Practicum 3: Diffusion Study in Pharma Preparations

PRACTICUM 3: DIFFUSION STUDY OF PHARMACEUTICAL PREPARATIONS (IN VITRO) Description of Theory Diffusion is a process of transferring the molecular mass of a substance carried by random molecular movement (Brownian movement) and is related to the presence of polymers, which is an easy way to investigate the diffusion process. The passage of a substance through a boundary usually occurs by simple molecular permeation or by movement through pores and holes (channels). Molecular diffusion or penetration through non-porous media depends on the dissolution of molecules that penetrate the entire membrane, while the second process concerns the passage of a substance through the pores of a membrane, which contains solvent and is influenced by the relative size of the molecules that penetrate it and the diameter of the pore. The first quantitative study proved that most chemical molecules are absorbed through the skin by passive diffusion. The rate of absorption across the skin is not steady but a latency time is always observed. Latent time reflects the delay in the penetration of the compound into the horn structure and the attainment of the diffusion gradient. This time varies from one compound to another. When balance is achieved, the number of compounds leaving the dermis surface membrane will be the same as the compounds that penetrate the epidermis layer. The permeability constant Kp reflects the ability to penetrate a compound across a particular membrane, the higher the value of this constant, the more pronounced its ability. The permeability constant of a compound that diffuses into all layers of the skin is the sum of several constants Kc, Ke, Kd which respectively are the permeability constants forstratum corneum,epidermis and dermis using appropriate methods. The designations c, e and d represent the diffusion resistance of the horn layer, epidermis and dermis, respectively. In most preparations, diffusion resistance across the horn layer (Stratum corneum) is very high and is a determining factor in percutaneous absorption. On the other hand, the resistance of the malformed epidermis and dermis is negligible. Based on this, it can be seen that water diffusion is a thousand times faster across the horn layer (stratum corneum) from the epidermis layer and the living dermis layer. Implementation of Practicum a. Tools and materials Tool:Tools used: Franz model diffusion tool (modification), membrane millipore, UV-Vis spectrophotometer, volume flask, beaker glass, measuring cup, volume pipette Material:Emulgel preparation (ketoprofen) and Methanol, Isopropyl myristrate, phosphate buffer solution pH 7.4 and Aquadest. b. Work procedures 1) Determination of active substance levels a) Preparation of a phosphate solution with a pH of 7.4 A pH 7.4 phosphate buffer solution can be made by mixing 500 mL of 0.1 M potassium dihydrogen phosphate solution and 39.1 mL of 0.1 N NaOH solution, then adding distilled water to the 1000 mL mark. b) Preparation of a mother standard solution of active substances in phosphate buffer pH 7.4. The active substance was weighed carefully in the amount of 50 mg then put into a 100 mL measuring flask, and added with a phosphate buffer solution of pH 7.4.up to the limit mark to obtain an active substance concentration of 500 µg/mL. c) Determination of the maximum wavelength of the active substance in a phosphate buffer medium pH 7.4 and creation of a calibration curve. 1.0 mL of the mother standard solution of the active substance that has been prepared is taken and then put into a 50 mL measuring flask, after which phosphate buffer pH 7.4 is added to the limit mark to obtain a working standard solution with a concentration of 10 µg/mL. Measure the absorption of the working solution with a UV-Vis spectrophotometer at a wavelength between 200-800 nm, to obtain the maximum wavelength value of the active substance. Solutions of the active substance in phosphate buffer were made in various concentrations from lowest to highest and the absorption at the maximum wavelength was measured and a regression line equation was created. The calibration curve is obtained from the plot between the absorption value and the concentration of the working standard solution. 2)Dosage diffusion testin vitro a) Optimization of millipore membrane impregnation time in isopropyl myristate The millipore membrane used was weighed and then impregnated in isopropyl myristate for 10, 30, 45, 60, 75 minutes. After that, the membrane was taken and dried on filter paper. The weight of the membrane before and after impregnation was weighed to obtain the same conditions for each membrane.The time with the smallest impregnation percentage value is determined as the optimum time and is then used to impregnate the membrane. b) Diffusion test of cream and nanoemulsion preparations The diffusion rate test was carried out using the flow through method using a modified Franz diffusion cell consisting of a diffusion cell, peristaltic pump, stirrer, beaker, water bath, receptor reservoir, thermometer and hose. The test formula is weighed ± 1 gram and then smeared on the impregnated membrane evenly and thinly. System temperature 37 ± 1 ° C with receptor fluid 330 mL phosphate buffer solution pH 7.4. The peristaltic pump will draw the receptor fluid from the beaker, then pump it into the diffusion cell and the fluid will flow directly to the receptor. The process was carried out for 2 hours, 10 mL samples were taken of the receptor fluid in a beaker and every time 10 mL of pH 7.4 phosphate buffer solution was taken, it was replaced with the same amount of solution. Samples were taken at 5, 10, 15, 30, 45, 60, 90, and 120 minutes. The absorption of the samples was measured at the maximum wavelength. From the absorbance data obtained, the value of the active substance content that diffuses is calculated. c) Determination of the diffusion rate The levels of active ingredients that diffuse are included in the kinetic model equation to determine the rate of diffusion of active ingredients. The linear regression equation for each diffusion rate kinetic model is determined by determining the linearity relationship from the data: 1. Zero order kinetics. A zero-order linearity relationship is shown between data on the levels of active ingredients that diffuse against time. 2. First order kinetics. A first order linearity relationship is shown between the data ln levels of active ingredients that diffuse against time. 3. Higuchi model kinetics. A linear relationship is shown between data on the levels of active ingredients that diffuse and the root of time. 4. Kinetics of the Korsmeyer-Peppas model. A linear relationship is shown between the data ln concentration of diffuse allicin against ln time. Determination of the kinetics of diffusion rate can be seen from the r value obtained from the linear regression equation. The release kinetics is based on the r value that is closest to 1 and the diffusion rate value is the slope value (b) in the linear regression equation