3.1.2. High-Performance Liquid Chromatography

High performance liquid chromatography (HPLC) is the usual methodology for the analyses of DGs with UV, PDA and MS as the most common detectors [56,57]. Several methods have been developed to separate DGs from complex S. rebaudiana extract mixtures. Since the most common aglycone is steviol, novel potential sugar-substitutes could be found based on the number of monosaccharides, type of sugar units and their arrangements. These slight structural differences often cause co-elution of DGs in a single HPLC method.

An overview of the advantages and disadvantages of some HPLC methods using different stationary phases (HILIC, NH₂, RP-C18, Sepaxdiol, Synergi, silica gel) is given herein, although detailed information can be found [40,58]. Several purified DGs containing different numbers of sugars and linkage arrangements to steviol were analyzed for this purpose. Interestingly, in all the methods reported, acetonitrile: water acidified elution gradients were described for the analyses of DGs [40,58]. The use of a common binary mobile phase is a practical methodology for the analyses of DGs with simple changes in the column adsorbent chemistry (Figure 4). There is no change in selectivity between the silica gel and the amino method. In silica gel, stevioside and rebaudioside C coelute while in an amino column, a good separation is achieved, but rubusoside and steviolbioside are better resolved in silica than in amino column. When a Synergi column is utilized, there is a big room for the separation of very polar steviol glycosides (more polar than rebaudioside O) and also between rebaudiosides A and N. There is no change in selectivity if HILIC column is compared with Sepax-diol one, although sharper peaks are gotten with a Sepax-diol column. The appropriate combination of HPLC methods which best resolved components in the range of retention times of interest give a good understanding of the DGs in the extract and chromatography fractions and provide guidance for further preparative methods to be used.

Selected HPLC methods used for the analysis of steviol glycosides: silica method (A), RP-C18 (B), Synergi (C), amino (D), HILIC (E) and Sepax-diol (F).