## 3 Results and Discussion  
### Optimization of Fluent Composition  
Picein, triandrin, fructose, and glucose standard compounds were used for HILIC optimization with the Phenomenex Luna(r) Omega Sugar column. This column has proved to be useful in carbohydrate separations [16,17] and was thus selected for the eluent composition optimization studies. The use of an ELSD detector for non-absorbing carbohydrates allows for the use of gradient elution, but our initial aim was to develop an isocratic method that could also be used with a refractive index detector wherever the ELSD detector is not available. Already, the preliminary trials showed that the separation of picein and triandrin from each other is the most challenging task. The method optimization for isocratic separation was started by testing the eluent composition of 80/20 (_v/v_; all the elements prepared in this work were _v/v_) ACN/water, which is recommended by the column manufacturer. Increasing the ACN content to 83% resulted in the near baseline separation between picein and triandrin (Figure 1). This preliminary method (ACN/water 83/17) was recently used for the screening of separated fractions from willow bark extract [18]. Further increase in ACN content in the eluent increased the resolution between picein and triadrin, but the retention time of the monosaccharides increased also, and the total analysis time was prolonged. Additionally, the glucose peak area was diminished drastically when the ACN content was above 83%. Figure 1: ELSD signals for picein (\(t_{\text{r}}\) = 3.45 and 3.49 min), triandrin (\(t_{\text{r}}\) = 3.66 and 3.77 min), fructose (\(t_{\text{r}}\) = 7.11 and 8.09 min), and glucose (\(t_{\text{r}}\) = 8.41 and 9.80 min) using the eluents: A = water, B = ACN (A/B = 17/83) and A = 30% MeOH in water, B = ACN (A/B = 20/80). The flow-rate was 1.3 mL/min and the injection volume 5 \(\upmu\)L. The use of MeOH in aqueous eluent clearly increased the resolution between the picein and triandrin, while the retention time of fructose and glucose decreased. The column used was Phenomenex Luna Omega Sugar (please see Materials and Methods section for further details). It has been reported that HILIC separation can be enhanced by adding organic modifiers to the eluent. In some cases, it is possible to replace water with organic protic solvent [19; 20]. To improve the resolution between picein and triandrin, we decided to test different organic modifiers, namely 2-propanol, tetrahydrofuran, and methanol, in the water phase (pump A being a mixture of organic modifier and water, and pump B always consisting of 100% acetonitrile). In addition, the effect of pH (pH 4.5) was tested, but mild lowering of the pH had no effect to the retention of the analytes. The preliminary trials with organic modifiers showed that 2-propanol and tetrahydrofuran did not increase the resolution between picein and triandrin (results not shown). Methanol, however, seemed promising, and thus a mixture of picein and triandrin as well as mixture of fructose and glucose were separated using different methanol contents in the eluent A (20%, 30%, 40%, 50%, 60%, and 80% of methanol in water; eluent B was ACN and A/B = 20/80). The corresponding ACN/water/MeOH ratios are 80/16/4, 80/14/6, 80/12/8, 80/10/10, 80/8/12, and 80/4/16. As shown in Figures 1 and 2, the resolution between picein and triandrin increased drastically when methanol content was increased. A resolution close to 1.5 was achieved when 20% methanol was added in the water phase. In addition, the other two compounds, fructose and glucose, eluted earlier when methanol was added to eluent A. The evolution of the retention time of the latest eluting component, glucose, is also presented in Figure 2. The retention time for glucose was below 9 min when a low concentration of methanol (20% and 30%) was used, but increased to over 15 min when 80% methanol was added in eluent A.