TLC常用展开剂及应用

1、 生物碱

（1）沉淀反应——碘化汞钾试剂 →白色或浅黄色沉淀 碘化铋钾试剂 →橘红色沉淀 碘—碘化钾试剂 →浅棕或暗棕色沉淀 硅钨酸试剂 →浅黄或黄棕色沉淀 磷钨酸试剂 →浅黄色沉淀

磷钼酸试剂 →白色或淡黄色沉淀 苦味酸试剂 →黄色结晶或非结晶形沉淀 鞣酸试剂 →棕黄色沉淀 氯化金试剂 →黄色结晶 氯化铂试剂 →白色结晶 雷氏铵盐 →红色无定形沉淀

（2）薄层层析检查：吸附剂——碱性氧化铝（Ⅲ级，干法铺板） 硅胶G（稀碱湿法铺板） 展开剂——氯仿：甲醇 显色——UV；碘化铋钾

2、 氨基酸、多肽、蛋白质

（1）加热沉淀试验：加热煮沸 →混浊或沉淀 （蛋白质）

+5%H2SO4（不加热）→混浊或沉淀

（2）双缩脲反应：+40%NaOH，1%CuSO4 →紫色、红色或紫红色（多肽、蛋白质） （3）茚三酮反应：+0.2%茚三酮试液 →蓝或蓝紫色（氨基酸、多肽、蛋白质） （4）吲哚醌反应：+吲哚醌试液 →各种颜色（氨基酸）

（5）Millon反应：+Hg，H2NO2 →红色（蛋白质分子中有酪氨酸组成）

（6）Hopkins-Cole反应：+乙醛酸，浓硫酸 →各色（蛋白质分子中有色氨酸组成） （7）氨基酸的薄层层析检查：吸附剂——硅胶G

展开剂—— n-BuOH，n-BuOH：HAc：H2O 显色剂——0.25%茚三酮试液 →紫红色斑点 3、 有机酸 （1）PH试纸检查

（2）溴酚兰试液：喷洒→蓝色背景黄色斑点

（3）薄层层析检查：吸附剂——硅胶G或酸性氧化铝 展开剂—— C6H6：EtOH

显色剂——0.1%溴酚兰试液→黄色 4、 酚类和鞣质

（1）FeCl3试剂：+1%FeCl3试液 →蓝、暗绿或蓝紫色 （2）三氯化铁-铁氰化钾试剂：喷洒→蓝色斑点

（3）香草醛-盐酸试剂：喷洒 →红色（间苯二酚、间苯三酚） （4）重氮盐试剂：+对硝基苯胺、亚硝酸钠 →红色 （5）薄层层析检查：吸附剂——硅胶G或纤维素

展开剂—— n-BuOH：HAc：H2O；15%HAc 显色剂——1% FeCl3试液

1%三氯化铁-1%铁氰化钾试液 →蓝、绿或黑色

鞣质与酚类的区别：+明胶 —— 沉淀

上清液 +1%FeCl3试液 →蓝、暗绿或蓝紫色

5、 糖和苷

（1）斐林试剂：+硫酸铜、酒石酸钾钠 —— 砖红色沉淀（还原糖） （—）+1%HCl +NaOH 沉淀（苷元） △30min 上清液（+）（多糖、苷） （2）Molish反应：+α-萘酚-浓硫酸 →紫红色环

（3）银镜反应：+0.1N硝酸银、5N氨水 →银褐色（还原糖） （4）薄层层析检查：：吸附剂——硅胶G或纤维素 展开剂—— n-BuOH：Pd：H2O；15%HAc 显色剂—— 苯胺-邻苯二甲酸 6、 皂苷

