2 ,7 -Dichlorofluorescein reagent

Vitamin E compounds can be detected (about 20 /tg) as dark spots in UV light. They appear violet and detection is appreciably more sensitive (0.02 /tg) on layers that contain 0.02% Na-fluorescein. Moreover, these are visible in daylight as reddish spots (limit of detection 2 fig). The same effect is produced by spraying with fluorescein or dichlorofluorescein reagent. Nonspecific visualization procedures for tocopherols and tocotrienols are based on spraying with sulfuric acid, molybdophosphoric add, antimony(V) chloride, dipyridyl-iron reagent, nitric add, and copper(II) sulfate-phosphoric add [1-4]. 

The following are amongst the reagents that have been reported as being added to the mobile phase acids for quinine alkaloids [184], ninhydnn for amino acids [185 — 187], fluorescamine for biogenic amines [188] Fluorescein sodium [189], dichlorofluorescein [190], rhodamine 6G [191], ANS reagent [192] and bromine [193] have all been descnbed as additives to mobile phases... 

Note The full fluorescence intensity usually only develops about 30 min after the dipping process it then remains stable for several days if the chromatograms are stored in the dark (1, 5]. Fluorescein sodium can be employed in the reagent in place of 2, 7 -dichlorofluorescein [5]. The detection limits lie in the lower nanogram to picogram range [1, 5]. 

Unless otherwise noted all reagents were used as received and all samples were prepared in distilled-deionized water or ethanol (Aaper Chemical Co.). Benzo[a]pyrene, fluorescein, dimethylPOPOP, rhodaralne B, rhodamine, 6G, Naflon, and rubrene were all from Aldrich, 2,7-dichlorofluorescein was purchased from Kodak. All samples were prepared in the appropriate solvent and sonicated for at least 15 minutes. 

Lead tetraacetate-2,7-dichlorofluorescein (A) lead tetraacetate (saturated solution) in glacial acetic acid (B) 2,7-dichlorofluorescein in water (1%, m/v, or lower, down to 0.2%, if background interferes with detection) A and B are mixed 1 1 (v/v), then toluene (19 1, v/v) is added Plate is dipped in reagent for 10s, then heated at 100°C for 3 min All carbohydrates with vicinal diol groups, oxidized by lead tetraacetate, are detected fluorimetric scanning (excitation 313nm, emission 366 nm) 50-100pmol... 

Preliminary identification of many lipids may be obtained by the use of specific sprays on tic plates. Reagents such as 2, 7 -dichlorofluorescein and rhodamine 6G permit the nondestructive revelation of lipid bands and, if sufficient lipid is present, a water spray can also be used. Exposure to iodine vapor can be employed provided fatty acid analysis is not required. More specific chemicals include ninhydrin, Schiff-periodate, and Zinzadze s reagent (cf. Kates, 1972 Stahl, 1969). 

Procedure 10—100 mg of the lipid sample in hexane solution are applied as a band to a 0.5 mm thick silica gel G layer (20 X 20 cm). The plate is then held about 15 cm above a basin containing warm (50 to 60° C) concentrated hydrochloric acid, so that the layer faces the acid reagent. After 5 min, the plate is developed twice with hexane-diethyl ether (95 + 5). The fractions are visualised by spraying with 2, 7 -dichlorofluorescein solution (Rgt. No. 63) and inspecting in UV light and the aldehydes (near the solvent front) eluted with diethyl ether. 

Fig. 129. Separation of neutral lipids and their hydrolysis products by adsorption-TLC on silica gel G [114]. Solvent petrol ether (BP 60—70° C)-diethyl ether-acetic acid (90 + 10 + 1) time of run 40 min spray reagent 2, 7 dichlorofluorescein in ethanol 20 p,g of each apphed. a 9-octadecene 6 oleyl alcohol c oleyl aldehyde d oleic acid e methyl oleate / cholesteryl oleate g monoolein Ji diolehi i triolein j trilinolein k trihnolenin I tricaproin(a) and tristearin(]5) m cholesterol n selachyl alcohol o selachyl diolein p oleyl oleate q dioleoy 1-lecithin...

Fig. 142. Separation of the methyl eaters of saturated and unsaturated erytJiro- and reo-dihydroxy fatty acids [136]. Adsorbents silica gel G, silica gel G-silver nitrate, sihca gel G-boric acid, silica gel G-silver nitrate-boric acid the layers were prepared by spraying with saturated aqueous silver nitrate solution and saturated methanolic boric acid solution, dried and activated solvent hexane-diethyl ether (40 + 60) time of run 40 min spray reagent 2, 7 dichlorofluorescein in ethanol (UV light) amounts ca 30 jxg of each. 1 ert/ ro-9,10-dihydroxystearate 2 reo-9,10-dihydro-xystearate 3 er ro-12,13-dihydroxystearate 4 reo-12,13-dihydroxystearate 5 er2/ ro-12,13-dihydroxyoleate 6 jireo-12,13-dihydroxyoleate (see also [138])...

Methods of Detection. Ozonides may often be detected as white zones on silica gel G layers without using any indicator. 2, 7 -Dichlorofluorescein solution (Rgt. No. 64) is a convenient spray reagent chromic acid-sulphuric acid (Hgt. No. 46) can be used for charring ozonides [170]. 

Fig. 149. Separation of the triglycerides of sunflower oil (Helianthus annuus) by combination of argentatic i -TLC and reversed phase partition-TLC [89]. A pre-fractionation of the triglycerides through argentation-TLC adsorbent silica gel G-silver nitrate (25%) solvent benzene-diethyl ether (80 + 20) time of run 3.5 h spray reagent 2, 7 -dichlorofluorescein in ethanol (UV light) amount 20 mg B separation of fractions 1—9 (A) through reversed phase partition-TLC stationary phase paraffin on kieselguhr G solvent acetone-acetonitrile (80 + 20) (80% saturated with paraffin), developed twice time of run 40 min each time visualisation aqueous-alcoholic a-cyclodextrin solution-iodine vapour amounts ca 12 [xg of each of the fractions 1—9 and 20 jxg sunflower oil...

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