Spotting Development and Examination of the TLC Plate

The plates most often used for aflatoxin analyses are 20 x 20 cm glass plates, pre-coated with a 0.25 mm layer of silica gel 60 (E. Merck, Darmstadt); plates from other manufacturers may work equally well. Spotting should be done in subdued incandescent light to avoid photodecomposition of the aflatoxins. Using a 10 |L syringe, on an imaginary line 4 cm from the bottom of the plate and 1 cm apart, 2, 5 and two 10 |L spots of the sample extract are applied together with 2, 5 and 10 |L spots of mixed aflatoxin standards; 5 | L of the standard is applied on top of one of the 10 |L spots of sample extract. It is possible to spot four samples on to each plate.

The plate is developed for less than 90 min with acetone-chloroform (1:9 v/v) until solvent is within 4 cm of the top of the plate. It may be necessary to adjust the acetone-chloroform ratio to obtain optimum resolution. The plate is removed from the tank and air-dried in the hood in the dark.

Plates are examined under long wave ultraviolet light at 365 nm in a cabinet equipped with a filter for protecting the eyes from the ultraviolet light. Aflatoxins appear in order of decreasing RF: B1, B2, G1 and G2. G1 and G2 are slightly greener than the blue B1 and B2. The RF values for the aflatoxins in the sample spots should be the same as those of the standard spots. The aflatoxins in the sample spot upon which the standard is superimposed should coincide exactly with the standard spots. The intensity of the fluorescence of each of the sample spots may be compared with that of the standard spots to estimate the amount of aflatoxin present in the extract. Separate estimates need to be made for B1, B2, G1 and G2. If the spots of the smallest portion of the sample are more fluorescent than the strongest standard spot it is necessary to dilute the sample extract and re-chromatograph. The plate may be run on a densitometer equipped with an ultraviolet light source set at 365 nm and an ultraviolet filter before the photomultiplier detector. Connecting the TLC densitometer to a computer permits the integration, calculation, printing, and storage of results. If more accurate quantitative results are necessary, the extract can be re-diluted to a concentration approximately equal to that of the standard and re-chromatographed in the same manner as above. The concentration of each aflatoxin in the extract can be calculated using the formula:

where S = |L of standard spot equal to sample; Y = concentration of standard in ng |L_1; V = |L of final dilution of sample extract; X = |L of sample spot equal to standard; and W = grams of sample that the extract represents.

Not all blue fluorescent spots in the extracts are necessarily aflatoxins. Sample extracts may contain interferences, especially at the RF values of G1 and G2. Respotting with an alternative solvent system such as the top phase benzene-ethyl alcohol-water (46 : 35 : 19 v/v) or with benzene-methanol-acetic acid (90 : 5 : 5 v/v) often resolves the aflatoxins from the interferences. Other solvents which are sometimes used are: ether-methanol-water (96 : 3 : 1 v/v), chloroform-acetone-water (88 : 12 : 1.5 v/v), or chloroform-acetone-isopropanol-water (88 : 12 : 1.5 : 1 v/v).

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