（1）泡末试验：振摇 →大量持续性泡末

+0.1M HCl 二管泡末高度相同（三萜皂苷） +0.1M NaOH 碱管高于酸管（甾体皂苷）

（2）溶血试验：+2%红血球悬浮液 →溶血

（3）Lieberman—Burchard反应：+醋酐-浓硫酸—— 紫红色（三萜皂苷） 黄-红-紫-污绿（甾体皂苷） 7、甾体

（1）Lieberman—Burchard反应：+醋酐-浓硫酸 →黄-红-紫-污绿 （2）氯仿-浓硫酸反应：+氯仿-浓硫酸 氯仿层→红或青色 硫酸层→绿色荧光

（3）五氯化锑或三氯化锑反应：+SbCl3或SbCl5 →红色 （4）薄层层析检查：吸附剂——中性氧化铝或硅胶G 展开剂—— C6H6-MeOH；CHCl3-MeOH 显色剂—— 10%磷钼酸 →蓝-蓝紫色 5%三氯化锑试液 →红、棕红或绿色 8、黄酮

（1）盐酸-镁粉反应：+HCl-Mg →红色 （2）三氯化铝反应：+AlCl3 →黄色

（3）浓氨水反应：+NH3 →亮黄或橙色

（4）薄层层析检查：吸附剂——聚酰胺或硅胶G 展开剂—— MeOH-H2O；EtOH-H2O 显色剂—— UV→亮黄或黄绿色荧光 1%三氯化铝试液→亮黄色

9、香豆素、内酯

（1）开闭环反应：+1%NaOH→澄清 +2%HCl→混浊

（2）异羟污酸铁反应：+7%盐酸羟胺、10%KOH △ +稀HCl、1%FeCl3 →红色 （3）重氮盐试剂：+对硝基苯胺、亚硝酸钠 →红色

（4）薄层层析检查：吸附剂——酸性硅胶G或硅胶G 或酸性氧化铝

展开剂—— 甲苯-乙酸乙酯-甲酸（5：4：1） 显色剂—— UV→蓝色荧光 异羟污酸铁试液 →红色 10、强心苷

（1）Kedde试剂：+3，5-二硝基苯甲酸试液 →紫红色 （2）Baljet试剂：+碱性苦味酸试液 →橙或橙红色 （3）Legal试剂：+亚硝酰铁氰化钠试液 →紫红色

（4）K-K反应：+FeCl3/冰HAc、浓H2SO4→ 上层绿~蓝色 （2-去氧糖） 界面红棕色

（5）薄层层析检查：吸附剂——硅胶G 或中性氧化铝 展开剂—— n-BuOH：HAc：H2O（4：1：5） 显色剂—— 碱性3，5-二硝基苯甲酸试液→紫红色 碱性苦味酸试液 →橙红色 11、蒽醌

（1）碱液反应：+10%NaOH →红色 +H2O2 →红色不褪 +H+ →红色褪去 （2）醋酸镁反应：+1%MgAc2 →红色 （3）薄层层析检查：吸附剂——硅胶G 展开剂——Pet：EtOAc 显色剂—— UV→黄色荧光 5%NaOH →红色 12、挥发油、油脂

（1）油斑检查：油斑挥发 →挥发油； 油斑不消失→油脂或类脂

（2）磷钼酸反应：喷洒5%磷钼酸试液 →蓝色（油脂、三萜、甾醇）

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, acquired any MSDS (material safety data sheets) 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 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 apply 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.

When information about the results of using the visualization reagents was available, this was put under each reagent as ‘Results’. If not give, the user will have to do a few experiments to see what the results might be.

Always remember to look under normal light and also short and long wavelength UV light

so as not to miss any possibilities.

Many of the reagents for these visualizers can be found in the EMD Chemicals catalog or on the website

(www.emdchemicals.com).

Aluminium chloride

For flavonoids

Spray plate with a 1% ethanolic solution of aluminum chloride. Results: Yellow fluorescence in long wavelength UV light (360nm)

4-Aminoantipyrine/potassium hexacyanferrate (III) (Emerson reaction) (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 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 diphenylborate, see Ethanolamine diphenylborate

Ammonium metavanadate, ammonium monovanadate, see Vanadium(V) / sulfuric acid 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-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 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 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 Lester.

