Linear Development

Linear development is usually performed in a rectangular vessel with ascending migration of the eluent through the adsorbent layer, from the bottom to the top of the chromatographic plate. The plate is usually positioned vertically in the developing chamber in a few millilitres of solvent. The separation of a standard lipid mixture by this mode of development is presented in Figure 1A and B by way of example. The separations were performed on a 10x10 cm pre-coated silica gel plate for high performance thin-layer chromatography (TLC), and on a conventional 20 x 20 cm plate (E. Merck) with eluent composed of methyl acetate-w-propanol-chloroform-methanol-0.25% aqueous potassium chloride (25 : 25 : 25 : 10 : 9). A conventional developing chamber (rectangular vessel) was lined with filter paper in order to ensure saturation of the vapour phase with the solvent. Samples were applied on the start line of the chrom-atographic plate in the form of streaks containing 0.5-3.0 igmL-1 phosphorous lipid. The chromato-graphic process was performed at room temperature and was stopped when the eluent reached the upper edge of the plate (50 min for high performance TLC and 150 min for conventional plates). Natural lipids, cerebrosides, sulfatides, phosphatidylethanolamine, phosphatidylinositol,

Figure 1 Chromatograms of standard lipids in the solvent methyl acetate-n-propanol-chloroform-methanol-0.25% aqueous KCI (25 : 25 : 25 : 10 : 9). (A) Separation on 10 x 10 cm HPTLC plate; (B) separation on classical pre-coated silica gel plate, 10 x20cm. NL, Neutral lipids; CER, cerebrosides; SULF, sulfatides; PE, phosphatidylethanolamine; PA, phosphatidic acid; DPG, cardiolipin; PI, phosphatidylinositol; PS, phosphatidylserine; PC, phosphatidylcholine; SPH, sphingomyelin. Staining: molybdate reagent. (Reproduced with permission from Vitiello F and Zanetta J-P (1973) Thin-layer chromatography of phospholipids. Journal of Chromatography 166: 637.)

Figure 1 Chromatograms of standard lipids in the solvent methyl acetate-n-propanol-chloroform-methanol-0.25% aqueous KCI (25 : 25 : 25 : 10 : 9). (A) Separation on 10 x 10 cm HPTLC plate; (B) separation on classical pre-coated silica gel plate, 10 x20cm. NL, Neutral lipids; CER, cerebrosides; SULF, sulfatides; PE, phosphatidylethanolamine; PA, phosphatidic acid; DPG, cardiolipin; PI, phosphatidylinositol; PS, phosphatidylserine; PC, phosphatidylcholine; SPH, sphingomyelin. Staining: molybdate reagent. (Reproduced with permission from Vitiello F and Zanetta J-P (1973) Thin-layer chromatography of phospholipids. Journal of Chromatography 166: 637.)

phosphatidylserine, phosphatidylcholine and sphingomyelin are well separated. In addition, cerebrosides with nonhydroxylated fatty acid chains are separated from those with hydroxylated chains. Similar resolution is observed for sulfatides. The minor brain phos-pholipids, phosphatic acid and diphosphatidyl-glycerol show the same migration distance but are well separated from phosphatidylethanolamine and phosphatidylinositol.

Slightly better efficiency can be obtained in horizontal chambers. Solvent migration is not dependent on gravity and equilibration between vapour and liquid is more rapid and uniform (inner chamber volume is small). A cross-section of the horizontal DS (Dzido, Soczewinski) chamber (Chromdes, Lublin, Poland) is shown in Figure 2. Eluent can be supplied to the chromatographic plate simultaneously from its opposite edges so that the number of separated samples can be doubled in comparison to development in a vertical chamber. An example of this type of linear development is illustrated in Figure 3. The samples of a test dye-stuff mixture were spotted along two opposite edges of the 10 x 20 cm high performance TLC plate coated with silica gel. The plate was developed with toluene from opposite directions simulta neously. The development stops when both eluent fronts meet each other in the middle of the plate.

Another variation of linear development can be performed by changing the eluent composition during the development process (stepwise or continuous gradient elution). Samples containing components of a wide range of polarity cannot be readily separated in a single isocratic development, but the application of a gradient mobile phase can improve the separation. Figure 4 demonstrates the application of a simple stepwise gradient to increase the separation efficiency of aromatic amines. The separation was performed in an equilibrium sandwich horizontal chamber which allows delivery of very small volumes of eluent to the plate. The glass plates (5 x 20 cm) were covered with a 0.25 mm layer of silica gel, dried in air and activated for 1 h at 80°C and 2 h at 130°C. The solutes were spotted 4 cm behind the solvent front as 0.5% benzene solutions to avoid solvent demixing. A marker (azobenzene, RF = 1 was spotted together with the samples to show the position of the solvent front. Two chromatograms were obtained isocratically; development with constant concentration of the mobile phase with (A) 5% methyl ethyl ketone in cyclohexane, (C) 50% methyl ethyl ketone

Figure 2 Cross-section of the horizontal DS chamber (Chromdes) (A) before and (B) during development from two opposite edges of the plate. 1, Reservoir cover plates; 2, eluent reservoirs; 3, eluent (black area); 4, chromatographic plate; 5, eluent distributor; 6, trough cover plates; 7, troughs; 8, body of the chamber; 9, glass cover plate.

