## 3 Results and Discussion
### Optimisation of the Chromatographic Conditions  
When optimizing a liquid chromatographic (LC) separation, there are several variables to consider, including analytical column chemistry and geometry, mobile phase composition, flow rate, and temperature. However, as far as analysis of thoroughly studied analytes is concerned, the critical previous findings should be adopted to avoid unnecessary repetitions in experimental design. Considering the earlier studies, C18 is the stationary phase that is most commonly used in the LC separation of OA and UA [8; 15; 16; 17; 18; 19; 20; 26; 38]. This is hardly surprising, as RP-18 columns are also the  
Figure 2: Content of OA, UA, and polyphenols (TPC) in the leaves of _A. uva__ursi_ as calculated per dry weight basis in extracts obtained with different solvents: S1, _n_-hexane (3 \(\times\) 30 mL); S2, chloroform (3 \(\times\) 30 mL); S3, chloroform-methanol 1:1 (3 \(\times\) 30 mL); S4, chloroform (2 \(\times\) 30 mL) + chloroform-methanol 1:1 (30 mL); S5, methanol (3 \(\times\) 30 mL); and S6, _n_-butanol (3 \(\times\) 30 mL). Values are presented as means (_n_ = 3) \(\pm\) standard deviation (error bars). Different superscripts (capitals, A–E) for each analyte indicate significant differences in the means at \(p\) < 0.05.  
most popular ones when it comes to plant material standardisation in general and, therefore, easily available in laboratories performing such studies. Moreover, in a study conducted by [PERSON] et al. [15], where different column chemistries (C8, C18, CN, phenyl) were compared, only C18-based stationary phases exhibited selectivity adequate for effective separation of both compounds. The best resolution was achieved on polymeric C18 bonded phase (polycyclic aromatic hydrocarbons, PAH), however, standard C18 phases gave superior peak shape and symmetry and, therefore, lower detection limits. We decided, thus, to use classic RP-18 UHPLC columns in further optimisation. Among of couple of columns assessed in the preliminary experiments, Zorbax Eclipse XDB C18 (100 mm \(\times\) 3.0 mm, 1.8 \(\upmu\)m) column was proven to be the most suitable for the task. The internal column diameter of 3.0 mm was chosen as less susceptible to the plugging problems than standard 2.1 mm UHPLC columns, especially for the plant samples with complex lipoidal matrices, such as the target extracts. With only two analytes to separate, a column shorter than 100 mm would seem to be a better choice as it could offer a significant reduction of retention times. However, the efficiency of 50 mm columns tested was strongly insufficient to separate so closely-related chemicals as OA and UA, which eluted then in one band independently from the analytical conditions. The mobile phase used to conduct the analysis is another important factor affecting the separation process. According to the literature [16; 18; 19; 20; 21; 28; 39; 40; 41; 42], isocratic binary elution systems containing acetonitrile or methanol are preferred in analysis of native triterpen acids as preventing baseline drifts often observed in gradient modes at detection wavelengths set below 220 nm, the region which the both organic modifiers exhibit also the highest absorption in. In the corresponding mobile phases, the concentrations of both solvents are similar, usually 80%-95% (\(v/v\)) for methanol and 85%-90% (\(v/v\)) for acetonitrile. In general, methanol-containing phases, due to the lower eluotropic strength, tend to give longer retention times, but better resolution. Our previous comparative studies [16] confirmed this finding and 90% (\(v/v\)) aqueous methanol with 1% (\(w/v\)) of orthophosphoric acid was demonstrated to be the optimal eluent for OA and UA separation. As it fits in the above-mentioned range of the most selective mobile phase compositions, it has been selected for the present analysis. The flow rate and temperature are further factors that can easily allow changing the separation efficiency.