Phd Practical Assay of Tetracycline PDF

Assay of Tetracycline by calibration curve method To use the calibration curve technique, several standards containing exactly known concentrations of the analyte are introduced into instrument, and the instrumental response is recorded, ordinarily this response is corrected for instrument output o...

Assay of Tetracycline by calibration curve method To use the calibration curve technique, several standards containing exactly known concentrations of the analyte are introduced into instrument, and the instrumental response is recorded, ordinarily this response is corrected for instrument output obtained with a blank, ideally, the blank contains all of the components of the original sample except for the analyte, always starting with the lowest concentration, from these absorbance values, the resulting data are then plotted to give a graph of corrected instrument response versus analyte concentration, and this graph (calibration curve) in turn can be used to find the concentration of an unknown. Colorimetric methods for tetracyclines evaluation There are 3 colorimetric methods for tetracyclines: 1-FeCl3methods 2-Acidic method 3-Basic method Ferric chloride method for tetracyclines assessment: Objective: determine the tetracycline content of a sample Principle: this method is a general method and is applicable to determine any of these antibiotics. All contain the same phenolic hydroxyl groups and they all react with ferric chloride in acidic solution to give an orange-brown color. The method may be used to determine the tetracycline sin such products as capsules, ointments and tablets.  Tetracycline colored chelate (Orange brown) Stable for 2 hrs. Procedure Note: Step 1 & 2 already prepared 3-Preparation of five standard solutions from stock solution and a blank as in the following table: Table 1: Standard solution Std1 Std 2 Std 3 Std 4 Std 5 Blank (Tet) Stock sol 1 ml 3 ml 5 ml 7 ml 9 ml ----- HCl sol (0.01N) 9 ml 7 ml 5 ml 3 ml 1 ml 10 ml FeCl3 sol 0.05 % 10 ml 10 ml 10 ml 10 ml 10 ml 10 ml Volumetric flask 50 ml 50 ml 50 ml 50 ml 50 ml 50 ml 50 ml (D.W) Mix and stand at room temperature for 10 min. orange-brown color will appear, measure the absorbance at λ max 490 nm. concentration of standard solutions C1x v1= C2x v2 1000 x 1=C2x 50 C2 = 20 ppm 1000 x 3= C2x 50 C2= 60 ppm 1000 x 5=C2 x 50 C2=100 ppm 1000 x 7= C2x 50 C2= 140 ppm 1000 x 9= C2x 50 C2= 180 ppm 4- tabulate the values of the absorbance as following: Table 2: Concentration Absorbance PPM 20 60 100 140 180 Example of Calibration Curve (using Molar concentration) 5- Finding the concentration of an unknown sample: You will be given a sample of tetracycline with an unknown concentration. The addition of the tetracycline stock solution and HCl has already been done, so you just need to complete the additions as specified in Table 1. Record the absorbance value and use the calibration curve to determine the concentration of the unknown sample.