Preparation of Chromatography Spray Reagents
Preparation of Chromatography Spray Reagents
Preparation of Chromatography Spray Reagents
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dilute
Acids organic
Bromocresol Green
Aldehydes
Fuchsin
Alkaloids
Dragendorff's; Iodoplatinate
Amines
Ninhydrin
Antioxidants
Phosphomolybdic acid
Barbiturates
Mercuric diphenylcarbazone
Flavanoids
Antimony trichloride
Glycopids
Lipids
Phenols
Steroids
Terpenes
Antimony pentachloride
Vitamins
H
U
J
V
K
W
L
X
M
Y
Aluminium chloride
For flavonoids
Dissolve 1 gm aluminum chloride in 100 ml 95% ethanol
Look for yellow fluorescence in UV light (360nm)
4-Aminoantipyrine/potassium hexacyanferrate (III) (Emerson reaction) for the
detection of phenols and aryl amines
Solution I: 1g aminoantipyrine (4-aminophenazone) in 100ml 95% ethanol
Solution II: 4g potassium hexacyanoferrate (III) in 50 ml water and 50ml 95% ethanol
Spray with solution I
Dry 5 minutes with warm air(heat gun)
Spray with solutionn II
Dry again for 5 minutes
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methanol.
H
Hydroxylamine / iron (III) chloride For detection of amides, lactones, carboxylic
acid esters and anhydrides
Solution I: Make up a solution of 7g hydroxylammonium chloride in 100ml methanol
and a solution of 7.2 g potassium hydroxide in 100ml methanol. Mix both solutions
together and filter.
Solution II: Dissolve 2gm of iron (III) chloride and 1ml conc. HCl in 100ml water.
Dry plate in air
Spray with solution I, then with solution II
I
Iodoplatinate For detecting alkaloids, amines, and organic nitrogen compounds.
Dissolve 0.15gm potassium chloroplatinate and 3gm potassium iodide in 100ml dilute
hydrochloric acid.
Iodoplatinic acid For detection of alkaloids, cocaine metabolites, amines, analgesics
and biotin.
Dissolve 3ml. of chloroplatinic acid (hydrogen hexachloroplatinate) in 100ml. water,
then mix with a solution of 6gm of potassium iodide in 100ml water.
Iron (III) chloride / potassium hexacyanoferrate / sodium arsenate (according to
Patterson & Clements)
For detection of iodine compounds, e..g., thyroid gland hormones
Solution I: Dissolve 2.7gm iron (III) chloride hexahydrate in 100ml of 2N hydrochloric
acid
Solution II: Dissolve 3.5gm potassium hexacyanoferrate in 100ml water
Solution III: Dissolve 3.8g arsenic trioxide in 25ml 2N sodium hydroxide solution
heating slightly, cool to 5C, and add 50ml 2N sulfuric acid, fill to 200ml with water
Immediately before use mix 5ml solution I, 5ml solution II and 1ml solution III
Isatin For detecting proline, hydroxyproline and other amino acids and peptides.
0.2gm isatin in 100ml methanol.
JKL
Lead tetraacetate/2,7-dichlorofluorescein for detection of vicinal diols, glycosides
and phenols, e.g. sugar acids
Solutions:
1. 2gm lead tetraacetate in 100ml glacial acetic acid
2. 1gm 2,7-dichlorofluorescein in 100ml ethanol
Mix 5 ml each of solution 1 and 2 and make up up to 200 ml with dry toluene. This
reagent solution is stable for only about 2 hours.
M
Mandelin reagent See Vanadium(V) / sulfuric acid
Manganese / salicylaldehyde For detection of organothiophosphorus pesticides
Solution I: Dissolve 100mg manganese chloride (MnCl2.4H2O) in 100ml ethanol.
Solution II: Dissolve 1.3g 2-hydrozine quinoline in a minimum volume of hot ethanol.
Dissolve 1 g salicyaldehyde in 5ml ethanol and add 1-2 drops glacial acetic acid.
Combine both solutions and reflux 30 minutes. The crystals of salicyl-2-aldehyde
C2-quinolinehydrazone precipitated during the cooling are recrystallized from ethanol.
For solution dissolve 50mg of the salicylate derivative in 100ml ethanol
Spray with a mixture of equal volumes of solutions 1 and 2.
Mercury (II) chloride / diphenylcarbazone For detection of barbiturates
Solution I: Dissolve 2gm mercury (II) chloride in ethanol and make up to 100ml with
water.
Solution II: Dissolve 0.2gm diphenlycarbazone in ethanol and make up to 100ml with
water.
Mix equal parts of both solutions together just before use.
Mercury (II) chloride / dithizone For detection of barbiturates
Solution I: Dissolve 1.5gm mercury (II) chloride in 100ml ethanol.
