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1. Interaction study by TLC/ FTIR.[30]

IR spectroscopic studies:-The IR spectra of the free drug and the microspheres are recorded. The identical peaks corresponding to the functional groups features confirm that neither the polymer nor the method of preparation has affected the drug stability.

Thin layer chromatographic studies:-The drug stability in the prepared microspheres canalso be tested by the TLC method. The Rf values ofthe prepared microspheres can be compared with theRf value of the pure drug. The values indicate thedrug stability.

UV-FTTR (Fourier transform infra red):-The drug polymer interaction and also degradation of drug while processing for microencapsulation can be determined by FTIR. In this method the pellets of drug and potassium bromide are prepared by compressing the powders at 20 psi for 10 min on KBr?press and the spectra are scanned in the wave number range of 4000?600 cm?1. FTIR study is carried on pure drug, physical mixture, formulations and empty microspheres [30]

2. Particle size distribution of prepared microspheres:[31]
The size of the prepared microspheres can be measured by the optical microscopy method using a calibrated stage micrometer for randomly selected samples of all the formulations.

Optical microscopy:-This method is used to determine particle size ofmicrospheres by using optical microscope (MeizerOPTIK) The measurement is done under 45x (10x eyepiece and 45x objective) and100 particles are calculated.

3. Surface topography by Scanning Electron Microscopy (SEM):- SEM of the microspheres shows the surface morphology of the microspheres like their shape and size.

Scanning electron microscopy (SEM):-Surface morphology of microspheres is determined by the method SEM. In this method microspheres are mounted directly on the SEM sample slub with the help of double sided sticking tape and coated with gold film under reduced pressure. Scanning Electron photomicrographs of drug?loaded microspheres are taken. A small amount of microspheres s spread on gold stub. Afterwards, the stub containing the sample is placed in the Scanning electron microscopy (SEM). A Scanning electron photomicrograph is taken at an acceleration voltage of 20KV and chamber pressure of 0.6 mm Hg [31].

 Particle size analysis:- The particle sizes and particles size distributions are further analyzed by using dynamic light scattering technique, Microspheres are dispersed into 100 ml of water and sonicated for 1 min to remove agglomerations. The mean volume diameter (Vd) is recorded and polydispersity is determined by the SPAN factor. A high value of SPAN indicates a wide distribution in size and a high polydispersity.

4. Swelling Index:[32]
Swelling index is determined by measuring the extent of swelling of microspheres in a particular solvent. To ensure the complete equilibrium, exactly weighed 100 mg of microspheres ares allowed to swell in solvent for 34 hrs. The excess surface adhered liquid drops are removed by blotting and the swollen microspheres are weighed by using microbalance. The Hydrogel microspheres then dried in an oven at 60° for 5 hrs. until there is no change in the dried mass of sample. The swelling index of the microsphere is calculated by using the formula:

Swelling index =mass of swollen microspheres – mass of dried microsphere/mass of dried × 100

5. Entrapment efficiency:[33]
Microspheres containing of drug (5mg) were crushed and then dissolved in distilled water with the help of ultrasonic stirrer for 3 hr., and was filtered then assayed by uv-vis spectroscopy. Entrapment efficiency is equal to ratio of actual drug content to theoretical drug content.

% Entrapment = Actual content/Theoreticalcontent x 100Swelling.[33]

6. Stability studies:[34]
By placing the microspheres in screw capped glass container and stored them at following conditions:
1. Ambient humid condition
2. Room temperature (27+/-2 0C)
3. Oven temperature (40+/-2 0C)
4. Refrigerator (5 0C -80C).

It was carried out of a 60 days and the drug content of the microsphere was analysed.[34]

7. Density determination:[3]
The density of the microspheres can be measured by using a multi volume pychnometer. Accurately weighed sample in a cup is placed into the multi volume pychnometer. Helium is introduced at a constant pressure in the chamber and allowed to expand. This expansion results in a decrease in pressure within the chamber. Two consecutive readings of reduction in pressure at different initial pressure are noted. From two pressure readings the volume and hence the density of the microsphere carrier is determined.[3]

8. Isoelectric point:[3]
The micro electrophoresis is an apparatus used to measure the electrophoretic mobility of microspheres from which the isoelectric point can be determined. The mean velocity at different PH values ranging from 3-10 is calculated by measuring the time of particle movement over a distance of 1 mm. By using this data the electrical mobility of the particle can be determined. The electrophoretic mobility can be related to surface contained charge, ionisable behaviour or ion absorption nature of the microspheres.[3]

9. Bulk density:[35]
The microspheres fabricated are weighed and transferred to a 10-ml glass graduated cylinder. The cylinder is tapped using an autotrap until the microsphere bed volume is stabilized. The bulk density is estimated by the ratio of microsphere weight to the final volume of the tapped microsphere bed.[35]

10. Angle of contact:[36]
The angle of contact is measured to determine the wetting property of a micro particulate carrier. It determines the nature of microspheres in terms of hydrophilicity or hydrophobicity. The angle of contact is measured at the solid/air/water interface. The angles of contact are measured by placing a droplet in a circular cell mounted above objective of inverted microscope. Contact angle is measured at 200C within a minute of deposition of microspheres.[36]

11.  X-ray diffraction:[1]
Change in crystalinity of drug can be determined by this technique. Microperticles and its individual components were analysed by the help of D & discover (Bruker, Germony).Scanning range angle between 8 0C - 70 0C. Scan speed - 4o/min Scintillation detector Primary silt=1mm, Secondary silt=0.6 mm. Thermal analysis of microcapsule and its component can be done by using-

o   Differential scanning calorimetry (DSC)
o   Thermo gravimetric analysis (TGA)
o   Differential thermometric analysis (DTA)

Accurately the sample was weighed and heated on alumina pan at constant rate of 10oc/min under nitrogen flow of 40 ml/min.[1]



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