Aniline phthalate（邻苯二甲酸胺） 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 almost colorless background. Some spots give

fluorescence at 365nm.

p-Anisaldehyde – sulfuric acid（对甲氧基苯甲醛-硫酸）

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 105°C until 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 acid in 18ml ethanol and heat at110°C.

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 in n-butanol Spray and heat at 100°C 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

For detection of carbohydrates and reducing sugars

Spray with a solution 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（三氯化锑）

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

antiomony (III) chloride in chloroform or carbon tetrachloride is used). Heat 10min at 100C, view under long wavelength light (360nm).

Bromine / Carbon tetrachloride（四氯化碳） For detection of organothiophosphorous pesticides

Place chromatogram in a chamber with a 10% bromine and tetrachloride without contact

with the liquid. 3

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（硫酸铜-磷酸）

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.

Chromosulfuric acid

See under Potassium dichromate / sulfuric acid 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（二氯荧光黄）

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（荧光钠）

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（二苯胺）

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（双硫腙）

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

Dittmer and Lester See Molybdenum blue

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

Ethanolamine diphenylborate (flavone reagent according to Neu) For detection of flavonoids

Spray with a 1% solution of ethanolamine diphenylborate in methanol

Spray with a 5% ethanolic solution of polyyethylene glycol for fluorescence stabilization Irradiate 2 minutes with intense 365nm UV light View under 365nm UV light Erhlich’s reagent

See p-Dimethylaminobenzaldehyde Emerson reagent

See 4-aminoantipyrine/potassium hexa-cyanoferrate (III) Fast Blue B reagent

For detection of cannabinoids, phenols, tanning agents, amines with can be coupled Spray with a solution of 0.5g Fast Blue B (tetraazotized di-o-anisidine) in acetone/water (9:1, v/v), always

prepared fresh

Then overspray with 0.1M sodium hydroxide solution Results: Cannabinoids turn dark red/purple in color Ferric Chloride / sulfuric acid

Used as a charring reagent for polymer bound TLC plates (the newer hard layer plates) Spray with a solution of 2g FeCl3 in 83ml n-butanol and 15ml conc. sulfuric 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. Flavone reagent according to Neu See ethanolamine diphenyl borate Fluorescamine

For detection of primary and secondary amines, peptides, sulfonamides, e.g., nitrosoamines after photolysis

Spray plate with a solution of 0.1mg/ml 4-phenyl-spiro[furan-2(3H),1-phthalan]-3,3-dione in acetone prepared

fresh daily

For stabilization of fluorescence at 366nm spray with 10g triethylamine, brought to 100ml with dichloromethane. Fluorescent Indicator

For detection of compounds which absorb UV light

Some TLC plates when manufactured have an inorganic fluorescent indicator added to the slurry poured to

make the final plates. This type of indicator will not dissolve or elute off. They are activated at 254nm or 360nm

(see recommendations of the manufacturer for that type of plate).

Results: When activated the fluorescent indicator will turn a green or white (depending on the indicator added)

and the compounds appear as dark spots or shadows against this background. If viewing at other than the

activation wavelength, the compounds might also have some fluorescence of their own, so various colors

against a dark background would be seen.

Formaldehyde / sulfuric acid

For detection of alkaloids, aromatic hydrocarbons, e.g., antihypertensive drugs

Spray with a solution of 37% formaldehyde in conc. sulfuric acid (1:10) immediately after taking the plate from

the developing chamber. Heating is not necessary. Results: various colored spots. Formaldehyde / phosphoric acid

For detection of steroid alkaloids, steroid sapogenins and phenothiazine derivatives 6

Spray with a solution of 0.03g formaldehyde in 100ml of 85% phosphoric acid with stirring at room temperature.

The reagent is stable for several weeks. Furfural / sulfuric acid

For detection of carbamate esters

Spray solution I: 1% solution of furfural in acetone Spray solution II: 10% solution of sulfuric acid in acetone Spray plate with I, then II. Gentian Violet – Bromine For detection of lipids

Spray 0.1% gentian violet (crystal violet) in methanol onto plate and place in a tank containing bromine vapor.

