Page 95 - Feed Lab

Estimation of Aflatoxin Modified Romer's Method Suguna Management System Ver 1.0 / SOP / FQL / P2 - 18 Page 3 of 4

FAfter spotting the standards and sample, develop
Preparation of activated TLC plate
the spots in an unsaturated developing tank
To prepare 2 plates (10cm x 10cm or 10cm x 5cm) containing chloroform: acetone: water in the
of 0.2mm thickness dissolve 16g of silica gel (G) in 88:12:1. (Picture - 22)
35ml of water, apply on the plates using applicators
and allow it to natural dry. Then keep the plates at FAfter developing three fourth of the plate, the plate
0
105 c for 1 hour and cool. Draw lines with 1cm space is carefully removed from the tank, dried well and
such that standards are at the middle of the plate and viewed in a UV cabinet viewer using long
four sample spots can be applied on either side of the wavelength (365 nm). (Picture - 23)
standard.

PROCEDURE
FTake 10g of the sample. Add 40ml of distilled water.
Beat it in the mixer for 2mts. Add 60ml of acetone
and again beat it for two minutes. Contents may
slightly be heated up. High temperature should be
avoided. (Picture - 16)
FFilter the contents. Take 30ml of the filtrate and add
Picture - 16 Picture - 17
approximately 0.6g of cupric carbonate in beaker
(A).
FIn another beaker (B), take 34ml of 0.2 M NaOH +
6ml of FeCl (0.41M) and swirl the contents. Add
3
the contents in the beaker (B) to beaker (A) and
again mix it slowly by swirling movements. (Picture -
17)

FFilter the contents through Whatman No.1 filter
paper. Take 40 ml of the filtrate in a 250 ml
Picture - 18 Picture - 19
separating funnel.
FAdd 40ml of (0.03%) H S0 and 10ml of
4
2
chloroform. Mix it slowly. (Picture - 18)
FCollect the chloroform layer in a 100ml beaker, add
again 10ml of chloroform, mix thoroughly, and allow
settling and collecting the chloroform in the same
100ml beaker. In a second separating funnel, take
40ml of 0.02 M KOH +1% KCl mixtures. (Picture -
19) Picture - 20 Picture - 21

FTo this, add the collected 20ml chloroform extract.
Mix it slowly and collect the layer through
anhydrous sodium sulphate bed drop by drop to
remove any traces of moisture. (Picture - 20)
O
FKeep the chloroform extract in an oven at 50 c till it
becomes dry. The dry aflatoxin film is re-diluted with
0.2ml chloroform and spot on the TLC plate taking
exactly 5 µ , 20 µ, and 40 µ besides the standards
spots of 5 µ and 10 µ. (Picture - 21) Picture - 22 Picture - 23

Prepared by : Approved by :
83 Director - Technical