TLC Stains

Thin-Layer Chromatography (TLC) is a chromatography using a thin layer plate prepared by fixing an adsorbent fixed in thin film.

When one end of the thin layer plate is immersed in a solvent, the solvent migrates upward through gaps (capillary phenomenon). When a sample chemical is present on the plate, the sample also moves in a way being dragged by the solvent migration. At this time, the distance (Rf value) of sample migration differs depending on the difference / balance between the degree of adsorption to the fixed layer (adsorbent) and the affinity to the mobile layer (solvent). This principle is utilized to separate and identify organic compounds.

Selection of TLC stain is also important to obtain good results in TLC analysis. For example, if you want to detect compounds with an amino group from multiple spots, you can selectively stain by using ninhydrin TS.

Common TLC stains, preparation method of stains, and stained compounds are shown below. FUJIFILM Wako offers ready-prepared product in addition to reagents as raw materials for stains. Utilize them according to the intended purposes.

Stains	Prepared Reagents	Reagents1,2)	Stained Chemicals	Features
p-Anisaldehyde		p-Methoxybenz-aldehyde (11 mL) + Ethanol (500 mL) + Sulfuric Acid (5 mL)	Common organic compounds	Colors developed change depending on the type of compound.
Basic Potassium Permanganate		Potassium Permanganate (5 g) + Potassium Carbonate (33 g) + Sodium Hydroxide (0.4 g) + Distilled Water (500 mL)	Common organic compounds	Effective for functional groups that are particularly susceptible to oxidation.
Ceric Ammonium Molybdate (Hanessian Staining Solution)		Ammonium Molybdate (VI) Tetrahydrate (25 g) + Cerium (IV) Sulfate Tetrahydrate (6 g) + Distilled Water (450 mL) + Sulfuric Acid (50 mL)	Common organic compounds	High- sensitivity stain (less background color than phosphomolybdic acid)
Vanillin		Vanillin (24 g) + Ethanol (500 mL) + Sulfuric Acid (100 mL)	Common organic compounds	Colors developed change depending on the type of compound.
Cerium Sulfate		Cerium (IV) Sulfate Tetrahydrate (35 g) + Distilled Water (400 mL) + Sulfuric Acid (36 mL)	Common organic compounds	Development of a yellow or brown color
Phosphomolybdic acid (PMA)		Sodium Phosphomolybdate n-Hydrate (36 g) + Ethanol (500 mL)	Common organic compounds	High- sensitivity stain
Iodine		Iodine	Common organic compounds	Development of a yellow color
Iron (II) Chloride		Ethanol solution Iron (III) Chloride (1 g) + Distilled Water (5 mL) + Ethanol (95 mL) Water solution Iron (III) Chloride (2 g) + Distilled Water (100 mL)	Phenols	Purple, green, and yellowish brown, etc. color developed by phenols (varies depending on the compound) on a pale yellow background.
Palladium Chloride		Palladium (II) Chloride (500 mg) + Hydrochloric Acid (2.5 mL) + Ethanol (100 mL)	Phosphorus containing compounds, sulfur containing compounds	Yellowish brown to black color developed by phosphorus containing compounds on a pale yellow background, yellow to orange and brown color developed by sulfur containing compounds on a pale yellow background.
Curcumin		Curcumin (100 mg) + [a mixture of Ethanol: 2 mol/L Hydrochloric Acid= (99: 1)] (→100 mL)	Boronic acid derivatives	Red color developed by boronic acid derivatives.
Dinitrophenylhydrazine (DNP)		2,4-Dinitrophenylhydrazine (1.5 g) + [a cold mixture of Distilled Water (10 mL) + Sulfuric Acid (10 mL)] + [a mixture of Ethanol: Distilled Water (1: 3)] (→100 mL)	Aldehydes, ketones	An orange color developed by aromatic ketones and aldehydes, a yellow color developed by aliphatics.
Dragendorff's Reagent		A. Bismuth (III) Nitrate Basic (0.85 g) + Acetic Acid (10 mL) + Distilled Water (40 mL) B. Potassium Iodide (8 g) + Distilled Water (20 mL) → A. 20 mL + B. 20 mL + Acetic Acid 20 mL	Nitrogen containing compounds	Especially effective for tertiary and quaternary amines.
Ninhydrin		Ninhydrin (1 g) + Ethanol (50 mL)	α-Amino acids, amino groups, amide groups	Effective particularly for primary and secondary amines
Bromocresol Green (BCG)	0.1w/v% Bromocresol Green Ethanol (50) Solution	Bromocresol Green (0.05 g) + Ethanol (20 mL) + 0.1 mol/L Sodium Hydroxide Solution [Sodium Hydroxide (0.04 g) + Distilled Water (10 mL)] (0.72 mL) + Distilled Water (→100 mL)	Carboxylic acid, sulfonic acid	Effective for compounds that containing functional groups at pH 5 or lower.
Rhodamine B		Rhodamine B (100 mg) + Ethanol (→100 mL)	Long alkyl chain compounds, polycyclic aromatic hydrocarbon compounds	Red color developed on a pink background. Deep red color developed by UV (365 nm).

1. Translated by Akio Uemura: "Classic recipes for organic synthesis used readily in laboratories", Maruzen (2009).
2. Japanese Pharmacopoeia 17th Edition

• Each prescription method is a example and does not guarantee.
• This article is provided with the cooperation of the chemical portal site "Chem-Station" that summarizes, organizes, and provides mixed chemical information on the web.