Practical one: Partition Coefficient of Fluconazole Drug Discovery & Development BHS005-6 Theory The octanol/buffer partition coefficient (P) is defined as the ratio of a chemical’s concentration in the octanol phase to its concentration in the buffer phase of a two-phase octanol/buffer system. This experiment determines the partition coefficient of fluconazole by measuring the amount extracted into phosphate buffer pH 7.4 and those remaining in octanol at the room temperature. With the aim to increase the accuracy of result, the measurement will be performed at 4 different concentrations of fluconazole in partitioned mixture (octanol – phosphate buffer pH 7.4). Method The stock solution (Solution A) containing 100µg/ml (10mg/100ml) of the fluconazole dissolved in phosphate buffer pH 7.4 was prepared. The Solution B was prepared containing 100µg/ml (10mg/100ml) of the fluconazole dissolved in octanol. Partitioning samples: Into four separating funnels add the following: 25ml phosphate buffer + 5ml Solution B + 20ml octanol25ml phosphate buffer + 10ml Solution B + 15ml octanol25ml phosphate buffer + 15ml Solution B + 10ml octanol25ml phosphate buffer + 20ml Solution B + 5ml octanol Shake gently the separating funnels at frequent intervals for 30-60mins.Put the funnels to the holders and wait for 2-3 hours until the mixture is apparently separated into two clear layers: lower layer is buffer (aq), and upper layer is octanol (org). While you are waiting, please perform the practical two! Pour carefully the buffer phase into a 50ml tube using the funnel’s valve, and pour the octanol phase into another 50ml tube.Measure absorbance of the buffer phase (use phosphate buffer as blank) and octanol phase (use octanol as blank) portions at λmax 260nm using UV-visible spectrophotometer.Repeat the procedures 3 and 4 for funnels 2-4. Data for calculation: Absorbance (PBS phase) Funnel A: 0.034 Funnel B: 0.066 Funnel C: 0.123 Funnel D: 0.133 Absorbance (Octanol phase) Funnel A: 0.056 Funnel B: 0.078 Funnel C: 0.125 Funnel D: 0.156 Calibration curves: 1. Using the Solution A, prepare a range of calibration standards each (2ml) containing 5, 10, 20, 40, 60, 80 and 100 μg/ml of the fluconazole in phosphate buffer pH 7.4 (see the tables below). Read the absorbance of each standard at the λmax 260nm using phosphate buffer pH 7.4 as the blank. Record the readings in the table. Use these readings to construct a calibration curve of absorbance versus concentration for the fluconazole in phosphate buffer. Concentrations of fluconazole in PBS Phase (µg/ml)Volume of Solution A(ml)Volume of phosphate buffer (ml)Absorbance at λmax 260nm50.11.90.006100.21.80.017200.41.60.040400.81.20.094601.20.80.145801.60.40.1881002.000.240 2. Using the Solution B, prepare a range of calibration standards each (2ml) containing 5, 10, 20, 40, 60, 80 and 100 μg/ml of the fluconazole in Octanol (see the tables below). Read the absorbance of each standard at the λmax 260nm using phosphate buffer pH 7.4 as the blank. Record the readings in the table. Use these readings to construct a calibration curve of absorbance versus concentration for the fluconazole in phosphate buffer. Concentrations of fluconazole in Octanol Phase (µg/ml)Volume of Solution B(ml)Volume of Octanol (ml)Absorbance at λmax 260nm50.11.90.016100.21.80.025200.41.60.045400.81.20.078601.20.80.170801.60.40.2131002.000.256 Calculations: Calculate the concentration of fluconazole in the buffer (Caq) and octanol phase (Corg) respectively, for each composition of the partitioned mixture from the standard curve.Calculate the partition coefficient P(fluconazole) at each of the partitioned mixture, P = Corg/Caq Express the average value of the partition coefficient P from four experiments, and compare it with values from literatures.
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