SiliaPlate - TLC Visualization Methods

TLC Plate Visualization

If components of the reaction are colored, no visualization method is required (spots can be seen directly on the silica layer). However, most of the time it is not the case, therefore one of the methods described below should be used to reveal the spots.

Non-destructive methods

As a general visualization procedure, before treating the TLC plate with any destructive methods, UV-active compounds can be viewed under an ultraviolet lamp (usually for polyconjugated compounds like benzophenones and anthracenes). Furthermore, an iodine chamber can be useful for thiols, phosphines, and alkenes but it works in about 50% of cases for alkanes. It is recommended to circle the spots with a pencil on the TLC plate prior to visualization by destructive methods.

Destructive methods

For compounds that are not UV-active, there are several varieties of stains that can be used depending on the nature of the compound of interest. To use a stain, simply dip the TLC plate into the staining solution as quickly as possible, and then immediately absorb the excess stain with paper and heat carefully with a heat gun or on a hot plate at 110°C until spots are revealed. See next two pages.

Chromatogram Interpretation

Retention factor (Rf) definition

Retention factor analysis is used to evaluate if the solvent system is adequate. Rf is defined as the distance traveled by the compound divided by the distance traveled by the solvent front. This means: the larger the Rf value of a compound, the larger is the distance traveled by the compound. In other words, when comparing Rf values of various compounds under identical chromatography conditions, the compound with the larger Rf is less polar because it interacts less strongly with the polar adsorbent on the plate.

Remember, a good solvent system is one that moves all components off the baseline with Rf values between 0.15 and 0.85 (ideal Rf is 0.2 - 0.4). Otherwise, when possible, it is preferable to chose another solvent system.

Retention factor (Rf) =

distance traveled by the compound
_____________________________

distance traveled by the solvent front

Rf calculation based on the example shown here:
Rf = 4.0 cm / 5.5 cm = 0.73

Prediction of Column Volumes (CV)

TLC data can be used to predict column elution based on the relationship between the retention factor and the column volume. CV is the number of column volumes required to elute the component from the column regardless of column dimensions [(bed volume) – (volume of packing)].

CV = 1 / Rf & ΔCV = 1 / Rf₁ – 1/ Rf₂

The greater the ΔCV, the greater will be the separation and resolution between the spots (easier separation). A bigger ΔCV will therefore allow more sample to be loaded onto the column.

Described below, are the most frequently used TLC visualization methods (also called stains) in alphabetical order.

