Reagents Solutions

Only established analytical reagent grade chemicals should be employed for the preparation of standards and reagent solutions and the treatment of samples. 

Reference to water implies the use of distilled or deionized water of equal quality. Unless otherwise specified, all dilutions to volume are done with water. 

Prepare 50pgml-1 stock solutions by diluting 50ml of each l.OOOgl-1 solution to 11. 

Lithium, sodium and potassium AES calibration standards. Pipet 5, 10, 20, 30, 40, 60 and 80-ml portions of the 50pgml-1 stock solutions into 1-1 

Calcium, AES and A AS, strontium AES and barium AES calibration standards. Pipet 5,10,15,20, 25, 30, 40 and 60-ml aliquots of the 50 /igml-1 stock solutions into 1-1 volumetric flasks. Add 500 ml of water, 10 drops of methyl orange solution, 40 ml of the LaCl3 + EDTA flame buffer and sufficient 15 M NH4OH (dropwise) to each flask to turn the color of the solution yellow and dilute to volume. Calcium, strontium or barium 0.25, 0.50, 0.75, 1.00, 1.25, 1.50, 2.00 and 3.00/igml-1. 

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Estrone, estradiol, estnol Dip silica gel foil 2 cm in saturated Fast Black Salt K. solution and dry in a stream of warm air. Apply sample solution, dip again in reagent solution and dry. Dip the TCL plate 2 cm in 4% pyridine-cyclohexane solution, dry at 100 to 200°C and develop the azo-dyestuffs that are formed. [294]... 

Until a few years ago the most common method of rendering colorless substances on chromatograms visible was to spray them with reagent solutions [115] An all-... 

The same applies to the use of spray pistols (spray guns and aerosol cans), the frequency of whose use ought probably to be reduced on account of the propellant gas (chlorofluorohydrocarbons) employed Manual depression of the button valve of the vertically held spray can shoots the propellant gas through a fine jet and drags the sucked-up reagent solution with it onto the vertically held chromatogram (water pump pnnciple)... 

The coating of the adsorbent layer with the reagent solution is more homogeneous than with even the most carefully carried out spraying process. 

TLC plate which is wetted with reagent solution when it leaves the dipping bath before laying it on the hotplate, laboratory bench or scanning stage. 

The developed chromatograms are briefly immersed in or evenly sprayed with the appropriate reagent solution. Solution I is employed for flavonoids [1, 3] and solution II for mycotoxins [5, 8, 12], phospholipids, triglycerides and cholesterol [14]. 

The chromatogram is freed from mobile phase and immersed for 1 s in the freshly prepared reagent solution and then heated to 105 to 110 °C for 5 to 10 min. Green, blue or purple fluorescence appears on a dark background under long-wavelength UV light (2 = 365 nm). 

The chromatogram is freed from mobile phase in the drying cupboard (10 min, 120°C) and immersed for 1 s in the reagent solution or sprayed homogeneously with it until the plate starts to appear transparent it is then dried briefly in a stream of warm air and heated to 125 —130 °C for 45 min. 

Storage The reagent solutions may be stored for several days. 

Detection and result The chromatogram was freed from mobile phase in a stream of warm air, immersed in the reagent solution for 1 s and heated to 120°C for 20 min. Intense yellow to brown zones of various hues were produced these appeared as dark zones on a fluorescent background under long-wavelength UV light (X = 365 nm). 

Note The developed chromatogram must be completely freed from nonpolar solvents before derivatization, otherwise an intense fluorescence will be stimulated over the whole plate. The fluorescence intensity of the chromatogram zones remains stable for ca. 40 min it decreases slowly as the layer dries out and can be returned to its original intensity by renewed immersion in the reagent solution or in water. 

Detection and result The developed chromatogram was freed from mobile phase by drying for 10 min at 110°C, allowed to cool and immersed for 1 s in the reagent solution. The plate was evaluated as rapidly as possible while it was moist since the fluorescent background increased in intensity as the plate dried out. Cholesterol appeared as a yellow-green fluorescent zone hR 20—25). 

Dipping solution Mix 2 ml antimony(V) chloride with 8 ml carbon tetrachloride. Storage The reagent solution should always be freshly prepared. 

The chromatograms are freed from mobile phase, immersed in the reagent solution for 1 s or sprayed homogeneously with it and then dried in a stream of cold air. 

Detection and result The chromatogram was freed from mobile phase and evenly sprayed with reagent solution. After a short time at room temperature violet-colored chromatogram zones appeared for the corticosteroids tetrahydrocortisol hRf 10-15), tetrahydrocortisone (hR( 15 — 20), prednisolone hRf 15-20), hydrocortisone (hR( 20 — 25), prednisone hRf 30 — 35), cortisone hRf 35 — 40), corticosterone hRf 45 — 50), cortexolone (Reichstein S., hRf 50 — 55), 11-dehydro-corticosterone hRf 60 — 65), 11-desoxycorticosterone hRf 75 — 80). 

Detection and result The chromatogram was dried in a current of warm air and either immersed in reagent solution I for 1 s or placed for 15 min in a twin-trough chamber in whose second trough 5 ml of dipping solution I had been placed ca. 10 min previously. If the chromatogram was derivatized by dipping it had to be dried for ca. 1 min in a stream of hot air and allowed to cool to room temperature. 

The chromatogram was then immersed for 1 s in reagent solution II to increase sensitivity and stabilize the fluorescence and then dried in a stream of hot air. 

The chromatograms stained with ninhydrin are immersed in the reagent solution for 1 s or sprayed evenly with it and then placed in the free half of a twin-trough chamber containing 25% ammonia solution. Apart from proline and hydroxyproline, which yield yellow copper complexes, all the amino acids yield reddish-colored chromatogram zones [3],... 

Detection and result The TLC plate was dried in the air for 30 min and heated to 110 °C for 10 min in order to remove the formic acid from the mobile phase, before immersing the chromatogram in the reagent solution for 10 s. 

Detection and result The chromatogram was freed from mobile phase (first dried for 5 min in a stream of cold air, then heated for 10 min at 110°C and allowed to cool), immersed for 5 s in the reagent solution and finally heated for 2 min at 110°C. Grey to grey-blue zones were formed on a light background. The following hRf values were obtained primidone QiR( 10-15) carbamazepine hRf 40 — 45) phenytoin hR( 50 — 55) phenobarbital hRf 60) ethosuximide hRf 75) hexobarbital hRf 90 — 95). 

The reagent solution may be kept for several days in the refrigerator. However, it should always be freshly prepared for quantitative analyses. 

DBA solutions should only be stored for a short time even in the refrigerator. On the other hand, DOOB reagent solution in bottles with ground-glass stoppers may be stored in the refrigerator for at least 2 weeks. 

The chromatogram is freed from mobile phase (stream of warm air, 15 min), immersed for 2 s in the reagent solution after cooling to room temperature and heated to 110— 120°C for 10—20 min. The chromatogram is then briefly immersed in liquid paraffin — n-hexane (1 + 6) in order to enhance and stabilize the fluorescence. 

Storage The reagent solutions should always be freshly made up and... 

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