for analyzing amino acids by HPLC is either pre- or post-column derivation or detection by fluorescence because the acids lack suitable chromophores. This approach yields excellent sensitivity, but the derivatization process is time consuming and can introduce variability between each test run. ELSD excels at analyzing nonchromophoric compounds and eliminates the need for derivatization.
ELSD works by making a light-scattering measurement of target analytes that have been dried of mobile phase via evaporation. The detector produces stable baselines during gradient elution chromatography, and its response is independent of the spectral properties of the analyte and solvent.
Polyethylene glycol is difficult to analyze with a UV detector because it has little UV absorbance. Usually, an RI detector is used to analyze this compound. However, achieving separation with isocratic elution while using an RI detector is limited when separating low-molecular-weight polyethylene glycol to the polymerization level using a reversed-phase mode.
Figure 2 shows an example of results obtained when researchers subjected polyethylene glycol 1000 to gradient elution using the reverse-phase mode. Detection was performed with ELSD. The graph shows how using ELSD makes it possible to separate polyethylene glycol according to the polymerization degree with a stable baseline using the gradient elution method.
According to the glucose polymerization degree, the normal-phase mode demonstrates superior selectivity in the separation of oligosaccharides. However, the time needed for elution increases as the number of glucose units increases.
With an RI detector, gradient elution cannot be performed. So, analysis must be carried out repeatedly using mobile-phase compositions that are suitable for the separation of each polymerization degree. Using an ELSD makes it possible to identify maltooligosaccharides with glucose polymerization degrees of up to about 20. Figure 3 shows an example of results from subjecting maltooligosaccharides to gradient elution using the normal-phase mode.