## 3 Results and Discussion
### Flash Chromatography and HPTLC
Fractions 13-19 were combined, and pure oleanolic acid was obtained with a 9.08% \(w/w\) concentration from 1.2 g of crude extract. Thus, flash chromatography of this sample provided 109 mg of pure oleanolic acid from 10 g of plant material (10.9 mg/g) within an hour. This is a significantly higher yield than has been previously reported. Previous work reported that optimized ultrasonic assisted extraction and then centrifugation isolation, yielded 3.6 mg/g of oleanolic acid [25], compared to solid-liquid extraction (maceration) that yielded 0.4-0.5 mg/g [19] and 0.015 mg/g [26]. Solid-liquid extraction (centrifugation) yielded 0.185-0.565 mg/g [27], ultrasonic assisted extraction yielded 0.838-1.003 mg/g [28], while solid-liquid, then ultrasonic-assisted extraction yielded 3.4 mg/g of oleanolic acid [29]. Please note that in these studies, oleanolic and maslinic acid were not isolated from the extracts. They were only quantified with either gas liquid or high performance liquid chromatography. In our work, the identity and purity of the isolated oleanolic acid compound was determined by ATR-IR spectroscopy and NMR. The presence of pure oleanolic acid was confirmed. However, the presence of maslinic acid could not be confirmed with NMR in fractions 27-30, due to the interference from other compounds that coeluted in these fractions. In order to selectively remove more polar compounds (lower \(R_{\rm F}\) values on the HPTLC plate) from the ethyl acetate extract while retaining others, water was used as the wash solvent. Aqueous washing is used to remove water soluble compounds from organic phase, since maslinic acid will be dissolved in the organic layer. The goal was to remove  
Figure 3: (**a**) Separation of ethyl acetate extract and (**b**) HPTLC assessment of collected fractions (13–19 high purity oleanolic acid and 27–30 low purity maslinic acid). interferents present in the fractions containing maslinic acid. Olive leaves were extracted using a mixture of ethyl acetate and water (1:1), with only the ethyl acetate phase separated using flash chromatography. Furthermore, 10% of acetic acid was added to the ethyl acetate mobile phase to improve the separation efficiency and peak shape of ionizable organic acids. When separating acidic compounds, adding a small amount of a volatile organic acid to the solvent system will minimize any secondary H-bonding interactions with the silica by keeping the acids protonated. Oleanolic acid started eluting at 8% of ethyl acetate/acetic acid (mobile phase D), while maslinic acid started eluting at 31% ethyl acetate/acetic acid (Figure 4). Maslinic acid was present in a pure form in the fractions 18-20. In a single chromatographic run, a yield of 6.25 mg/g pure maslinic acid was obtained from the crude extract. This is comparable to previously reported maslinic acid yields of 9.2 mg/g using optimized ultrasonic assisted extraction and then centrifugation [25], and 8.5 mg/g using solid-liquid, then ultrasonic-assisted extraction [29]. Moreover, our yield was significantly higher than previously reported yields of 1.2-1.5 mg/g [19] and 0.034 mg/g [26] using solid-liquid extraction (maceration), and yields of 0.295-0.904 mg/g using solid-liquid extraction (centrifugation) [27] and ultrasonic assisted extraction [28]. The presence of maslinic acid in fractions 18-20 was confirmed with ATR-FTIR (Figure 5b) and NMR. The ATR-FTIR spectrum of the isolated acids show the presence of OH (broad peak at around 3400 cm\({}^{-1}\)) along with a characteristic secondary alo  
Figure 4: (**a**) Separation of ethyl acetate fraction from the water-ethyl acetate extract and (**b**) HPTLC assessment of collected fractions (9–13 oleanolic acid and 18–20 maslinic acid). hol peak (1092 cm\({}^{-1}\)) (Figure 5). Strong peaks of C-H in the methyl stretching region (2850\(-\)3000 cm\({}^{-1}\)) of methyl and cyclic alkane can be attributed to terminal methylene and methyl groups. A strong sharp peak of carbonyl from a carboxylic acid (C=O, COOH) was observed at around 1690 cm\({}^{-1}\)[30].