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HPLC CERTIFICATE OF ANALYSIS (COD) FOR A.I. CONTENT

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HPLC CERTIFICATE OF ANALYSIS (COD) FOR A.I. CONTENT

  1. 1. CERTIFICATE OF ANALYSIS (COA) Strategic Business Unit (SBU): ECOFRIENDLY NATURAL BIO-PESTICIDE Extraction date: 23th March 2006 Analysis date: 24th March 2006 EXTRACTION PROCESS1) Method of extraction - Normal Soaking Extraction (NSE)2) Solvent of extraction - Acetone [Purity of 99.8 % (v/v)]2) Solvent-to-solid ratio - 10 ml/g3) Procurement of sample - Rubber and palm oil plantations estate at Kota Johor Lama, Johor4) Particles size - Coarse size (≅ 2.0 mm to 5.0 mm in diameter) + fine size (≅ 2.0 mm in diameter)5) Weight of sample (dried roots) - ≅ 140 g6) Sample ID: Standard of rotenone (SIGMA-Aldrich™, 95-98 %); Cstd = 0.0256 mg/ml - Dilution 1/10 ER1 - Dilution 1/100 - Before concentration ER2 - Dilution 1/100 - After concentration7) Time of extraction - 24 hours8) Extraction temperature - Ambient room temperature (26 ± 2 0C) CONCENTRATION PROCESS1) Time of total solvent evaporation ≅ 30 mins2) Operating temperature (Rotary evaporator: Heidolph-Laborata 4001) - 40 0C3) Vacuum pressure - 0.3 mbar (Vacuumbrand GmBH+CO, 2.20 m3/hour)4) Solvent recovery - ≅ 80 % of the total volume Liquid Crude Extract (LCE) QUANTITATIVE ANALYSIS (EXTERNAL STANDARD METHOD)1) Liquid chromatographic column - µBondapak™ C18 125Å 10µm, 3.9 mm × 150 mm RP-HPLC Column2) RP-HPLC parameters setting based on Baron and Freudenthal (1976): Parameters Setting Column temperature (0C) Ambient Flow rate 0.4 ml/min UV wavelength (λ) 294 nm Injection volume 10 µl Amplitude Unit Full Scale (AUFS) 2.0Prepared and analyzed by:------------------------------------SAIFUL IRWAN ZUBAIRIB. Eng. Chemical-Bioprocess (Hons.) UTM, M. Eng. Bioprocess, UTMResearch Officer (QA41)Head of Ecofriendly Natural Bio-pesticide SBU,Chemical Engineering Pilot Plant (CEPP),Universiti Teknologi Malaysia,81310 UTM Skudai, Johor.Tel. no.: +607-5532595Fax no.: +607-5569706e-mail: saiful@cepp.utm.my OR saiful-z@utm.my
  2. 2. Figure A-1 Chromatogram of rotenone standard (SIGMA-Aldrich™, 95-98 %)Standard of rotenone (SIGMA-Aldrich™; purity of 95-98 %):1) Concentration prepared: 0.256 mg/ml (6.4 mg rotenone dissolves in 25 ml of ACN:H20 [60:40])2) Diluted to [1/10]: 0.256/10 = 0.0256 mg/ml3) Rotenone standard concentration (Cstd) [10.68 min] = 0.0256 mg/ml4) Peak area (A) = 3018.995 [mV.s]5) Sensitivity factor (SF) = (A)/(Cstd) = 3018.995/0.0256 = 117,929.49 [mV.s. ml/mg]
  3. 3. Figure A-2 Chromatogram of ER1 of Liquid Crude Extract (LCE) before concentrationYield and concentration of rotenone in the Liquid Crude Extract (LCE):1) Rotenone peak area (Arotenone) [10.61 min] = 2641.68 [mV.s]2) Concentration of rotenone (Crotenone) = (Arotenone)/SF = 2641.68/117,929.49 = 0.022 mg/ml3) Dilution factor; DF = Flask volume/pipette volume = 100/1∴Actual concentration of rotenone = 0.022 mg/ml × 100/1 = 2.24 mg/ml4) Yield of rotenone in dried roots, mg = 2.24 mg/ml × 700 ml (volume of LCE) = 1,568.03 mg5) Yield of rotenone in dried roots, % (w/w) = 1,568.03 mg/100 g × 100 % = 1.57 % (w/w)
  4. 4. Figure A-3 Chromatogram of ER2 of Concentrated Liquid Crude Extract (CLCE)Yield and concentration of rotenone in the Concentrated Liquid Crude Extract (CLCE):1) Rotenone peak area (Arotenone) [10.64 min] = 13,580.63 [mV.s]2) Concentration of rotenone (Crotenone) = (Arotenone)/SF = 13,580.63/117,929.49 = 0.115 mg/ml3) Dilution factor; DF = Flask volume/pipette volume = 100/1∴Actual concentration of rotenone = 0.115 mg/ml × 100/1 = 11.52 mg/ml4) Yield of rotenone in dried roots, mg = 11.52 mg/ml × 100 ml (volume of CLCE) = 1,152 mg5) Yield of rotenone in dried roots, % (w/w) = 1,152 mg/100 g × 100 % = 1.15 % (w/w)

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