Thin-layer chromatograms, cellulose

Flavones (fluorescence enhancement). Flavones have been used to detect pesticides on cellulose thin-layer chromatograms (2 1). Fluorogenic reagents such as Fisetin (III) are relatively non-fluorescent on a non-polar background like cellulose. The presence of... 

Figure 21.12. Comparative TLC and PC separation of nucleotides. A Cellulose thin-layer chromatogram—development distance, 10 cm in 91 min. B Paper chromatogram run under identical conditions—development distance, 10 cm in 134 min paper Schleicher and Schull 20436. The solvent used for both was saturated ammonium sulfatejl M sodium acetate isopropanol (80 18 2). Each vertical column of spots corresponds to separate mixtures separated. Samples (1) 3 -AMP (2) 2 -AMP (3) 3 -GMP (4) I -GMP (5) 2 - and3 -GMP (6) 2 - and3 -UMP (7) 5 -AMP (8) 5 -ADP (9) 5 -ATP. (,A = adenosine, G = guanine, C = cytidine, M = mono-, D = di-, T = tri-, P = phosphate.) From K. Randerath, Biochem. Biophys. Res. Comm., 6, 452 (1961-62), by permission of Academic Press.

Yisualisation on cellulose thin-layer chromatograms can be carried out just as in paper chromatography. 

The blue-violet stain which forms on thin-layer chromatograms when amino acids are stained with ninhydrin is only stable for a short time. It rapidly begins to fade even on cellulose layers. The stability can be appreciably enhanced by complex formation with metal ions [3]. 

Fig. 2.11. Thin-layer chromatogram and corresponding peaks for some nucleotides separated on purified cellulose. The arrows indicate the direction of the reflectance scan.

Rump [17] has described a cellulose thin layer method for the detection of phenolic acids such as iw-hydroxybenzoic acid, iw-hydroxyphenylacetic acid and m-hydroxyphenylpropionic acid, in water samples suspected to be contaminated with liquid manure. The phenolic acid is extracted with ethyl acetate from a volume of acidified sample equalling lmg of oxygen consumed (measured with potassium permanganate). The ethyl acetate is evaporated and the residue dissolved in ethanol. After spotting of a lpm aliquot on a cellulose plate the chromatogram is developed by capillary ascent with the solvent n-propanol-w-butanol-25% NH3-water (4 4 1 1 by vol). The solvent front is allowed to advance 10cm. The air-dried plate is sprayed with a diazotised p-nitroanilinc reagent to make the phenolic acids visible. 

Likewise, the luminescence properties of many analytes can be altered in the presenoe of surfactant aggregates (4,7.,8.). Consequently, addition of micelle-forming surfactants (present either in the LC mobile phase or added post-column) can improve the sensitivity of fluorimetric LC detectors (49,482). Micellar spray reagents have been utilized to enhance the fluorescence densitometric detection of dansylamino acids or polycyclic aromatic hydrocarbons (483). The effect was observed for TLC performed on cellulose or polyamide stationary phases with the micellar spray reagent being either CTAC, SB-12, or NaC (483). More recently, use of nonionic Triton X-100 has been found to improve the HPLC detection of morphine by fluorescence determination after post-column derivatization (486) as well as improve the N-chlorination procedure for the detection of amines, amides, and related compounds on thin-layer chromatograms (488). 

Fig. 176. Thin-layer chromatogram of rat bile (1 hour collection) after intravenous injection of 20 mg/kg bromosulphalein. Layer cellulose solvent butanol-acetic acid-water (40 H- 10 -f 50) [186]...

Adenine may be detected specifically on silica gel and cellulose by using the Dragendorff-reagent (No. 98). All the reactions for detection of pentoses in nucleosides are useful for identifying these compounds on thin-layer chromatograms. 

Conventional thin-layer chromatography (TLC) in our experience, known under the name planar chromatography, uses horizontal or vertical glass or Teflon chambers for the development of chromatograms. As stationary phases, commonly known adsorbents or supports based on silica gel, aluminium oxide, magnesium silica, cellulose, and so forth are used particle sizes are about 20 jitm. The migration of the mobile phase is based on the phenomenon of capillary forces. This chromatographic method is described, in detail, in other sections of this volume. 

A very complex mononucleotide mixture can be separated by 2-D TLC on poly(ethyleneimine)-cellulose anionexchange thin layers. After applying the nucleotide solution at the starting point, the chromatogram is developed in a closed rectangular jar by stepwise elution with LiCl solution of 0.2, 1.0, and, finally, 1.6 M. In... 

Fig. 3. Schematic chromatogram of the ion-exchange thin-layer chromatography of free and conjugated bile acids. Ion exchanger polyethylenimine-cellulose (250-/ -thick layer, 20 X 20 mm plate) solvent 100 ml 1 iV acetic acid, 50 ml absolute ethanol development time 120-150 min. Amount applied 5-10 fxg of each acid detection 0.05% 2,7-dichlorofluorescein in 50% methanol, under UV light. Bile acid conjugates identified as in Fig. 2 C, D, and L free cholic, deoxycholic, and lithocholic acids, respectively FA common fatty acids.

In addition to the references cited above (83-91), Kirchner (147) has presented considerable information on TLC analysis of DNP-amino acids based on the literature available up to 1970. Grant and Wicken (148) prepared thin layers (5 plates of 20 x 20 cm x 0.25 mm) from a mixture of 10 g of cellulose MN-3(X) and 4 g of silica gel H (Merck), homogenized in 80 ml of water. The plates were dried overnight at 37 C and developed in the first dimension in two solvents successively, viz., I ro-propanol-acetic acid-H20 (75 10 15) for 15 min and n-butanol-0.15N ammonium hydroxide (1 1, upper phase). The dried chromatograms were developed in 1.5 M sodium phosphate buffer (pH 6.0) in the second dimension. 

Thin Layer Chromatography.- The first separation of all the constituent sugars (seven neutral, two acidic) of plant cell-wall polysaccharides on a single chromatogram was achieved by two-dimensional t.l.c. on cellulose. The values for 12 sugars in 15 solvent... 

The thin-layer chromatography was carried out on plates coated with silica gel or cellulose containing a fluorescent indicator. A chloroform-methanol-15 M ammonium hydroxide (volume ratios, 100 30 5) system was used for developing the chromatograms. 

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