Summary
Highlights
Brent Kadrowski introduces part three of the organic chemistry lab video series on TLC of analgesic drugs. This video will cover visualizing TLC plates, calculating RF values, and identifying unknowns. The previous video covered sample preparation, plate preparation, spotting, and plate development.
TLC plates contain a fluorescent indicator, allowing spots to appear as dark circles on a bright background under UV light. The presenter dims the lights and uses a UV lamp to visualize the spots on the first TLC plate, which contains standards of acetaminophen, aspirin, caffeine, ibuprofen, and unknown #9. The spots are then lightly circled in pencil for later visibility. Safety precautions, such as wearing gloves, are mentioned when handling UV light.
The video explains how to calculate RF (retardation factor) values for each spot. The RF value is the ratio of the distance the spot traveled from the origin (d1) to the distance the solvent front traveled from the origin (d2). An example calculation for acetaminophen shows d1 as 1.9 cm and d2 as 5.4 cm, resulting in an RF value of 0.35. RF values are unitless numbers between 0 and 1.
The same process of UV visualization and RF value calculation is applied to the second TLC plate, which contains an unknown #10. It is noted that unknown #10 shows two distinct spots, indicating it contains two different molecules. Similar RF values between some spots (e.g., top three spots) highlight the difficulty in identification solely based on RF values, leading to the introduction of staining.
To aid in identification, a ceric ammonium molybdate stain is introduced. The plate is dipped in the solution up to the solvent front, excess is blotted, and then heated with a hot air gun. Heating causes the spots to appear, and differential staining (different colors) further helps distinguish compounds. It is important to avoid overheating and be aware that not all spots will stain. Unknown #9 is identified as acetaminophen due to similar RF value and identical staining properties.
The second plate, containing unknown #10, is stained and heated. Differential staining clearly shows that the upper spot in lane E (unknown #10) is more similar to aspirin (spot 'b') than ibuprofen (spot 'd'), based on color, despite similar RF values. This demonstrates the strength of differential staining in making definitive identifications. The video concludes by encouraging viewers to watch the next or previous videos in the series and to subscribe.