Question

1, Why use ninhydrin reagent and iodine crystals as visualizing agent in TLC?

2. Differentiate TLC and Column Chromatography based on a) separation of components in mobile and stationary phases b) solvent mitigation c) type of support used

Answer 1

Ninhydrin is the reagent used for the identification of amino acids. The reagent is well-known and widely used for its high sensitivity. The identification of amino acids is important during the evaluation of protein structure. The amino acids are colorless compound and it can be detected on the chromatogram by the spraying of Ninhydrin reagent.It reacts with amino acids and produces purple violet color with all amino acids. But some exceptions are for proline and hydroxyl-proline due to the presence of secondary cyclic amine groups .
Iodine is also used for the identification of aromatic compound.The mostly used semi-destructive and visualization method is that to expose a developed TLC plate by iodine (I2) vapor.An iodine chamber can make by adding some iodine crystal into the cahmber of TLC.Then TLC plate has to put iodine chamber ,so it will expose to it and form yellow spot on it.The yellow spot is frming due to it forming complex with many organic compound present on TLC.This method can use many organic compound .It is considered to be a "semi-destructive" m ethod due complexation process is reversible, and so that iodine will easily evaporate from the TLC plate.So the original compound left on TLC plate.

Difference between TLC and Column Chromatography
The chromatography is the technic to separate the individual from compound.But sometimes the product may be degraded due to some distraction happening. Thin layer chromatography and column chromatography is one of the most useful method. Thin-layer chromatography is normally using for separating the number of mixes in a sample just as basis on the relative polarities rather than separating them by physically, this can be practiced with column chromatography. Two separating method are used to separate the analytes that are generally non-volatile.
•   TLC is having a stationary phase of silica gel and alumina.
•   Column chromatography is packed uses its stationary phase having material, like as silica.
•   TLC is worked against the gravity.
•   Column chromatography is carried under gravity.
•   TLC is used for analytical purposes.
•   Column chromatography is used for the preparative purpose.
•   TLC is needed very less time to separate the individual from the compound than the column chromatography.
•   Column chromatography has taken more time to separate individual from the compound than the TLC.
•   TLC uses very less amount of solvent to separate the analytes than column chromatography.
•   Column chromatography has taken more amount of solvent than TLC
•   TLC required more polar solvent compared to the column chromatography.
•   Column chromatography needed less polar solvent compared to the TLC.