Stains for Thin Layer Chromatography
Name	Visualization of...	Conversion / Selectivity (%)	Comments
p-Anisaldehyde #1	Universal Stain Good for nucleophiles and oxygenated compounds	Prepare stain as follows 2 mL of glacial acetic acid 5 mL of p-anisaldehyde 7 mL of conc. sulfuric acid 185 mL 95% ethanol Tip: Add dropwise the acid at the end and stir vigorously.	Visualization Colors Spots: Various colors BG: Orange to pink Appropriate Storage Aluminum wrapped at 0°C
N.B.: Tends to be insensitive to alkenes, alkynes and aromatic compounds unless other functional groups are present.
p-Anisaldehyde #2	Acronycine Cineoles Terpenes	Prepare stain as follows [1:10:20:80] p-anisaldehyde perchloric acid 7 mL of conc. sulfuric acid acetone water	Visualization Colors Spots: Various colors BG: Orange to pink Appropriate Storage Aluminum wrapped at 0°C
Bromocresol Green	Acidic groups (pK_a < 5) Carboxylic Acids	Prepare stain as follows 0.04 g of bromocresol green 100 mL of 95% ethanol 0.1 M solution of sodium hydroxide Tip: Add the base slowly at the end until the solution turns pale blue.	Visualization Colors Spots: Yellow to green BG: Blue Appropriate Storage Aluminum wrapped at 0°C Heating NOT required
Cerium Molybdate (CAM or Hanessian’s Stain)	Universal Stain Good for peptides	Prepare stain as follows 12 g of ammonium molybdate 0.5 g of ceric ammonium molybdate 15 mL of conc. sulfuric acid 235 mL of water	Visualization Colors Spots: Blue BG: White Appropriate Storage Aluminum wrapped
N.B.: Highly sensitive stain; very low concentration of product may appear as a significant impurity.
Cerium Sulfate (Ce(SO₄)₂)	Difficultly stainable compounds	Prepare stain as follows 15% aqueous sulphuric acid saturated with ceric sulfate	Visualization Colors Spots: Black BG: Yellow to white
Chromic Acid	Difficultly stainable compounds	Prepare stain as follows 2.5 g of potassium chromate 100 mL of 20% sulfuric acid in water	Visualization Colors Spots: Orange to green BG: Yellow to red
Cobalt Chloride (CoCl₂)	Universal stain Used in conjunction with PMA when this one is not effective enough	Prepare stain as follows 2 g of cobalt chloride 100 mL of water 10 mL of conc. sulfuric acid Tip: simply dip PMA treated plate in CoCl₂ solution.	Visualization Colors Spots: Various colors BG: Pink Heating NOT required
p-Dimethylamino- benzaldehyde (PDAB or Ehrlich’s Reagent)	Amines Indoles	Prepare stain as follows 0.5 g of p-dimethylamin-benzaldehyde 10 mL of conc. hydrochloric acid 40 mL of acetone (or 95% ethanol)	Visualization Colors Blue
2,4-Dinitrophenyl-hydrazine (DNP)	Aldehydes Ketones	Prepare stain as follows 12 g of 2,4-dinitrophenylhydrazine 60 mL of conc. sulfuric acid 80 mL of water 200 mL of 95% ethanol	Visualization Colors Spots: Yellow to red BG: Light orange DO NOT HEAT dipped plate
Dragendorff Reagent	Nitrogenous Compounds Alkaloids, amines, organics bases, etc. Phenols	Prepare stain as follows Solution A 1.7 g of bismuth nitrate 80 mL of water 20 mL of acetic acid Solution B 40 g of potassium iodide 100 mL of water Tip: mix 5 mL of each solution A and B to a solution of 20 mL of acetic acid in 70 mL of water.	Visualization Colors Spots: Orange to red BG: Yellow Appropriate Storage Aluminum wrapped Stain Shelf-Life One or two weeks Solutions A and B are long term storable DO NOT HEAT dipped plate
Ferric Chloride (FeCl₃)	Phenols	Prepare stain as follows 2 g of ferric chloride 102 mL of 0.5N hydrochloric acid	Visualization Colors Spots: Red BG: Yellow
Iodine	Unsaturated & Aromatic Compounds	Prepare stain as follows Iodine crystals in an amber bottle	Visualization Colors Spots: Dark brown BG: Light brown
N.B.: iodine stain can be removed by heating.
Morin Hydrate (Hydroxy Flavone)	Universal stain Fluorescently active	Prepare stain as follows 0.1% of morin hydrate in methanol Tip: by weight.	Visualization Colors Spots: Various colors BG: White
Ninhydrin (Indanetrione Hydrate)	Amino Acids Amino Sugars Amines	Prepare stain as follows 1.5 g of ninhydrin 3 mL acetic acid 100 mL of n-butanol	Visualization Colors Spots: Various colors BG: White
Phosphomolybdic Acid (PMA)	Universal stain Very effective against dilute sample	Prepare stain as follows 10 % of PMA solution in ethanol or 10 g of PMA in 100 mL ethanol	Visualization Colors Spots: Dark green to black BG: Light green
N.B.: Can be used for detection of alcohols, amines, sulfides and mercaptans groups when gently heated.
Vanillin	Universal stain Very effective for same polarity products (Rf)	Prepare stain as follows 15 g of vanillin 250 mL of 95% ethanol 2.5 mL of conc. sulfuric acid	Visualization Colors Spots: Various colors BG: Light tan
(N.B.: Shaded lines refer to “universal stains”. Occasionally, spots can be seen more clearly from glass side with glass backed TLC plate. Otherwise mentioned, stains are long-term stable when stored in a tightly-closed container to prevent solvent evaporation. “BG” stands for “background”.)

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SiliaPlate - TLC Visualization Methods

TLC Plate Visualization

Non-destructive methods

Destructive methods

Chromatogram Interpretation

Retention factor (Rf) definition

N.B.: Tends to be insensitive to alkenes, alkynes and aromatic compounds unless other functional groups are present.

N.B.: Highly sensitive stain; very low concentration of product may appear as a significant impurity.

N.B.: iodine stain can be removed by heating.

N.B.: Can be used for detection of alcohols, amines, sulfides and mercaptans groups when gently heated.

Glass-backed

Analytical TLC

Analytical HPTLC

Preparative TLC

Aluminum-backed

Analytical TLC

Analytical HPTLC

Plastic-backed

Analytical TLC

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