Results: lipids produce blue spots on a yellow background. Gibb’s reagent

For detection of phenols. For further applications see 2,6-dichloroquinone-4-chloroimide Spray with a solution of 3% 2,6-dibromo-N-chloro-p-benzoquinone imine in toluene or methanol.

Hydroxylamine / iron (III) chloride

For detection of amides, lactones, carboxylic acid esters and anhydrides

Solution 1) Mix 1 vol part of 7g hydroxylammonium chloride in 100ml methanol w 1 vol part of a solution of 7.2 g

potassium hydroxide in 100ml methanol. Filter from precipitated potassium chloride. Solution 2) 2% solution of iron (III) chloride in 1% aqueous hydrochloride acid Spray air dried plate first with solution 1, then with solution 2

Iodine containing compounds

Detection by decomposition under UV Dry plates at 100 C

After cooling spray with a small amount of 50% acetic acid Irradiate some minutes with unfiltered UV light.

Results: Iodine compounds show weakly violet to brown spots. The color can be enhanced by spraying with

10% acetic acid and irradiation with UV light (sudden appearance of blue spots).

Iodine vapor

Relatively unspecific universal reagent for many organic compounds Charge chamber with some crystals of iodine Place developed, dried chromatogram in iodine vapor Results: spots turn tan-brown in color

Iodoplatinate

For detection of organic nitrogen compounds, alkaloids, e.g., cocaine metabolites Spray with a freshly prepared mixture of 3ml hexachloroplatinic (IV) acid solution (10%) in 97ml/min water and

100ml aqueous potassium iodide solution.

Note - use 5% ethanol or methanol in water to prepare these solutions for the new

polymer bound TLC plates.

Iron (III) chloride / potassium hexacyanoferrate / sodium arsenate (according to Patterson & Clements)

For detection of iodine compounds, e..g., thyroid gland hormones Solution I: 2.7% iron (III) chloride hexahydrate in 2N hydrochloric acid

Solution II: 3.5% potassium hexacyanoferrate in water

Solution III: dissolve 3.8g arsenic trioxide in 25ml 2N sodium hydroxide solution heating slightly, cool to 5 C, 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. Spray onto the dry layer and dry carefully (temp below 50 C) Cover with glass plate and leave 15min in the dark

Results: Iodine containing compound show light blue spots on a yellowish background. Lead tetraacetate / 2,7-dichlorofluorescein

For detection of vicinal diols, glycosides and phenols, e.g., sugar acids Solution I: 2% (w/v) lead tetraacetate in glacial acetic acid Solution II: 1% (w/v) 2,7-dichlorofluorescein in ethanol

Mix 5ml of each solution 1 and 2, fill to 200ml with dry toluene. This reagent solution is stable for only about 2 hours.

Manganese / salicylaldehyde

For detection of organothiophosphorus pesticides

Solution I: dissolve 100mg manganese chloride (MnCl2.4H2O) in 100ml 80% alcohol Solution II: dissolve 1.3g 2-hydrozine quinoline in the lowest possible 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 –2-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.

Mandelin’s reagent

See Vanadium(V) / sulfuric acid

Mercury (II) chloride / diphenylcarbazone For detection of barbiturates

Solution I: 2% ethanolic mercury (II) chloride Solution II: 0.2% ethanolic diphenlycarbazone

Mix freshly before use in equal parts Results: Pink spots on a violet background

Mercury (II) chloride / dithizone For detection of barbiturates

Spray with a freshly prepared 1:1 mixture of 1-2% mercury (II) chloride in ethanol and 0.1-0.2% dithizone in ethanol.

