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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
Dinitrophenyl-hydrazine (DNP) 2,4-Dinitrophenyl- hydrazine (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. Basic Bismuth (III) nitrate (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.

  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.

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