EXTRACTION OF PENICILLIN FROM BROTH BY SOLVENT EXTRACTION

AIM :

To produce penicillin on a laboratory scale and extract it using an organic solvent.

THEORETICAL BACKGROUND :

The exact recovery and purification methods employed for the extraction of penicillin depends on the production media employed and on the final penicillin yields. If the medium does not interfere with recovery and purification, it greatly simplifies the procedure required to obtain a pure product. The recovery and purification process of penicillin at the industrial level can be divided into a number of steps :

1. Removal of mycelia :

Penicillin is released into the broth during fermentation. It remains in solution extracellularly, together with other metabolites and media components. The first step is to remove the mycelium from the fermentation broth by filtration. A rotary vacuum filter is employed for this purpose . It should be carried out under proper sterile conditions to avoid contamination from the filter used by penicillinase producing microorganisms, which may cause serious or total loss of the antibiotic.

2. Countercurrent solvent extraction :

The countercurrent solvent extraction of penicillin from the fermentation broth. The pH of the filtrate is adjusted to 2-2.5 with the help of phosphoric acid or sulphuric acid. This converts penicillin to its anionic form which simplifies the extraction procedure. But the penicillin in this form should be immediately extracted because it is unstable at low pH values. Solvent extraction is carried out in PODBIELNIAK countercurrent solvent extractor. Organic solvent such as amyl acetate, methyl isobutyl ketone or butyl acetate are used. The penicillin inorganic solvent is back extracted into aqueous buffer at pH 7-7.5. The resulting aqueous solution is again acidified and re-extracted with an organic solvent. These shifts between water and organic solvents helps in the purification of penicillin.

3. Purification :

The final solvent extract is carefully back extracted with aqueous KOH or NaOH to allow formation of respective salt of penicillin. This is given, charcoal treatment to eliminate pyrogens (fever causing agents). The resulting penicillin is sterilised with Seitz filter to remove bacteria, if present. The ultimate solution of penicillin is then subjected to crystallisation. The crystalline penicillin salt is then washed and dried.

In the following protocol the penicillin produced is extracted under acidic conditions in an organic solvent like ether. The extracted penicillin is brought back into the aqueous phase by adding sodium carbonate solution. This extract is used as a sample for bioassay and chemical assay and the concentration of penicillin can be estimated.

REQUIREMENTS :

20% phosphoric acid (20 ml), solvent ether (20 ml), 1% sodium bicarbonate solution (20 ml), pH papers to check pH during extraction, St. 10 ml and 1 ml pipettes, St. large test tubes (40 ml with cap), shaker (vortex), etc

PROCEDURE :

1. Transfer 20 ml of broth culture from each of the two flasks (static and shaker) into separate test tubes and centrifuge at 4000 rpm for 10 minutes.

2. Decant out 10 ml of clear supernatant containing the antibiotic into separate large test tubes and proceed with each of the supernatant as follows.

3. Add 5 ml of solvent ether and 7 ml of 20% phosphoric acid to this 10 ml of fermented broth. Ensure that the pH is not lower than 3.0-3.5.

4. Shake the tube for 10 minutes using a vortex mixture so as to extract the penicillin into the ether layer.

5. Pipette out the ether layer (the upper layer) into another test tube and add equal volume of aq. Sodium bicarbonate solution to the extract.

6. Agitate the tube on vortex to evaporate all the ether.

7. Use the aqueous extract to estimate the yield of penicillin using bioassay and chemical assay.

8. Compare the potency of penicillin produced under both the conditions of incubation.