View under 360nm UV light

4-Methoxybenzaldehyde / sulfuric acid / ethanol

For detection erythromycin and metabolites

Spray with 4-methocybenzaldehyde/sulfuric acid/ethanol (1:1:9) Heat 1 minute at 100 C Methyl yellow

For detection of chlorinated insecticides and antimicrobial compounds

Spray dried plate with a solution of 0.1g methyl yellow (N,N-dimethyl-4-phenylazoaniline) in 70ml ethanol, add

25ml water and fill to 100ml with ethanol. Dry at ambient temperature

Irradiate 5 min with UV light without a filter Results: Red spots on a yellow background

Molybdatophosphoric acid

See under Phosphomolybdic acid

Molybdenum blue reaction according to Dittmer and Lester For detection of phospholipids and phosphoric acid derivatives

Solution I: Boil 40.11g MoO3 in 1 liter 25N sulfuric acid for 3-4 hours until the molybdenum oxide is completely

dissolved. Let the light yellow solution slowly cool to ambient temperature overnight. The solution will turn light blue.

Solution II: Boil 1.78 g molybdenum powder and 500ml of solution I for 15min, cool and decant from the remaining residue. 8

For preparation of the spray reagent, add equal volumes of solutions I and II to 4.5 volume parts water. A dark

green solution is formed.

Solutions I and II are stable for several months when stored in the dark. The spray reagent has to be prepared weekly. Ninhydrin

For detection of amino acids, amines, amino sugars.

Spray with a solution of 0.2g ninhydrin in 100ml ethanol and heat to 110 C until spots appear.

Results: reddish spots appear

Ninhydrin / cadmium acetate

For detection of amino acids and heterocyclic amines

Dissolve 1g ninhydrin and 2.5g cadmium acetate in 10ml glacial acetic acid and fill to 500ml with ethanol.

Spray and heat 20min at 120 C

Results: Red, pink, or purple spots are seen.

Ninhydrin / pyridine / glacial acetic acid For detection of peptides

Spray with a 1% ninhydrin in pyridine/glacial acetic acid (5:1, v/v) Heat 5 min at 100 C

Nitric acid / ethanol

For detection of amines and alkaloids

Spray with a solution of 50 drops 65% nitric acid in 100ml ethanol (higher acid concentrations are also possible). If necessary, heat to 120 C for some time. Orcinol (Bials reagent)

For detection of glycosides, glycolipids

Reagent: dissolve 0.1g orcinol in 40.7ml conc. HCl, add 1ml 1% ferric (111) chloride, and dilute to 10ml

Spray and heat at 80°C for 90 minutes. Results: Glycolipids produce violet spots.

Patterson and Clements

See under Iron (III) chloride/potassium hexacyanoferrate/sodium arsenate Paraffin oil

For enhancement of fluorescence spots – more stable and greater intensity 1% paraffin oil in hexane

Spray evenly over the TLC plate

Results: spots should be more stable (no fading with time) for scanning and are of greater intensity

m-Phenylenediamine

For detection of reducing sugars

Spray with a solution of 3.6g m-phenylenediamine dihydrochloride in 100ml 70% ethanol and heat briefly at

105°C

Results: Intensely fluorescence colors in UV light (wavelength not specified, so check 254 and 366nm).

o-Phenylenediamine – trichloroacetic acid For detection of alpha-keto acids

Spray with a solution of 0.05g 1,2-phenylenediamine in 100ml 10% aqueous trichloroactic acid and heat plate at

100C for no more than 2 minutes.

Results: Green fluorescence spots in long wavelength UV light. p-Phenylenediamine – phthalic acid For detection of conjugated 3-ketosteroids 9

Spray with a solution of 0.9 p-phenylenediamine and 1.6g phthalic acid in 100ml 1-butanol saturated with water

and heat plate at 100-110°C Results: Yellow to orange spots

Phenylhydrazine sulfonate

For detection of some antimicrobial compounds

Solution I: dissolve 3.5g phenylhydrazine 4-sulfonic acid hemihydrate in 10ml water and 20ml 1N NaOH solution

Solution II: mix 30ml 1N sodium hydroxide solution with 40ml acetone The spray reagents have to be prepared fresh each time. Procedure:

Wet chromatogram evenly with spray solution 1

After air drying the plate, shake spray solution 2 and spray plate. Phosphoric acid

For detection of sterols, steroids, and bile acids

Spray heavily until the layer appears transparent with a solution of 85% phosphoric acid with water (1:1, v/v)

Then heat 10-15minutes at 120°C

Results: Sterols, steroids and bile acids and bile acids produce various colors under visible and UV light.

