TLC分析分离之总结.docx

1、TLC分析分离之总结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 she

2、ets) to see 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

3、remove these 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 a

4、pply moderate 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

5、 wavelength 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 (I

6、II) in 20ml 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 diphenylbora

7、te, see Ethanolamine 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

8、35ml semi-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 pla

9、te slowly 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

10、also be detected 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

11、Lester. Aniline 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 colo

12、rs on an almost 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 un

13、til maximum 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 a

14、cid in 18ml 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 hyd

15、rochloride 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 solutio

16、n of 1.23 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 (I

17、II) 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 satura

18、ted solution of antiomony (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

19、 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.

20、1% bromthymol blue in 10% aqueous ethanol made just alkaline with NH4OH Spray dried plate. Results: Compounds above produce blue-green colors; sensitivity 0.1-1g. Chloranil reagent For detection of phenols Spray with a solution of 1% tetrachloro-p-benzoquinone in toluene Chlorine / o-tolidine For de

21、tection 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/

22、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).

23、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% cop

24、per (II) sulfate in 10% phosphoric acid Heat 5-30min at 110C 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. Chromosulfuric acid See under Potassium dichromate / sulfuric a

25、cid 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 reagent For the detection of sweeteners saccharine & cyclamate Spray with a 0.2% solution of dichlorofluorescein in 96% ethanol

26、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-D

27、ichloroquinone 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-chlo

28、roimide 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-Dimethylamin

29、obenzaldehyde / hydrochloric acid reagent (Ehrlichs 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 S

30、pray 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

31、another glass plate, heat 30-40min at 110C 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-Diphenylpicrylhydrazy

32、l For detection of aldehydes and ketones Reagent: dissolve 15mg of 2,2-DPPH in 25ml chloroform Spray, heat 5-10min at 110C; 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