For informations about installation of the image analysis tool, access the “Simulator” tab
Step 1: Import the image of the Thin Layer Chromatography to be analyzed into the software.
The image will open in a new pop up window as shown.
Step 2: Subtract the background of the TLC image, to reduce the background effects on image analysis. Follow, Process---> Subtract background. A pop up window for adjusting the image backgrounds will appear in the screen. Enter the values in the provided box, select Light background and Preview options and Click OK to view a preview of the image.
Step 3: Using the rectangular selection tool, draw a rectangle around the first spot of the Thin Layer Chromatography image.
Step 4: When the first spot is selected in a rectangle, then select Analyze --->Gels ---> Select first Lane from the menu (or click “control-1″ in Windows).
The image will be displayed as below.
Step 5: After selecting the first spot, take the cursor and move it within the box set in the first section. This helps to click-and-hold, and drag the box to the second spot in the TLC plate.
Step 6: To analyze the selected spot, follow Analyze ---> Gels---> Select Next Lane (OR click “control-2″ in Windows). This will select an area in the second lane that is exactly equal to the area selected in the first spot.
The image will be displayed as shown below.
Step 7: Repeat the steps 5and 6 to select the spots in the third and fourth position of the TLC plate. Click within the yellow box and drag the box to cover the spots in the TLC plate.
Step 8: After selecting the last spot in the TLC plate, select the Analyze ---> Gels---> Plot Lanes menu in the menu bar or click “control-2″ in Windows.
A new pop window will open which depicts the densitometry measurements of the amino acids detected in the TLC plate. This graphically depicts the intensity of the spots in TLC plate.
Step 9: Using the line tool close off all of the peak area that rises above the background level.
Step 10: Select the wand tool and click inside each peak, starting from the top portion. The selected peak will change into yellow color.
This help to determine the area under the curve of the selected peak.
Step 11: After all the peaks are selected, follow Analyze --->Gels---> Label Peaks. This labels each peak with its size, expressed as a percentage of the total size of all of the highlighted peaks.
Step 11: Another window that depicts the “Results” will open that will help to analyze the intensity of each spots in the TLC plate.
Step 12: Copy and paste the values in an Excel sheet.
Step 13: To calculate the relative density of each spots in the TLC,here the percentage of Sample 1 is taken as a standard value. The Relative Density is calculated by dividing the Percent value for each sample by the Percent value for the standard.
Here,the percent value of the first sample, that is, 38.325 is taken as the standard value.
38.325/38.325 = 1
21.179/38.325 = 0.552
3.814/38.325 = 0.099
36.682/38.325 = 0.96
The value indicates that the sample 1 and sample 4 have the almost same relative density. Sample 3 is less dense compared to the other samples in the analyzed amino acids.
The relative values obtained from the image analysis show that the sample 1 and sample 4 have same band intensity and hence they may be similar amino acids.