4.3 Development of Thin-Layer Chromatograms
4.3.2 Two-Dimensional Thin-Layer Chromatography
In an attempt to maximize the utilization of the space available on an existing layer, Consden et al. [69] experimented with a two-dimensional technique as early as the era of paper chromatography, both the direction of development and the solvent being changed after the first development.
This gives a theoretical increase in the capacity of the spots, so that this method should be ideal for the identification of mixtures with a large number of components.
Even in the very early years of TLC on precoated plates, two-dimensional TLC(2D- TLC)was of great importance in the analysis of metabolic diseases. With the aid of this TLC technique it was possible to carry out screening tests for a large number of pa- tients in a relatively short time, to test urine for anomalies in the amino acids present, and to detect hyperaminoacidity in urine [70].
The principle of the procedure is described below:
An amino acid standard and a small amount of urine are applied in turn to the same point on a cellulose-precoated plate. This is then developed two-dimensionally using two different solvent systems, causing all the amino acids to be separated from each other. These are then visualized by derivatization with ninhydrin. The amino acids are identified from the known pattern of spots produced by the standard which is applied at the same time. Anomalies are detected by the significant intensification of certain spots compared with the chromatogram of a normal urine [71]. Figure 68 gives a sche- matic diagram of the positioning of the urea amino acids after a 2D-TLC.
Substance identification by 2D-TLC is also often performed in the investigation of phytopharmaceuticals, which usually have complex compositions. In the analysis of ginseng roots, for example, development is performed in the 1st dimension with n- butanol + ethyl acetate + water and in then in the 2nd dimension with chloroform + methanol + water. After derivatization with the vanillin-sulfuric acid reagent, it was found that the critical substance pairs had been separated [71a].
From a logical point of view, 2D-TLC using the same solvent in two directions should be the best system. However, this does not usually lead to any additional in- formation, as all the substances would lie on the diagonal, as shown in the diagram in Fig. 69. This method of 2D-TLC only becomes interesting if a reaction has occurred between the two developments, and deviations from the diagonal line can be observed after the second development.
108 4 Solvent Systems, Developing Chambers and Development
Figure 68. Position of the urine amino acids on a 20 × 20 cm TLC plate after a two-dimensional development and subsequent derivatization with the ninhydrin reagent(from [71])
× Start point
Position of substances present in the standard solution and not visible after the first heating process
Position of substances present in the standard solution and in general only visible after the second heating process
Position of substances that become visible in most urine chromatograms but are not present in the standard solution for technical reasons
Position of substances that do not usually occur in urine and are also not present in the stan- dard
109 4.3 Development of Thin-Layer Chromatograms
Figure 69. Schematic diagram of a 2D-TLC in which with the same solvent system was used for developing in both dimensions The direction of flow in the two runs is indi- cated by arrows.
An example of this is described in a paper on greater celandine [72]. To demonstrate that the action of high-energy light on pure chelidonine leads to a chemical change, the development in the 1st dimension was followed by exposure of part of the plate to 254- nm UV light for 15 min, and the plate was then developed in the 2nd dimension using the same solvent system. After removal of the solvent, several decomposition products with a yellow fluorescence could be seen in 365-nm UV light. The plate was then ex- posed to short-wave UV light for a further 5 min. Only after this were zones with a yel- low fluorescence detected at the height of chelidonine on the reference lanes and the diagonals produced by development in two dimensions. The detection steps described
Figure 70. Scheme of a 2D-TLC in which part of the plate (chelidonine) was irradiated with UV light (254 nm) after the first run(showing all points fluorescing in 365-nm UV light)
× Application points
C Chelidonine
F1 and F2 Solvent system fronts
The flow direction in the two runs is indicated by arrows.
(a) The dark area shows the part of the plate irradiated with short-wave UV light after the first run
(b) After the second run, before further irradiation with UV light (c) After further irradiation with UV light
110 4 Solvent Systems, Developing Chambers and Development
here are represented graphically in Fig. 70a–c. Further details on the analysis of greater celandine are given in Section 6.2 “Irradiation with High-Energy Light”. See also [72a].
In analogy to greater celandine, the TLC described below is also a so-called SRS technique(separation-reaction-separation). Whereas in the first example there is a photochemical reaction in which the sorbent layer is unchanged, there is now a chemi- cal reaction with decomposition of any fluorescence indicator present. Heisig and Wichtl describe the use of this technique in a TLC reaction chamber [73]. In the deter- mination of plant glycosides, using the example of marigold flowers (Calendula offici- nalisflos), this very selective identification by 2D-TLC with an intermediate chemical change of the substance is described. As the procedure can be used with all flavonoid- containing drugs, it is briefly described below:
A sample is applied in spot form to the bottom edge of a TLC plate and in band form to the right-hand edge. The plate is first developed in a solvent system suitable for flavone glycosides (shown from bottom to top of Fig. 71). The left-hand chromato- gram is hydrolytically decomposed by treatment with 5 N hydrochloric acid followed by heating (8 min at 120 °C) in the TLC reaction chamber. Rotation through 90° gives the starting band. A reference solution containing the appropriate aglycons is now ap- plied to both sides of this and is then chromatographed in a solvent system suitable for these substances. Residues from the incomplete hydrolysis can be seen on the left- hand side of the plate. The solvent front of the second development run is vertical and is on the left of the right-hand chromatogram. If one imagines horizontal lines be- tween the two chromatograms of the first run, the aglycons lie on these lines on the right of the corresponding glycosides. Based on the reference substances applied before the second development, aglycon can be assigned to glycoside with certainty. In Fig. 71a sample of birch leaves is used to illustrate the SRS technique described here
A further example of the technique is the two-dimensional separation of the ginkgo flavonol glycosides with intermediate glycoside decomposition [73a].
Figure 71:see Photograph Section.