) Calculate the Rf val ue for each spot on the TLC p l ate

1 1 ) Calculate the Rf val ue for each spot on the TLC p l ate

O nce the d i stances are tabu lated, the Rf val ue is calculated as follows:where

de is the distance that the spot traveled from the origin (see step 1 0 above) and ds is the d istance traveled by the solvent front (see step 9 above) . The Rf value can be easily calculated i n the spreadsheet program Excel .

1 2 ) Compare the u n known sample to the reference materials that were chro­

matographed on the same TLC plate Exam i n e the chromatographic patterns of each u n k nown and reference

m aterial . Com pare the Rf values of each material. The Rf values should match with i n about 3 % for a positive identification . Next, look at the color and

i ntensity of each separation zone fo r both the u n k nown and the reference chromatograms. The final identification should take into consideration the Rf val ue, color, and intensity of each separation zone. I t is possible that

Striegel . Hill

of the m i xture. N I H I mage can also be used to calculate the peak area for each peak in the chromatogram. A basel ine is d rawn by the operator for each peak . The basel ine for unresolved peaks m ust be esti mated . The wand tool is then used to select the peak . This method is sti l l in developmental stages. The following steps may be i m proved in the future.

1 3) Select the Peri meter/length measurement function of the Image program U nder the Analyze men u , select the Options submenu. Select the Perimeter/length measurement and deselect the Area measurement.

1 4) Draw a baseline for each peak

Select the line tool . Draw a baseline for any peak that does not touch the existing baseline. To do so, place the cross hair on the left side of the

peak and hold down the mouse button while d rawing the line to the right side of the peak. Repeat this step until all peaks have a baseline.

1 5) Measure area for each peak

Select the magic wand tool from the tools wi ndow (it looks like a wand and is the n i nth tool in the first column). Place the wand end of the tool i n the center of the peak and click once on the mouse. A running stripe will surround the selected area, and the measured area will appear in the values window.

In addition to tabu lati ng the areas, the value of the area can be placed on the plot by selecti ng the type tool (a big A) and placi ng it in the image .

Choose where you want the value on the plot, then hold the option key and click once on the mouse button . The last measured val ue will automati­ cally be placed on the plot.

Again , care should be taken to keep track of the o rder in which the peaks are measured. The measurements are viewed by selecti ng Show Resu lts from the Analyze window. Print results by selecti ng Print Measurements command under the File menu. These resu lts can be exported to a spread ­ sheet or saved i n N I H I mage.