Phosphoric acid – bromine

For detection of digitalis glycosides

Spray solution I: 10% aqueous phosphoric acid solution

Spray solution II: Mix 2ml saturated aqueous potassium bromide, 2ml saturated solution aqueous potassium

bromate and 2ml 25% hydrochloric acid.

Procedure: Spray plate with I and heat 12 min at 120°C.

Results: Digitalis glycosides of the series B, D, and E show blue fluorescence in long wavelength UV light

Procedure continued: Heat the plate again at 120°C and spray lightly with II

Results: Glycosides of the series A show orange, of the series C show grey-green to grey-blue fluorescence in

UV light.

Phosphomolydbic acid

For detection of reducing substances, e.g, alcohols, bile acids, lipids, fatty acids, steroids Also used as a charring reagent for polymer bound TLC plates (the newer hard layer plates) Spray with a solution of 250mg molybdatophosphoric acid in 50ml ethanol Heat to 120 C until spots appear (oven or heat gun)

If necessary, treat with ammonia vapors to remove some background coloration. The reagent solution is stable for only 10 days even in the dark.

Results when using as a charring reagent: 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.

Phosphotungstic acid

For detection of cholesterol and its esters, reducing compounds, lipids, sterols, and steroids

Spray with 20% phosphotungstic acid in ethanol, heat at 110°C for 5-15min or until maximum visualization of the spots occurs

Results: Cholesterol, esters will produce red spots. Pinacryptol yellow

For detection of sweetners, surfactants, alkyl- and arylsulfonic acids

Dissolve 100mg pinacryptol yellow in 100ml hot water or ethanol (or some combination for polymer bound plates)

Spray with reagent solution

Results: Yellow to orange fluorescence spots under long wavelength UV light (366nm) Potassium dichromate / sulfuric acid (chromosulfuric acid) - see below

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Potassium permanganate / sulfuric acid – see below Rhodamine B

For detection of a wide variety of compounds

Dry the developed chromatogram and spray with 0.025-0.25% ethanolic rhodamine B reagent.

Results: In most cases red-violet zones with an intense fluorescence at 365nm develop on a pink background.

RP phases are less suited, because in this case the environment of the spots also forms an intense color. By

placing the sprayed chromatograms into an ammonia atmosphere, the detection sensitivity can be improved. Rhodamine 6 G

For detection of lipids

Spray plate with a solution of 1mg rhodamine 6 G in 100ml acetone

Inspect at long wavelength UV. Silver nitrate / hydrogen peroxide

For detection of halogenated hydrocarbons

Spray plate with a solution of 0.1g silver nitrate in 1ml water, add 10ml 2-phenoxyethanol, fill to 200ml with

acetone and add 1 drop hydrogen peroxide (30% solution)

Irradiate with unfiltered UV light until optimal contrast is obtained. For alumina plates, about 50 minutes, for

silica gel plates about 15 minutes

Results: dark spots are formed.

Luckow [Fensenius Z. Anal. Chem., 294, 288, (1979)] uses this combination without the addition of hydrogen

peroxide for the detection of pesticide ioxynil (3,5-diiodo-4-hydroxybenzonitrile). Sodium azide

For detection of antibiotics (penicillins and cephlosporins) Solution I: 0.5% solution of soluble starch

Solution II: 3.5% sodium azide in 0.1N iodine solution Procedure: spray with 1), dry, spray with 2)

Results: Detects penicillin, sensitivity 0.2μg. Penicillin and penicilloic acids are also detected by starch-iodineiodine

reagents, as are cephalosporins.

Sodium 1,2-napthaquinone-4-sulfonate (NZS reagent)

For detection of thiazide drugs, basic drugs with primary amino groups Solution I: 0.1N NaOH

Solution II: saturated solution of reagent in 1:1 ethanol: water Spray with I, then II.

