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1、TLC Visualization Reagents This is a brief selection of the many available TLC visualization reagents. Below each title is the type of compounds or structure which can be detected with the specific reagent. When beginning work with these reagents, acquire any MSDS (material safety data sheets) to se
2、e if there are any extra precautions needed in safely using them. Before spraying, plates should be well dried in the hood of residual solvents and components. Amines and organic acids used in the mobile phases may adversely affect the visualization reaction being attempted. If heating to remove the
3、se components is done, consideration should be given so that loss of components or their decomposition is avoided (by lowering the temperature or using a shorter time in the oven). Always spray any of these reagents onto plates in a well ventilated hood while wearing safety glasses. Also apply moder
4、ate amounts to the plate so it always appears dull and flat (if it looks wet, you have sprayed too much). You can always overspray to enhance the detection. Aluminium chloride For flavonoids Spray plate with a 1% ethanolic solution of aluminum chloride. Results: Yellow fluorescence in long wavelengt
5、h UV light (360nm) 4-Aminoantipyrine/potassium hexacyanferrate (III) (Emerson reaction) check something missing (Emerson reagent) for the detection of phenols and arylamines Solution I: 1g aminoantipyrine (4-aminophenazone) in 100ml 80% ethanol Solution II: 4g potassium hexacyanoferrate (III) in 20m
6、l water, fill to 100ml with ethanol Procedure: Spray with solution I Dry 5 minutes with warm air Place chromatogram in a chamber with vapor from 25% ammonium solution, making sure that the layer does not contact the liquid. Results: Red-orange to salmon pink spots 2-Aminoethyl diphenylborate, see Et
7、hanolamine diphenylborate Ammonium metavanadate, ammonium monovanadate, see Vanadium(V)sulfuric acid reagent Ammonium molydbate For detection of phosphoric acid derivatives Solution I: 1M perchloric acid in water/acetone (1:1) Solution II: ammonium molybdate soln: 5g (NH4)6Mo7O24.4 H2O in 35ml semi-
8、conc. Nitric acid and 65ml water. Solution III: Tin(II) chloride soln: 0.5g SnCl2.2 H2O in 100ml 0.5M hydrochloric acid: Dry developed chromatogram and heat to 60 C Hydrolyse di- and triphosphates by spraying perchloric acid (solution I) onto the warm plate. After spraying 2 times, dry plate slowly
9、at 50 C. Amidophosphates might not be decomposed. In any case, spray the still warm plate with ammonium molybdate solution (solution II) Then spray the still wet plate with tin (II) chloride solution (solution III) Results: Phosphates appear as blue to blue-green spots. Polyphosphates can also be de
10、tected by dipping the plates in a solution of ammonium molybdate (1g) dissolved in water (8ml) and perchloric acid (3ml, ca. 70%), filled up to 100ml with acetone. Then phosphates appear as yellow-green spots on a blue background. Also see Molybdenum blue reaction according to Dittmer and Lester. An
11、iline phthalate For the detection of reducing sugars Dry the developed chromatogram Spray with 0.93g aniline and 1.66g o-phthalic acid dissolved in 100ml n-butanol saturated with water. Briefly dry with hot air, then heat to 105 C for 10 minutes Results: Substance spots show different colors on an a
12、lmost colorless background. Some spots give fluorescence at 365nm. p-Anisaldehyde sulfuric acid For detection of phenols, sugars, steroids, and terpenes Spray with a solution of freshly prepared 0.5ml p-anisaldehyde in 50ml glacial acetic acid and 1ml 97% sulfuric acid. and heat to 105C until maximu
13、m visualization of spots. The background might be brightened by water vapor. Results: Lichen constituents, phenols, terpenes, sugars, and steroids turn violet, blue, red, grey or green. For detection of sugars Spray with a solution of freshly prepared 1ml p-anisaldehyde, 1ml 97% sulfuric acid in 18m
14、l ethanol and heat at 110C. Results: Sugar phenylhydrazones produce green-yellow spots in 3 min. Sugars will produce blue, green, violet spots in 10min. Also detects digitalis glycosides. p-Anisidine Hydrochloride For detection of carbohydrates / sugars Mix a solution of 3% p-anisidine hydrochloride
15、 in n-butanol Spray and heat at 100C for 2-10min. Results: Aldohexoses are seen as green-brown spots, ketohexoses as yellow spots, aldopentoses as green spots, and uronic acids as red spots. Anisidine phthalate reagent For detection of carbohydrates and reducing sugars Spray with a solution of 1.23
16、g p-anisidine and 1.66g phthalic acid in 100ml 95% ethanol. Results: Hexoses, green; pentoses, red-violet sensitivity 0.5ug; methylpentoses, yellow-green; uronic acids, brown sensitivity 0.1-0.2ug. Antimony (III) chloride For detection of flavonoids Spray with a 10% solution of antimony (III) chloride in chloroform Results: Fluorescing spots in long wavelengt
