## 3 Results and Discussion
### Optimization of Fluent Composition
The peak area of glucose decreased as a function of increasing methanol content, and the peak became almost undetectable when the methanol content was 80%. Solubility issues might explain the low peak area of glucose when high organic content in the eluent was used. Glucose is known to be sparingly soluble in alcohols such as methanol and ethanol [21]. According to the graph (Figure 2) in which the resolution between picein and triandrin, retention time of glucose, and peak area of glucose are presented as a function of methanol content in eluent A, 30% of methanol in the aqueous phase was considered the most optimal for the separation of phenolic glycosides and monosaccharides. An even lower methanol content than 30% would have separated the standard compounds well, but because the method is aimed at the separation of willow bark extracts with tens of different compounds, conditions that enabled baseline separation between the two phenolic glycosides were chosen for the further studies. As discussed in the literature [14], the retention mechanism of HILIC is not unambiguously understood; therefore, unambiguous explanation of the effect of methanol cannot be given here. In HILIC separations, water is the strongest eluent while acetonitrile is the most common weak eluent. Methanol is slightly weaker than water [22]. In HILIC, it is  
Figure 2: Optimization of eluent conditions (detection ELSD). We chose 30% of MeOH in eluent A (eluent B was ACN, A/B = 20/80) for further studies. assumed that water forms a thin layer on the silica surface of the column stationary phase. It can also be speculated that methanol would be part of the aqueous layer that forms on top of the silica surface. The presence of methanol in the water layer might explain the behavior observed here. The aromatic compounds can have more interactions with a column stationary phase when part of the water is replaced with an organic modifier. Thus, the addition of methanol increases the resolution between the two aromatic compounds. Monosaccharides likely have interactions with water, and when methanol is embedded in the water layer, monosaccharides have less interaction with the water layer and thus the retention times of fructose and glucose decrease [22]. The ternary eluent consisting of ACN, water, and methanol seems promising mobile phase in HILIC, and could also be tested for other applications. To date, the eluents containing three components have not been widely used in HILIC separations. As a comparison with the Phenomenex Luna(r) Omega Sugar column, another HILIC column, Waters XBridge(r) Amide, was tested with the optimized method to see if similar retention behavior was observed for picein, triandrin, fructose, and glucose standard compounds. Only partial separation between picein and triandrin was obtained (results not shown). The difference between the retention behaviors likely arose from differences in the stationary phases. Both columns contain amide groups, but exact knowledge of the stationary phases is not available. Apparently, the Luna(r) Omega Sugar column stationary phase also contains polyols. All of the samples were initially dissolved in water and diluted with ACN (1:2). The addition of the weaker solvent ACN was necessary in order to avoid peak splitting. Solvent mismatch is typical, especially in HILIC where the amount of organic solvent in eluent is high [23]. Pure organic solvent is rarely the best option, however, if the analytes are highly polar. In our case, peak splitting was observed for phenolic glycosides when the injection solvent was pure water. After adding 50% of ACN, splitting was not observed.