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For detection of thiazide drugs, basic drugs with primary amino groups
Solution I: 0.1N NaOH
Solution II: saturated solution of reagent in 50 ml ethanol and 50 ml water.
Spray with I, then II.
Thiazide drugs appear as orange spots within 15min; basic drugs with primary amino
groups also react. However, barbiturates do not react.
Sodium nitrite / hydrochloric acid For detection of indoles and thiazoles
Spray plate with a freshly made solution of 1g sodium nitrite in 100ml hydrochloric
acid, and heat at 100C.
Indoles turn red and thiazole derivatives turn light green.
Sodium nitroprusside / hydrogen peroxide For detection of guanidine, urea,
thiourea and derivatives, creatine and creatinine.
Mix 2ml of 5% aqueous sodium nitroprusside, 1ml of 10% aqueous sodium hydroxide
and 5ml of 3% aqueous hydrogen peroxide. Dilute to 15 mls. with water.
Sodium nitroprussate / potassium hexacyanoferrate (III) For the detection of
aliphatic nitrogen compounds, cyanamide, guanidine, urea, thiourea and derivatives,
creatine, and creatinine.
1. Make up a solution with 15 mls 10% aqueous sodium hydroxide, 15 mls of 10%
sodium nitroprussate, 15 mls of 10% potassium hexacyanoferrate (III), and 45 mls of
water. Let the solution stand at room temperature for about half hour before use.
2. Mix the reagent solution with an equal part of acetone and spray.
Stannic Chloride See under Tin (IV) chloride
Sulphuric acid spray reagent
Charring reagent for organic compounds. For detection of bile acids
5% w/v of the acid in ethanol.
T
Tetracyanoethylene - TCNE reagent For detection of aromatic hydrocarbons and
heterocycles, aromatic amines, and phenols
Dissolve 0.5 - 1.0g tetracyanoethylene in 100ml dichloromethane or toluene.
Heat at 100C for a short time.
Tetranitrodiphenyl For detection of cardiac glycosides
Solution I: Saturated solution of 2,3,4,4-tetranitrodiphenyl in toluene
Solution II: Dissolve 10gm potassium hydroxide 50 ml water and 50 ml methanol
Spray with solution I, dry at room temperature, then spray with solution II.
Blue spots are observed.
Tetrazolium blue For detection of corticosteroids and other reducing compounds.
Solution 1. Dissolve 0.5gm tetrazolium blue 50ml water and 50 ml methanol.
Solution II. Make up 100 mls 6M NaOH.
Spray with an equal mixture of both solutions.
Violet spots at room temperature or with slight warming.
Thymol / sulfuric acid For detection of sugars
Spray with a solution of 0.5g thymol in 95ml ethanol, and add 5ml conc. sulfuric acid
with caution.
Heat 15-20min at 120C
Sugars show pink spots.
Tin (IV) chloride For detection of triterpenes, sterols, steroids, phenols, and
polyphenols
Spray with 10ml tin (IV) chloride in 80 mls of chloroform and 80 mls of glacial acetic
acid.
Heat the layer for 5-10min at 100C and inspect in visible and long wavelength UV
light.
TNF reagent (trinitrofluorenone) for detection of phenols
Spray with a solution of 2gm 2,4,7-trinitrofluorenone in 100 ml toluene
o-Tolidine, diazotized For detection of phenols
Tolidine solution - Make up 5g o-tolidine and 14ml conc. hydrochloric acid in 100ml
water
Nitrate solution - Dissolve 10gm sodium nitrate in 100 ml water, prepared fresh
Mix 20ml tolidine solution and 20ml nitrate solution at 0C stirring constantly.
The spray solution is stable for about 2-3 hours.
Note: After spraying it can take several hours until colored spots are formed.
p-Toluenesulfonic acid For detection of steroids, flavonoids and catechins
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WXYZ
None available
Study References:
1. Thin Layer Chromatography, 2nd edition, E. Stahl, ed, Springer-Verlag, NY, 1969
2. TLC Reagents & Detection Methods Physical & Chemical Detection Methods:
Fundamentals, Vol 1a, H. Jork, W. Funk, W. Fishcer, & H. Wimmer, Wiley, NY, 1990
3. Practice of Thin Layer Chromatography, 3rd edition, J.C. Touchstone, WileyInterscience, NY, 1992
4. Vogel's Textbook of Qualitative Inorganic Analysis by Vogel, A.I., 3rd, Ed.,
Longman (1961).
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Signature: Dhanlal De Lloyd, Chem. Dept, The University of The West Indies, St. Augustine campus
The Republic of Trinidad and Tobago.
Copyright: delloyd2000 All rights reserved.
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