TLC of Oleuropein and other compounds in olive mill waste water lab write up

Methodology: For extracting OMW: 1. 2. 3. 4. 5. 6. Add 25 mg of OMW to a test tube Add 1.25 mL of H20 and 2.5 mL of ethyl acetate Place on rotary spinner for 5 minutes Centrifuge for 5 minutes. Retrieve ethyl acetate phase. Perform TLC analyses using revelator and analyse under fluorescence. For TLC: One TLC chamber contained mobile phase amyle acetate/diethyl ether/ acetic acid in a 7/2/1 ratio. The other chamber in a 5/4/1 ratio. Revelators were made by: Spray 1: 20% sodium carbonate in H20 = 10g in 50m2 Spray 2: Dilute freshly 1/3 folin/H20. Added 15mL of H20 to 5mL of folin. Had TLC comparing to a calibration curve of 50 mg/L, 100, 200, 400 and 600 of oleacein. Also did TLC comparing to standards of Hydroxytyrosol, Tyrosol, Oleuropein, Oleacein and caffeigue. Figure 1 shows how to make oleacein calibration curve standards: Purple highlight = amount taken from previous solution. Red highlight = amount of MeOH/H20 (50:50) solution added. Stock Oleacein Solution 900 µ/L g/L 50 µg/L 600 µg/L 50 µL + 850 µL 400 µg/L = 900 µL 200 µL + 250 µL 400 µL + 200 µL = 600 µL = 450 µL 100 µg/L 100 µL + 300 µL = 400 µL 200 µg/L 150 µL + 300 µL = 450 µL Figure 1. Oleacein calibration method Results: TLC Results for Samples (Presence of Compounds) • Noted whether each sample had Oleacein, Hydroxytyrosol, Tyrosol, Oleuropein, or Caffeic acid present. Quantitative Results for Oleacein Content • Measured using MS, UV, and TLC methods. 5/4/1 mobile phase Rf Oleacein = 0.71 Rf Hydroxytyrosol = 0.66 Rf tyrosol = 0.75 Rf Oleuropein = 0.1 Rf Cafeic acid = 0.77 7/2/1 mobile phase Rf Oleacein = 0.69 Rf Hydroxytyrosol = 0.59 Rf tyrosol = 0.70 Rf Oleuropein = 0.09 Rf Cafeic acid = 0.72 TLC 5/4/1 Marked X if present Sample B2,5V19 Oleacein Hydroxytyrosol Tyrosol Oleuropein Cafeic acid B3N19 A2,5V19 A3V19 L3V18 L3N18 L3V19 L3N19 O3V18 O3N18 O3V19 O3N19 P3V18 P3N18 P2,5V19 P2,5N19 P3V19 P3I19 TLC 7/2/1 Sample B2,5V19 B3N19 A2,5V19 A3V19 L3V18 L3N18 L3V19 L3N19 O3V18 O3N18 O3V19 O3N19 P3V18 P3N18 P2,5V19 P2,5N19 P3V19 P3I19 X X X X X X X X X X X X X X X X Oleacein Hydroxytyrosol Tyrosol X X X X X X X X X X X X X X X X X X X X Oleuropein Cafeic acid Method Sample B2,5V19 B3N19 A2,5V19 A3V19 L3V18 L3N18 L3V19 L3N19 O3V18 O3N18 O3V19 O3N19 P3V18 P3N18 P2,5V19 P2,5N19 P3V19 P3I19 MS UV TLC 5/4/1 Oleacein cont. (mg/g) SD (mg/g) Oleacein cont. (mg/g) SD (mg/g) Oleacein cont. (mg/g) Diff (mg/g) MS 9.47 0.53 12.2 0.5 [5-10] 4.47 0.41 4.12 0.24