Results: Thiazide drugs appear as orange spots within 15min; basic drugs with primary amino groups also react but barbiturates do not.

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, 1mol/L and heat at

100°C.

Results: Indoles turn red and thiazole derivatives turn light green. Sodium nitroprusside / hydrogen peroxide

For detection of guanidine, urea, thiourea and derivatives, creatine and creatinine.

Spray with a solution made up of 2ml 5% aqueous sodium nitroprusside, 1ml 10% aqueous sodium hydroxide

and 5ml 3% aqueous hydrogen peroxide and dilute with 15ml water. This solution can be stored several days in the refrigerator.

Sodium nitroprussate / potassium hexacyanoferrate (III)

For the detection of aliphatic nitrogen compounds, cyanamide, guanidine, urea, thiourea and derivatives,

creatine, and creatinine. 12

Spray solution III – for digitalis glycosides – dissolve 3.3g trichloroacetic acid in 10ml chloroform and add 1-2 drops hydrogen peroxide.

After applying the appropriate solution, heat 5-10min at 120°C. Inspect the spots in daylight and in long wavelength UV light. Trifluoroacetic acid

For detection of steroids

Spray with a solution of 1% trifluoroacetic acid in chloroform and heat 5min at 120°C Triethanolamine

For enhancement of fluorescence species 20% solution of reagent in isopropanol Tungstophosphoric acid

See under Phosphotungstic Acid Ultraviolet Light - fluorescence

For detection of various compounds which have native fluorescence.

With a plate containing a fluorescent indicator (F254), examine the dried chromatogram under 360nm UVlight.

With a TLC plate containing no fluorescent indicator, examine the dried chromatogram under both 254 and 360nm UV light.

Ultraviolet Light - quenching

For detection of various compounds which absorb 254nm UV light; quenching of TLC plate fluorescence.

Examine a fluorescent indicator (F254) containing plate after dried, under 254nm UV light. Compounds absorbing this wavelength light will appear as dark spots against a green or white fluorescence

caused by the UV activation of the fluorescent indicator in the plate. Urea / hydrochloric acid

For detection of sugars

Spray with a solution of 5g urea in 20ml 2M hydrochloric acid, with 100ml ethanol added and heat to 100°C.

Results: Ketoses and oligosaccharides containing ketoses turn blue.

Vanadium (V) / sulfuric acid

For detection of carbohydrates, glycols, reducing carboxylic acids, steroids, antioxidants, vitamins, phenols,

aromatic amines, antihistamines

a) Ammonium monovanadate (ammonium metavanadate) /sulfuric acid reagent

Spray with a solution of 1.2g ammonium monovanadate in 95ml water and 5ml conc. sulfuric acid or for beta

blockers: spray with saturated solution of ammonium monovanadate in conc. sulfuric acid. b) Vanadium pentoxide / sulfuric acid reagent

Spray with a solution of 1.82g vanadium pentoxide in 30ml 1M sodium carbonate, sonicate to achieve

complete dissolution, after cooling add 46ml 2.5M sulfuric acid and fill to 100ml with acetonitrile.

Vanillin / potassium hydroxide

For detection of amines and amino acids

Spray with a solution of 1g vanillin in 50ml 2-propanol and dry 10min at 110 C

Then spray with 1ml 1M potassium hydroxide solution filled up to 100ml with ethanol and dry 10 min at 110 C View at 365nm UV

Results: After spraying with a) ornithine will be seen as a bright green-yellow fluorescence, lysine as a greenyellow

fluorescence. After spraying with b) ornithine will show a salmon color that will fade; proline,

hydroxyproline, pipecolinic (pipecolic) acid and sarcosine will turn red after a few hours. Glycine will produce a

green brown spot, and other amino acids will turn faint brown. Vanillin / phosphoric acid

Used as a charring reagent for polymer bound TLC plates (the newer hard layer plates) Spray plate with 1g vanillin in 100ml 50% aqueous H3PO4

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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. Vanillin / sulfuric acid - see immediately below

The charring visualization reagents given below can only be used with glass TLC plates in

which a G (gypsum) binder has been used in the formulation to hold the silica gel on the plate:

Potassium dichromate / sulfuric acid (chromosulfuric acid)

Universal visualization reagent for organic compounds (e.g., alcohols, also for bile acids, lipids) – note: do not

use this reagent on polymer bound TLC plates since it will char the binder; use only with G

(gypsum) binder

plates.

