TLC stains

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A TLC stain is used in TLC development to reveal compounds that are not visible by UV. Also, selective detection of compounds is possible by choosing the appropriate TLC stain.

Figure 1.A TLC plate seen under UV light

Contents

Introduction

After eluting a TLC plate, the first analysis technique should always be UV light. This technique is fast, non-destructive and allows staining to be done afterwards. Compounds that will typically appear are aromatic groups, α,β-unsaturated carbonyls, and other compounds containing extensive conjugation. When using "F254 silica gel" TLC plates (silica gel that fluoresces with a 254 nm absorption) these compounds will appear as dark spots (because they "block" the fluorescence by absorbing the UV light) on a green background. Spots should be lightly circled with a pencil while under the UV lamp as they will not be visible once taken out from under the UV lamp. The plate can then be stained using an adequate stain from the following list.

Recipe table

The following table lists TLC stains, along with their general usage, the components and a few notes on the stains. For detailed preparation procedures along with example TLCs scroll to the bottom.

Stain Used for Recipe Comments
p-Anisaldehyde (PAA) General purpose (especially nucleophiles)
  • 12.5 mL p-anisaldehyde
  • 5 mL AcOH
  • 465 mL EtOH
  • 17.5 mL conc. H2SO4

Requires heating
Range of colors on light pink background
Medium shelf life

Bromocresol Green

Acidic groups (pKa < 5)
Carboxylic acids

  • 0.04 g Bromocresol green
  • 100 mL EtOH
  • 0.1 M solution of NaOH (added until blue)

Typically doesn't require heating
Bright yellow spots on blue background

Ceric Ammonium Molybdate (CAM) General Purpose
  • 5 g Ce(SO4)2
  • 25 g (NH4)6Mo7O24·4H2O
  • 50 mL conc. H2SO4
  • 450 mL H2O

Requires heating
Dark blue spots on white or light blue background
Very long shelf life

Ceric Ammonium Sulfate Vinca alkaloids
  • 1 g (NH4)4Ce(SO4)4·2H2O
  • 100 mL 50% H3PO4
Ceric Sulfate

General purpose
(good for alkaloids)

  • 8 g Ce(SO4)2·2H2SO4
  • 100 mL 15% aq. H2SO4
Black spots on yellow-white background
Chromic acid Difficultly stainable compounds
  • 2.5 g potassium chromate (K2Cr2O7)
  • 100 mL 20% aq. H2SO4
2,4-Dinitrophenylhydrazine (DNP)

Aldehydes
Ketones

  • 12 g 2,4-dinitrophenylhydrazine
  • 60 mL conc. H2SO4
  • 80 mL H2O
  • 200 mL 95% EtOH

Requires heating
Yellow to red spots on light yellow background
Long shelf life

Dragendorff

Amines
Organic base

A:

  • 1.7 g Bi5O(OH)9(NO3)4
  • 80 mL H2O
  • 20 mL AcOH

B:

  • 40 g KI
  • 100 mL H2O

5 mL A + 5 mL B in 20 mL AcOH + 70 mL H2O

Dragendorff-Munier Amines
  • 10 g KI
  • 1.5 g Bi(NO3)3
  • 20 g Tartaric acid
  • 120 mL H2O
Ehrlich Reagent Amines
  • 0.5 g dimethylaminobenzaldehyde
  • 10 mL conc. H2SO4
  • 90 mL 95% EtOH
Ferric chloride Phenols
  • 1 g FeCl3
  • 50 mL MeOH
  • 50 mL H2O
Iodine

Unsaturated and
aromatic compounds

  • A few Iodine crystals
  • Silica Gel

Doesn't require heating
Orange to brown spots on light orange background
Spots fade away very rapidly
Very long shelf life

Morin hydrate General Purpose
  • 0.1 g C15H10O7·xH2O
  • 100 mL MeOH
Ninhydrin

Amino acids
Amines

  • 0.3 g Ninhydrin
  • 3 mL conc. H2SO4
  • 100 mL n-butanol
Phosphomolybdic acid (PMA) General purpose
  • 7 g phosphomolybdic acid
  • 100 mL EtOH

Requires heating
Dark green/black spots on light green background
Long shelf life

