- Isolation and purification of stevioside
Isolation and purification of stevioside content from crude glycosides using methanol, the ratio of crude glycosides and methanol was investigated in the range of 1:2 to 1:7. Determination of stevioside content by HPLC, we obtained the results in Table 4.3.1. Through the results obtained, we can see that the stevioside content fluctuated from 74.3% to 91.4% and the difference between the treatments was statistically significant at the 95% confidence level. Accordingly, at the ratio of 1:2 to 1:4, when the methanol content gradually increased, the content of pure stevioside also increased and between the treatments from 1:2 to 1:4, there was a statistically significant difference at the 95% confidence level. At the ratio of 1:5 to 1:7, although the methanol content gradually increased, the stevioside content did not increase and there was no statistically significant difference between these treatments. Overall, the above results showed that the best ratio for the isolation and purification of stevioside content was 1:5 and the purity of stevioside was 91.1% - 91.5%.
Table 4.3.1 Results of isolation and purification of stevioside
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Ratio of powder and methanol (w/v) Stevioside content (%)
1:2 74.3a ± 0.1528
1:3 74.7 b ±0.0577
1:4 80.8 c ±0.1528
1:5 91.3d ± 0.2000
1:6 91.2d ± 0.1528
1:7 91.4d ± 0.0707
The figures in the table are the average values of 3 repetitions.
Values with the same letter are not statistically different at the confidence level.
95% confidence
- Isolation and purification of rebaudioside A
The filtrate from the stevioside purification experiment was placed in a drying oven to remove methanol and purify rebaudioside A. The filtrate after removing methanol was diluted with water and passed through a polysulfone ultrafiltration membrane. The filtrate was dried to obtain syrup. Ethanol was added to the syrup and the temperature was maintained at 45-50°C for about 30 minutes. During this time, a precipitate was formed, the precipitate was filtered and dried. The sweetenercontent obtained was used to determine rebaudioside A by high-performance liquid chromatography. The purity of Rebaudioside A obtained is shown in Table 4.3.2.
Table 4.3.2 Results of isolation and purification of rebaudioside A
Ratio of syrup and ethanol (w/v) Rebaudiosid A content (%)
1:2 88.5a ± 0.0577
1:3 90.5 b ±0.0577
1:4 90.6 c ±0.0001
1:5 90.8d ± 0.1000
1:6 90.7d ± 0.1155
1:7 90.8d ± 0.0577
The figures in the table are the average values of 3 repetitions.
Values with the same letter are not statistically different at the confidence level.
95% confidence
During the process of isolating and purifying the rebaudiosid A content, the ratio of syrup and ethanol mixture used was limited to about 1:2, 1:3, 1:4, 1:5, 1:6, 1:7. The results presented in Table 4.3.2 show that the rebaudiosid A content ranged from 88.5% to 90.8%, with statistically significant differences between the different treatments at the 95% confidence level. In addition, it was also found that at the ratios of 1:2, 1:3, 1:4, the methanol content increased, and the rebaudiosid A content also increased. At the ratios of 1:5, 1:6, 1:7, the ethanol content increased but the rebaudiosid A ratio did not increase. Therefore, in the process of isolating and purifying rebaudiosid A, the best ratio of powder and ethanol is 1:5 and the purity of rebaudiosid A is 90.7% - 90.9%.
CHAPTER V. CONCLUSION AND RECOMMENDATIONS
5.1 Conclusion
Through this study, the investigation of the appropriate ratio of Stevia leaf powder and water in the extraction process was limited to about 1:20 to 1:25 w/v. The best extraction temperature and time were 75 o C for 240 minutes and 100 o C for 30 minutes. The isolation and purification of stevioside from the crude glycoside mixture was carried out by using methanol for precipitation, the ratio of crude glycoside and methanol was between 1:2 to 1:7 w/v, the best was 1:5 w/v, the stevioside content obtained was in the range of 90.1% -
91.5%. The isolation and purification of rebaudioside A was carried out by using ethanol to precipitate rebaudioside A, the ratio of syrup and ethanol was 1:2 to 1:7 w/v, preferably 1:5 w/v, rebaudioside A with 90.7% - 90.9% purity was obtained. The two sweet substances obtained were non-caloric, non-bitter, applicable in food and pharmaceuticals.
5.2 Recommendations
- Continue to purify the above steviosid and rebaudiosid A compounds to meet the standards of reference standards used in the field of analytical chemistry and testing.
- Continue to isolate the remaining substances rebaudiosid B, C, D, E and Dulcosid
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APPENDIX 1. PHYSICAL AND CHEMICAL ANALYSIS METHODS
High performance liquid chromatography (HPLC)
Analytical HPLC was performed using a HIQSIL 100 C18 column (25 cm x 4.6 mm ID, 5 micron). The column temperature was maintained at 27–28°C and the UV detector was adjusted at 210 nm. The mobile phase was HPLC grade acetonitrile and deionized water (80:20) and the pH was adjusted to 3.0 with 85% phosphoric acid. Samples were filtered through a 0.22 µm filter or equivalent. The injection volume was set to 10 μL with a flow rate of 1 mL/min. Stevioside hydrate (98%) and rebaudioside A (96%), (Sigma–Aldrich) standards were used.
Statistical analysis
The experiments were carried out in triplicate. Analysis of variance (ANOVA) was performed and significant differences (p < 0.05) between mean values were assessed by Duncan, using SPSS version 19.0 (SPSS, Chicago, IL, USA).