Spray with a solution of 5g potassium dichromate in 100ml conc. Sulfuric acid. If necessary heat plate to 150 C

Results: Brown, grey, or black spots result Potassium permanganate / sulfuric acid

Universal reagent for organic compounds, e.g., fatty acid derivatives

Note: Do not use this reagent on polymer bound TLC plates since it will char the binder; use only with G (gypsum) binder plates.

Spray with a solution of 1.6% potassium permanganate in conc. sulfuric acid (make this solution very carefully,

and slowly to prevent any accidents). Heat plate for 15-20min at 180 C

Vanillin / sulfuric acid

For detection of steroids, Note, this reagent can only be used with G (gypsum) binder plates since it will char the

polymer binders in the harder layer plates.

Spray with a 1% solution of vanillin in conc. sulfuric acid and at 120 C until maximum color formation.

Another formulation of this reagent: 0.5g vanillin in 100ml sulfuric acid/ethanol (40:10). TLC Texts with Visualization Reagent Information:

Thin Layer Chromatography, 2nd edition, E. Stahl, ed, Springer-Verlag, NY, 1969

TLC Reagents & Detection Methods – Physical & Chemical Detection Methods: Fundamentals, Reagents,I, Vol

1a, H. Jork, W. Funk, W. Fishcer, & H. Wimmer, Wiley, NY, 1990

Practice of Thin Layer Chromatography, 3rd edition, J.C. Touchstone, Wiley-Interscience, NY, 1992

TLC Reagents & Detection Methods – Physical & Chemical Detection Methods: Activation Reactions, Reagent

Sequences, Reagents, II, Vol 1b, H. Jork, W. Funk, W. Fishcer, & H. Wimmer, Wiley, NY, 1994

Important Notes – Please read carefully :

1) When using the older texts, if they suggest putting the components into benzene, always substitute

toluene since it is much less toxic. Likewise, as mentioned above, if the components are added to pure

water, these solutions cannot penetrate well into the polymer bound layers (hard layers) now sold.

Substitute 5% methanol in water or 5% ethanol in water as the makeup solvent in these instances.

2) In past years various visualization reagents were made up with benzidine. It had been used for the

detection of terpene aldehydes, flavoinoids, carbohydrates and phenols. This reagent is no

longer

recommended for use since it is now classified as a carcinogen. Other suitable visualizers for the

detection of the above compounds can be found in this and the newer references listed above.

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3) A few words about the supports the layers might be coated onto and the use of these visualization

reagents (and subsequent heating). Most of these reagents can be used on silica gel, bonded silica gel,

or cellulose plates with glass, plastic, or aluminum supports. The exceptions are strong acid containing

visualizers which cannot be used with aluminum supports (which obviously would react with the

aluminum to dissolve it). Also when heating, plastic supports are limited to about 110°C only. Plastic

supported plates when heated at any temperature should be placed on a metal or glass plate in an oven

so they heat evenly. Some ovens have a metal mesh or grid, which would heat the plastic support

unevenly leading to warping and lifting of the layer. If you have questions, please consult the

manufacturer about the suitability of any procedure you intend on using.

ChromNotes related to TLC plate visualization (click on topic to link to it):

1) Charring for Detection on Glass, Aluminum, and Plastic Backed TLC/HPTLC Plates 2) Improving Detection Limits in TLC with Spot Compression and Fluorescence Enhancement

3) TLC Plate Visualization by Spraying and Dipping.