Potassium Permanganate (KMnO4)

Olefins
Readily oxidized groups

  • 3 g KMnO4
  • 20 g K2CO3
  • 5 mL 5% aq NaOH
  • 300 mL H2O

Alkenes/alkynes/aromatics usually stain without heating
Other oxidizable groups require heating
Yellow spots on purple background
Very long shelf life

Vanillin General purpose
  • 15 g vanillin
  • 250 mL EtOH
  • 2.5 mL conc. H2SO4

Requires heating
Range of colors
Medium shelf life

Procedures and examples

p-Anisaldehyde (PAA)

To a cold (0 °C) mixture of AcOH (5 mL) and absolute EtOH (465 mL) was added p-anisaldehyde (12.5 mL) followed by slow addition of sulfuric acid (17.5 mL). The resulting clear solution was warmed to rt and used as is. The excess was stored in the fridge.
This stain is light and oxidation sensitive and will gradually turn pink/orange. The stain should be kept in an aluminum-covered jar while in use, and the excess should be kept cold and in the dark. Once the stain turns dark red, it should be discarded and made fresh again.

Ceric Ammonium Molybdate (CAM) - Hanessian's Stain

In a dark hood, ceric sulfate (5 g) was added to a cold (0 °C) clear colorless stirring solution of ammonium molybdate (25 g) in water (450 mL). The resulting bright yellow cloudy solution was vigorously stirred and sulfuric acid (50 mL) was slowly added over 90 min resulting in a very exothermic reaction. The resulting clear gold solution was warmed to rt, poured in a jar for immediate use and the excess was stored in the fridge.
This stain can be kept for months on the bench and in the fridge. Decoloration to very pale yellow usually occurs but does not affect the stain.

Chromic acid (K2Cr2O7)

To a cold (0 °C) solution of sulfuric acid (100 mL, 20% v/v aq.) was slowly added potassium chromate (2.5 g). The resulting clear bright red/orange solution was warmed to rt and used as is. The excess was stored in the fridge.

2,4-Dinitrophenylhydrazine (DNP)

To a cold (0 °C) mixture of water (80 mL) and absolute EtOH (200 mL) was added 2,4-dinitrophenylhydrazine (12 g). To the resulting cloudy bright orange solution was slowly added sulfuric acid (60 mL) resulting in a clear orange solution that was warmed to rt and used as is. The excess was stored in the fridge.

Iodine (I2)

In a mortar, grind a few crystals of iodine with some silica gel. Transfer to a jar with a plastic screw cap.
Spots are visualized by leaving the plate in the chamber for a couple minutes until spots turn brown. Once taken out of the chamber, spots will rapidly fade away.
This stain can be kept for months on the bench. Once it fades to white (by I2 sublimation) it should be made fresh again.

Phosphomolybdic acid (PMA)

Phosphomolybdic acid (10 g) was added to cold (0 °C) absolute EtOH (100 g) and then warmed to rt. The resulting cloudy yellow suspension was filtered over filter paper to give a clear bright yellow solution that was used as is. The excess was stored in the fridge.
This stain can be kept for months on the bench and in the fridge. PMA stain will gradually turn green but does not affect the stain.

Potassium Permanganate (KMnO4)

In a 1000 mL erlenmeyer flask was added sequentially water (600 mL), 5% aq. NaOH (10 mL) and K2CO3 (40 g). Once the solution became clear, KMnO4 (6 g) was added resulting in a dark purple solution. The solution was stirred 1 h at rt. The resulting purple solution was poured in a jar for immediate use and the excess was stored in the fridge.
This stain can be kept for months on the bench and in the fridge.

Vanillin

To a cold (0 °C) clear colorless solution of vanillin (15 g) in absolute EtOH (250 mL) was slowly added sulfuric acid (2.5 mL). The resulting clear solution was warmed to rt and used as is. The excess was stored in the fridge.
This stain is light and oxidation sensitive and will gradually turn dark. The stain should be kept in an aluminum-covered jar while in use, and the excess should be kept cold and in the dark. Once the stain turns black, it should be discarded and made fresh again.

Figure 2. TLC plates stained with CAM, PMA, DNP, Vanillin, KMnO4, p-anisaldehyde and I2
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