TLC Visualization Reagents Part 2

Dear Friends,
I am going to post in this week my collection of a list of several derivatizing agents/ visualization indicators which will prove helpful to you also.

continued from last post......................

Antimony (III) chloride
For detection of flavonoids
Spray with a 10% solution of antimony (III) chloride in chloroform
Results: Fluorescing spots in long wavelength light (360nm).

Antimony (III) chloride
For detection of vitamins A & D, carotenoids, steroids, sapogenins, steroid glycosides, terpenes
Spray with a solution of 25g antimony (III) chloride in 75ml chloroform (generally a saturated solution of antimony (III) chloride in chloroform or carbon tetrachloride is used).
Heat 10min at 100C, view under long wavelength light (360nm).

Bromine / Carbon tetrachloride
For detection of organothiophosphorous pesticides
Place chromatogram in a chamber with a 10% bromine and tetrachloride without contact with the liquid.

Bromocresol green
For detection of organic acids
Dip chromatogram in a solution of 0.1g bromocresol green in 500ml ethanol and 5ml 0.1M NaOH
Results: Acids yield yellow spots on a blue background.

Bromthymol blue
For detection of lipids and phospholipids
Reagent: 0.1% bromthymol blue in 10% aqueous ethanol made just alkaline with NH4OH
Spray dried plate.
Results: Compounds above produce blue-green colors; sensitivity 0.1-1μg.

Chloranil reagent
For detection of phenols
Spray with a solution of 1% tetrachloro-p-benzoquinone in toluene

Chlorine / o-tolidine
For detection of of compounds forming chloroamines, e.g., urea derivatives, carbamated, antibiotics
Solution I: 160mg o-tolidine in 30ml glacial acetic acid, filled to 500ml with distilled water, plus 1g KI solution
Solution II: saturated solution of o-tolidine in 2% acetic acid/0.85% KI solution (1:1, v/v)
Procedure A
Place chromatogram 15-20min in a chlorine atmosphere (e.g., Potassium permanganate +10% Hydrochloric acid)
Leave 5 minutes at ambient temperature until the chlorine is evaporated completely (spray corner of plate to insure no blue color is seen, showing complete absence of chlorine).
Spray with solution I
Procedure B
Spray with 2% potassium hypochlorite solution in water
Leave 1-1.5hr at ambient temperature
Spray with solution II

Copper sulfate / phosphoric acid
Used as a charring reagent for polymer bound TLC plates (the newer hard layer plates)
Spray with a solution of 10% copper (II) sulfate in 10% phosphoric acid
Heat 5-30min at 110°C
Results: View frequently (every 5-10min) to see if colored or fluorescent spots (at 254 and 360nm) can be seen. Charring can be continued until spots are brown, grey or black.

DDQ Reagent (Dichlorodicyanobenzoquinone)
For detection of phenols
Spray with a solution of 2% 2,3-dichloro-5,6-dicyano-1,4-benzoquinone in toluene

Dichlorofluorescein
For the detection of sweeteners saccharine & cyclamate
Spray with a 0.2% solution of dichlorofluorescein in 96% ethanol
Dry with warm air; if necessary, spray with water
View under 360nm UV light

Dichlorofluorescein / fluorescein sodium salt
For detection of N-substituted barbiturates
Spray with a 0.1% ethanolic solution of dichlorofluorescein
Then spray with a 0.1% ethanolic solution of fluorescein sodium salt

2,6-Dichloroquinone -4- chloroimide
For detection of antioxidants, phenols, primary and secondary aliphatic amines, secondary and tertiary aromatic amines, aromatic hydrocarbons, pharmaceuticals, phenoxyacetic acid herbicides, etc
Spray with a freshly prepared 0.5-2% solution of 2,6-dichloroquinone-4-chloroimide in ethanol (reagent stable for 3 weeks if refrigerated).
Heat 10min at 110 C; treat with ammonia vapor

p-Dimethylaminobenzaldehyde
For detection of sulfonamides
Spray with a solution of 1% p-dimethylaminobenzaldehyde in 5% hydrochloric acid; add 5% ethanol
Detects sulfonamides

p-Dimethylaminobenzaldehyde / hydrochloric acid reagent (Ehrlich’s reagent)
For detection of amines, indole derivatives
Spray with a solution of 1% p-dimethylaminobenzaldehyde in conc. hydrochloric acid/methanol (2:2)
Heat plates for 20min at 50 C

2,4-Dinitrophenylhydrazine
For detection of aldehydes and ketones
Spray plate with solution of 0.4 g 2,4-DNPH in 100ml 2N hydrochloric acid, add 1ml ethanol
Results: Yellow-red spots will be seen.

Diphenylamine
For detection of glycosides, glycolipids
Reagent: 10ml 10% diphenylamine in ethanol, 100ml HCl and 80ml glacial acetic acid
Spray lightly, cover plate with another glass plate, heat 30-40min at 110°C until positive areas appear
Results: Glycolipids produce blue spots.

s-Diphenylcarbazone
For detection of barbiturates
Spary with a solution of 0.1% s-diphenylcarbazone in 95% ethanol
Results: Barbiturates will produce purple spots

2,2’-Diphenylpicrylhydrazyl
For detection of aldehydes and ketones
Reagent: dissolve 15mg of 2,2’-DPPH in 25ml chloroform
Spray, heat 5-10min at 110°C;
Results: Yellow spots on a purple background will be seen.

Dithizone
For detection of heavy metal ions
Dissolve 20mg dithizone in 100ml acetone, store in a brown bottle in a refrigerator
Procedure:
Spray with dithizone solution
Spray with 25% ammonia solution

Dragendorff reagent
For detection of nitrogen compounds, alkaloids, antiarrhythmic drugs, surfactants
Solution 1) 1.7g basic bismuth nitrate and 20g tartaric acid in 80ml water
Solution 2) 16 g potassium iodide in 40ml water
Stock solution (stable for several weeks in a refrigerator):
Mix equal volumes of solutions 1 and 2
Procedure:
Spray with a solution of 10g tartaric acid, 50ml water and 5ml stock solution