Question

1. A solution of the sample to be analyzed has been made in a suitable solvent and wil l be provided for you. With this solution, a small spot is applied at a distance hear the bottom of the edge of a TLC plate using an applicator. (see Figure 1) This spot must be above the level of solvent in the TLC chamber. Why? 2. The plate is placed in a developing chamber containing the appropriate solvent (eluent), covered, and removed when the solvent has traveled near the top of the plate. Why should you NOT allow the solvent to run all the way off the top of the plate? The solvent front is marked with pencil upon removal from the solvent and the spot is illuminated under UV light and circled with pencil. Why is the spot illuminated with UV light. [Reminder: look at structure of molecules. Refer to a textbook or online resource for necessary information.] 3.

Answer 1

1.
The sample should be above the solvent level. If the solvent level covers the sample, the sample spot will be washed off into the solvent before it travels up the TLC plate.

2.
If the solvent front reaches the top of the plate in Thin-Layer Chromatography (TLC), the spots for your compounds/mixture will start to diffuse. Since solvent has no further place to move, the paper remains saturated with the mixed solvent, and the compounds you have running up your paper will start to diffuse and spread out, giving you a very messy chromatograph.
Again, it basically means your R_f value would be inaccurate as you will not be able to accurately determine the distance the solvent has travelled.

3.
For this question, you have not given any specific information. The general explanation is as follows:
The silica gel used for TLC plates have an inorganic fluorescent agent (< 0.5%) impregnated into the adsorbent layer. When illuminated with an ultraviolet (UV) lamp, the absorbent then glows the blue colored fluorescent light. However, dark spots show up at the places where UV absorbing organic compounds are located on the TLC plate because they quench the fluorescence.
​Again, to be detectable under the UV lamp, compounds must have strong UV absorption in the 220 – 280 nm range.