ultraviolet detection methods for TLC analysis and HPLC

directory of Chem Help ASAP videos: https://www.chemhelpasap.com/youtube/ Almost every organic chemistry student runs a TLC at some point during a teaching lab experiment. The most common, simple method for detecting compounds on a TLC plate is with a UV lamp. Here is an image of a TLC plate (a three-lane TLC plate used to monitor a reaction). What is happening with this plate? The TLC plate has a colorless dye. Under a UV lamp (specifically a UV lamp with a wavelength of 254 nm), the dye on the TLC plate glows green. Why do the compounds on the plate look like dark spots? Well, if there is a compound on the plate that can absorb UV light then, at that position, the compound is absorbing the UV light instead of the dye on the plate. Therefore, the plate in that location does not glow green, and it appears dark. It is a wonderfully simple application of UV spectroscopy, and many users do not realize that they are using the ideas of spectroscopy. Note that this method only works if the compound on the plate absorbs UV light with a wavelength of 254 nm. If the compound or impurity does not absorb UV light, it will be present on the plate but remain invisible to the detection method. The compound will not absorb the UV light, and the plate in that position will glow brightly. Fortunately, many organic molecules are UV-active at 254 nm. Often a single benzene ring in a molecule is enough for UV detection. Also, in case your molecule is not UV-active, there are many other methods for detecting molecules on a TLC plate, for example, dip stains or an iodine chamber. The second use of UV spectroscopy is closely related to TLC. People also use UV spectroscopy to detect the elution of molecules in different types of chromatography. Let’s discuss HPLC – high-performance liquid chromatography. In HPLC, people often analyze a mixture of compounds. They take a mixture of compounds or maybe a pure compound and pass the compound through a tiny pipe (a “column”) with high solvent pressure to push everything through the tiny column. Based on their different properties, each compound in the mixture will take a different amount of time to pass through the column. So, the column separates the components in the mixture. A detector at the end of the column checks the liquid coming through for the presence of the desired compound or impurities. You end up getting a readout – called a chromatogram or maybe a trace – with peaks at different times corresponding to the elution of each compound. How do you detect each compound? It is often UV detection. The elution solvent typically is not UV active. As the liquid exits the column, a beam of UV light (normally 254 nm) passes through the solution. If a UV active compound is dissolved in the solution, the absorbance is detected and converted into a signal on the chromatogram. Just like with TLC, the compound needs to be UV active to be detected, but that is generally not an issue. Often, when people use an HPLC, they do not even think about the role of UV spectroscopy for generating the chromatogram.