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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

Retention-factor-tlc75

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 (pKa < 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

  • 1.5 g of potassium permanganate
  • 10 g of potassium carbonate
  • 1.25 mL of 10% sodium hydroxyde
  • 200 mL of water

Visualization Colors

  • Spots: Yellow to light brown
  • BG: Purple to pink

Stain Shelf-Life

  • Three months
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”.)