Figure 2 Cross-section of the horizontal DS chamber (Chromdes) (A) before and (B) during development from two opposite edges of the plate. 1, Reservoir cover plates; 2, eluent reservoirs; 3, eluent (black area); 4, chromatographic plate; 5, eluent distributor; 6, trough cover plates; 7, troughs; 8, body of the chamber; 9, glass cover plate.

Figure 3 Separation of dyestuff mixture from opposite directions on silica gel high performance TLC plate with toluene. 1, 4-chloro-4'-dimethylaminoazobenzene; 2, fast yellow; 3, 2-nitroaniline; 4, 4-nitroaniline; 5, phenol red.

Figure 3 Separation of dyestuff mixture from opposite directions on silica gel high performance TLC plate with toluene. 1, 4-chloro-4'-dimethylaminoazobenzene; 2, fast yellow; 3, 2-nitroaniline; 4, 4-nitroaniline; 5, phenol red.

in cyclohexane and chromatogram (B) with a two-step gradient was performed in the following manner. The plate was first developed with 5% methyl ethyl ketone. When the azobenzene spot has reached the middle of the plate the development was continued with 50% methyl ethyl ketone until the eluent front (with the azobenzene spot) has migrated to the end of the plate which protruded from the chamber. The plate

Figure 4 RF values of aromatic amines obtained on silica gel plate. (a,c) Isocratic development with 5 and 50% solutions of methyl ethyl ketone in cyclohexane, respectively; (b) stepwise development with both solvents. Open squares, V/,V/-dimethylani-line; open triangles, /V-ethylaniline; open circles, aniline; diamonds, 2-phenylenediamine, filled squares, 3-phenylenediamine; filled triangles, 4-phenylenediamine; filled circles, 3-amino-pyridine. (Reproduced with permission from Soczewinski E and Czapinska K (1979) Stepwise gradient development in sandwich tanks for quasi-column thin-layer chromatography. Journal of Chromatography 168: 230.)

Figure 4 RF values of aromatic amines obtained on silica gel plate. (a,c) Isocratic development with 5 and 50% solutions of methyl ethyl ketone in cyclohexane, respectively; (b) stepwise development with both solvents. Open squares, V/,V/-dimethylani-line; open triangles, /V-ethylaniline; open circles, aniline; diamonds, 2-phenylenediamine, filled squares, 3-phenylenediamine; filled triangles, 4-phenylenediamine; filled circles, 3-amino-pyridine. (Reproduced with permission from Soczewinski E and Czapinska K (1979) Stepwise gradient development in sandwich tanks for quasi-column thin-layer chromatography. Journal of Chromatography 168: 230.)

was dried and the spots were detected by spraying with aqueous sodium hydrogen carbonate and then with bis-diazotized benzidine. All the spots are well separated using the two-step gradient development as opposed to separation by isocratic development.

Figure 5 shows the densitogram of a mixture of glycosides obtained by stepwise gradient development with seven eluent fractions which were applied consecutively to the plate (pre-coated silica gel glass plate for HPTLC, 10 x 10 cm, E. Merck) using a horizontal DS chamber. The volumes and compositions of eluent fractions as solutions of methanol in ethyl acetate were as follows: (1) 0.22 ml 0.0%; (2) 0.11 ml 20%; (3) 0.11 ml 30%; (4) 0.11 ml 2%; (5) 0.11 ml 10%; (6) 0.11ml 35%; (7) 0.11ml 100%. Each fraction was introduced into the mobile phase reservoir of the chamber with a micropipette after the previous one had been completely absorbed by the adsorbent layer. The plate was developed for a distance of 8 cm and the glycosides were detected by spraying with a solution of chloramine in trich-loroacetic acid, heating for 5-10 min at 100-110°C and scanned with a Shimadzu CS-930 densitometer at 360 nm. The densitogram shows relatively good resolution of the glycosides.

This kind of simple stepwise gradient elution can help solve difficult separation problems, especially for mixtures consisting of solutes with a wide range of polarity, e.g. plant extracts. However, for more complicated stepwise gradients, poorer retention repro-ducibility is obtained.

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