Extraction of Aflatoxin
- Grind dry kernels in a Romer mill and mix well
- Weigh 20g ground kernels
- Add 40ml 50% ethanol and shake for 2 minutes
- Store at 4C until use
Dry sample and re-suspend
- Add 200ul to 1.5ml tube and dry
- Re-suspend in 20ul chloroform
- Spot on TLC plate along with standards
Quantification of Aflatoxin using TLC
- Spot standards of aflatoxin along with the sample to be quantified on the TLC plate
- 10, 20, 50, 100, 500 and 1000 ng of aflatoxin can be used as a standard
- Every plate should contain both the standard and the sample
- After running the TLC, take a picture of it and use Imagej to quantify it
Using Imagej to quantify toxin
- Use imagej to open the picture
- Draw a rectangle over each spot on the TLC
- On the tool bar, click on gel and open analyze
- Using that, designate each lane as Lane 1,2,3 etc
- After plotting the lanes, click on analyze
- A peak will be obtained for each spot on the TLC, the base line of the peak can be adjusted to get the exact area
- Go to the small wand on the toolbar (Next to the A) and click on it
- It will give the area under each plot
- On an excel sheet, plot the areas against known concentrations of aflatoxin to get a “Standard graph”
- Use the standard graph to plot the area of the test (whose concentration is not known) and using the standard graph bring it down to the X-axis to find its concentration.