Thin Layer Chromatography - The Assay of Sulfamethazine in Pork Carcasses 2

 

The silica plates that were employed were 10 x 10 cm with a pre-absorbent strip for spot focusing. 40 ml aliquots of extracts and standards were applied to the plate using micro capillary pipettes. The plate was dried and placed in a chromatographic tank that contained methanol. The solvent was allowed to advance to the top of the pre-absorbent strip and was then removed and dried. The plate was then placed in another tank filled with ethyl acetate and the solvent allowed to advance 2 cm beyond the pre-absorbent strip; the plate was then again removed and sprayed with a solution of 4-phenylspiro(furan-2-(3H),1'-phthalan)3,3'-dione (a fluorescent reagent named Fluorescamine manufactured by Roche Products). After allowing a 15 minute reaction time the plate was viewed under UV light (366 nm) to locate the fluorescent bands. For those samples for which it was necessary to resolve, sulfadiazine, sulfathiozole and sulfurmerazine from sulfamethazine, the ethyl acetate was allowed to advance 3.5 cm beyond the pre-absorbent strip.

 

The technique was shown to recover about 80% of the antibacterial agents at when present at levels between 50-100 ppb in porcine tissue. The minimum level of detection (which could be considered as the sensitivity of the test) was about 30 ppb. The standard used contained 0.2 mg of the antibiotic per ml; less dense spots that contained the antibacterial agent at a level below that permissible were simply reported as negative; more dense spots that indicated that the antibacterial agent was present at a level above that permissible were simply reported as positive. This example could be considered as typical of the more everyday use of quantitative TLC analysis. The analysis merely determines whether a component is above or below the level of a standard and is, thus, a pass or fail type of test. It employs the eye as a comparator (for which the eye is most efficient) to render the test as fast and as simple as possible.

 

Aflatoxins are highly toxic metabolites produced by the molds Aspergillus flavus and Aspergillus parasiticus. These molds thrive on substrates rich in lipids and carbohydrates and particularly under the damp conditions resulting from the high relative humidity and elevated ambient temperatures common in tropical countries. The oil palm Elaies guineensis Jacquin occurs both wild and cultivated in the equatorial areas of Africa, South East Asia and South America. Palm oil is obtained from both the fruit and the nut, the latter containing as much as 50% of its mass as oil. The oil finds commercial use in the food and detergent industries and the residual pressed 'cake' provides a protein source for animal food. The aflatoxins are generated both before and after harvesting and their production is aggravated by cracking, insect activity and poor storage conditions. Due to the high toxicity of the aflatoxins, maximum levels of contamination have been set by most user-countries and consequently, efficient accurate and precise methods for their measurement had to be developed. Nawaz et al. [13] devised a practical analytical procedure that involved an efficient extraction procedure followed by a TLC finish. The plate was finally quantitatively assessed by automatically scanning the aflatoxin fluorescence.