Chromatograms liquid

Example of the application of liquid-solid chromatography to the analysis of amphetamines. (Chromatogram courtesy of Alltech Associates, Inc. Deerfield, IL). 

Typical (a) gas and (b) liquid chromatograms. The charts show amounts (y-axis) of substance emerging from a column versus time (x-axis). The time taken (measured at the top of a peak) for a substance to elute is called a retention time. 

A single peak from an ordinary liquid chromatogram (a) is revealed as two closely separated peak.s by resolution enhancement (b). 

Fig. 52 Fluorescence intensity of porphyrin chromatogram zones as a function of time after dipping in 50% liquid paraffin solution and storage in darkness. —...

Further examples pf fluorescence stabilization and intensity augmentation as a result of treatment of the chromatogram with viscous, lipophilic liquids are listed in Table 22. The alteration of the pH [293] or the addition of organic acids or bases [292] have also been found to be effective. Wintersteiger [291] has also described the effect that the TLC layer itself (binder) can influence the fluorescence intensity. 

Liquid paraffin digitoxm, digoxin, methyldigoxin enhancement the chromatogram was coated with a film of paraffin liquid [243]... 

Hydrophilic liquids can also cause stabilization and amplification of fluorescence Thus, Dunphy et al employed water or ethanol vapor to intensify the emissions of their chromatograms after treatment with 2, 7 dichlorofluorescein [260] Some groups of workers have pointed out that the layer matenal itself can affect the yield of fluorescent energy [261 —263] Thus, polyamide and cellulose layers were employed m addition to silica gel ones [245] The fluorescence yield was generally increased by a factor of 5 to 10 [264], but the increase can reach 100-fold [234, 265]... 

Detection and result The chromatogram was freed from mobile phase and dipped for 1 s in solution I and after drying for 1 min in a stream of cold air it was dipped in a solution of liquid paraffin — -hexane (1 + 2) in order to stabilize and increase the intensity of fluorescence by a factor of 1.5—2.5. The derivatives which were pale yellow in daylight after drying fluoresce pale blue to turquoise in long-wave... 

Note Subsequent immersion of the chromatogram in a mixture of liquid paraffin — n-hexane (1+2) leads to an increase in the fluorescence by a factor of 2.5 to 4.5 for some carbohydrates. 

The fluorescence intensity of the chromatogram zones could be stabilized and increased by a factor of 2.5 to 3.5 by subsequent immersion in liquid paraffin — -hexane (1 + 2). 

Detection and result The chromatogram was heated in the drying cupboard to 110 —120°C for 25 min and immediately placed — while still hot - in a twin-trough chamber, whose second trough contained 10 ml 25% ammonia solution, for 15 min. The chromatogram was then immersed for 2 s in a solution of liquid paraffin — n-hexane (1 + 2). 

Note If the chromatogram developed by method B was exposed to ammonia vapors for 10 min before being immersed in liquid paraffin— -hexane (1 -I- 2) the fluorescence of the chromatogram zones became deep red. Glucose and fructose also appeared red. 

The chromatogram was then immersed for 1 s in a mixture of liquid paraffin and -hexane (1 -I- 2) to stabilize and enhance the fluorescence (by a factor of ca. 1.5). The detection limits were then ca. 1 ng substance per chromatogram zone. 

Note A 5% solution of polyethylene glycol 4000 in ethanol can be sprayed onto the chromatogram [2, 4] for the purpose of increasing and stabilizing the fluorescence instead of dipping it in liquid paraffin - -hexane (1 - - 2). If this alternative is chosen the plate should not be analyzed for a further 30 min since it is only then that the full intensity of the fluorescence develops [6]. 

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. 

Additional dipping in a 0.001% solution of dansyl semipiperazide or bis-dansyl piperazide in dichloromethane — liquid paraffin (75 -F 25) stabilized the color of the chromatogram for a period of months. 

Note The reagent can be employed on silica gel, kieselguhr. Si 50 000 and RP layers [6]. The fluorescence intensities of the chromatogram zones can be increased by dipping in a solution of liquid paraffin in hexane or chloroform [8,11]. 

In situ quantitation The chromatogram was then dipped into liquid paraffin — -hcxanc (10 + 20) to increase the intensity of the fluorescence by a factor of ten and to stabilize it. The detection limit for testosterone is less than 500 pg per chromatogram zone (X c = 365 nm Xn > 430 nm). 

The result was that light blue fluorescent zones were visible under long-wavelength U V light (2 = 365 nm). Before fluorimetric analysis the chromatogram was dipped for 1 s into liquid paraffin — n-hexane (1 + 2) to enhance (by a factor of 2 to 8) and stabilize the intensity of the fluorescence and then dried for 1 min in a stream of cold air. The quantitation (2e,c = 365 nm An > 430 nm) was carried out after 1 h since it was only then that the fluorescence intensity had stabilized. 

Detection and result The chromatogram was dried in a stream of warm air for 1 min, after cooling it was immersed for 1 s in the reagent solution. After redrying in a stream of warm air it was dipped into a mixture of chloroform — liquid... 

In situ quantitation The chromatogram was immersed twice for ca. 1 s with brief intermediate drying in a mixture of chloroform — liquid paraffin and triethanolamine (60 -H 10 + 10) to stabilize the fluorescence (for ca. 24 h) and increase its intensity (by a factor of ca. 3). The analysis was made in UV light... 

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