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GC and HPLC are commonly used as methods for highly sensitive separation analysis. Novel detection methods and high resolution columns have been developed, thereby highly-sensitive and highly-selective separation analysis is now routinely performed. Furthermore, the substances that are being analyzed are of a wide variety, ranging from such things as trace biological constituents to trace environmental pollutants. GC and HPLC are playing important roles in the advancement of today’s microanalytical technology.
Thin-layer chromatography (TLC) is also an important analytical tool, being used to confirm the progress of reaction, and for evaluation of HPLC separation conditions. Furthermore, the Japanese Pharmacopoeia (JP) has established that the identification tests by TLC must be conducted for many of the natural medicines, such as Scutellaria Root, Phellodendron Bark and Rhubarb, etc. As mentioned, TLC is still an important method of simple separation analysis, which widely used in the many fields.
The Rf-value is a very important piece of data that is provided by TLC. When TLC development conditions are same, the Rf-value is characteristic for a substance. Therefore, the Rf-values are often used for identification of substances. Additionally, selective detection of compounds is possible by choosing the appropriate TLC stains. For example, after development of the TLC plate, if one seeks to identify a compound containing an amino group, the TLC plate is treated with ninhydrin solution which stain only those compounds with an amino group. There are many types of TLC stains reported and used. To obtain accurate results, it is important to select the appropriate TLC stains.
The below table indicates typical TLC stains and the corresponding functional groups. Each of them is prepared so that can be used straight away after the TLC development.
|Prod.#||Prod. Name||Treatment||Target Compounds|
|A1674||p-Anisaldehyde (Ethanol Solution) (contains Acetic Acid, H2SO4)||Heat||Versatile-type, effective with almost all functional groups, esp. nucleophile ones such as phenols, sugars|
|P1484||Phosphomolybdic Acid (Ethanol Solution) (PMA)||Heat||Versatile-type, effective with almost all functional groups|
|P1483||Potassium Permanganate Solution (contains K2CO3, NaOH)||-||Versatile-type, effective with oxidizable functional groups, multiple-bond, alcohols, amines, sulfides, mercaptans|
|N0719||Ninhydrin (Ethanol Solution) (contains Acetic Acid)||Heat||Amines, amino acids, Boc protected amino groups after deprotection while on the TLC plate|
|D2968||2,4-Dinitrophenylhydrazine (Ethanol Solution) (contains HCl)||Heat||Aldehydes, ketones|
|B2401||Bromocresol Green (BCG) (Ethanol Solution) (contains NaOH)||-||Compounds with acidic functional groups, carboxylic acids, sulfonic acids, etc.|
|C1794||Ceric Ammonium Molybdate Solution (CAM) (contains H2SO4)||Heat||Effective with almost all functional groups|
|V0080||Vanillin (Ethanol Solution) (contains H2SO4)||Heat||Alcohols, phenols|