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  Section: Plant Lab Protocols
 
 
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Methodology for Separation Procedures

 
     
 
Analysis of aflatoxins
 
A wide spectrum of fungi infest most of the agricultural produce including food grains and feedstuffs under warm humid conditions, especially when the moisture content of the stuffs is high. Storage fungi such as Aspergillus sp., Penicillium sp., Fusarium sp. etc. grow very commonly on the moist materials and make them unfit for human/animal consumption. During their growth on the materials, fungi elaborate certain metabolites called mycotoxins which are toxic to the consumer. Continuous intake of fungus-contaminated materials leads to the damage of liver and kidney by the toxic substances. In severe cases, liver cancer and nephropathy of kidney are produced, ultimately resulting in death. Asperilli sp. are the most widely contaminating fungi producing aflatoxins B1? B2, G1 and G2. Because of the potential health hazards produced by these toxins even in minute amounts, a watch over their presence in various food materials is very much inevitable. Although a large number of procedures and chemical methods are available for the identification and estimation of aflatoxins, the following method is simple and can be performed without any sophisticated equipment.
 
 
Materials
Thin Layer Chromatography (tic) Kit
Ultra-Violet (UV) Chamber
Mechanical Shaker
'Quick-fit' Distillation Set
Toluene
Ethyl Acetate
Formic Acid
Chloroform
Silica Gel G (tlc Grade)
 
 
Procedure
Extraction of Toxins
1.
Weigh exactly 50g of ground sample material and transfer it into a 250mL conical flask.
2.
Moist the material uniformly by adding 10-15mL of distilled water and add about 200mL chloroform, stopper the mouth with a cotton plug in aluminum foil.
3.
Shake the flask for one hour mechanically. (It is important that the oil-containing materials are defatted prior to extraction.)
4.
Filter the slurry through a Buchner funnel under mild suction. Equal amount of a filtering aid such as celite may be mixed before filtering in order to ease filtration. Wash the flask and the slurry thoroughly with additional chloroform (25mL) and collect the filtrate.
5.
Transfer the filtrate quantitatively to a separatory funnel and shake with water one-half volume of chloroform. After the phases separate, drain the bottom (chloroform) phase into a flask containing about 10g sodium sulphate (anhydrous) to absorb any water.
6.
Concentrate the clear, chloroform extract 'under vacuum' over a warm water bath using quick fit distillation set. Make up the concentrate to a known volume with chloroform and store in amber-colored vials under refrigeration until analysis.
 
 
Preparation of tlc Plates
1.
Place 30g silica gel G (with CaSO4 as binder) in a stoppered flask, share vigorously with 60-65mL distilled water for about one minute, transfer to the applicator and spread uniformly on five clean glass plates (20 x 20cm). The exact quantity of water required to get a good slurry will vary from batch to batch of silica gel G. The thickness of layer should usually be 0.25mm.
2.
Allow the plates to dry for 1-3h in dust-free conditions. Activate the gel, prior to use, for 30 min at 110°C in a hot-air oven. The activated gel plates should be stored in a desiccator chamber.
3.
Divide the gel into a number of lanes by drawing lines on the gel with a sharp needle.
4.
Spot different known volumes (5, 10mL etc.) of the sample extract in various lanes carefully with a microsyringe on an imaginary line 2.5cm away from one end of the plate. Similarly spot standard aflatoxins (B1, B2, G1 and G2) mixture in the concentration range 0.0025-0.0125mg in parallel lanes.
5.
Develop the plate in a solvent system of toluene: ethyl acetate: formic acid (6 : 3 :1) in a chromatographic tank for about 50 min. By then, the solvent front might have moved up to 20mm below the top end of the plate.
6.
Dry the plate at room temperature to remove the solvent. Visualize the fluorescing spots of toxins under UV light in a cabinet. Protective glass should be worn while viewing under UV light; otherwise, eye sight will be affected.
7.
Identify each fluorescing spot of the sample extract by comparing with the authentic toxin spot co-chromatographed. Determine the Rf value of each spot.
8.
For quantitative estimation of the toxins in the sample extract, match the intensity of spots of the sample with that of standard toxin spots, by diluting both to extinction. Calculate the amount of toxin in a kg sample material.
 
 



Notes

1.
Care to collect a representative sample of the experimental material.  Conveniently, small portions of samples from various points are collected, mixed thoroughly and quartered. Sampling procedure varies from commodity to commodity (for details read the reference below).
2.
Pre-run of activated plates in diethyl ether is useful to eliminate UV fluorescing substances, if any in the gel.
3.
A variety of solvent systems are used for developing the plates. Choose the system to get all the spots clearly resolved.
4.
The Rf value is in the order of B1>B2>G1>G2.
5.
Under UV light B1 and B2 fluroese blue and G1 and G2 green. B1 content is usually greater than the other toxins.
6.
Dilution to Extinction: The standard or sample is diluted serially. Each dilution is spotted in equal volume on the plate. After developing, at one dilution a particular toxin (say B1) will be visible under UV light while at its next high dilution the spot will not be visible. The dilution at which the spot is visible is termed 'dilution to extinction'. For each toxin, the
dilution to extinction is different from the other.

 
 



References

1. Jones, B 3D (1972) Tropical Products Institute Report G 70 London p 13.
 
 
     
 
 
     




     
 
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