Chromatogram linear development

Isocratic linear development is the most popular mode of chromatogram development in analytical and preparative planar chromatography. It can be easily performed in horizontal chambers of all types. The mobile phase in the reservoir is brought into contact with the adsorbent layer, and then the movement of the eluent front takes place. Chromatogram development is stopped when the mobile phase front reaches the desired position. Usually 20 X 20 cm and 10 X 20 cm plates are applied for preparative separations, and this makes the migration distance equal to about 18 cm. Due to the fact that the migration distance varies with time according to the equation Z, = (Z, c, and t are the distance of the solvent front traveled, constant,... 

Separation of dTTP, dTDP-D-glucose, dTDP, and dTMP (where dTMP, dTDP, and dTTP are the deoxy forms of thymidine mono-, di-, and triphosphate, respectively) occurred on a Supelcosil, LC-SAX column (4.6 mm x 250 mm). The chromatogram was developed at room temperature with a 20 mL linear gradient from 50 to 400 mM potassium phosphate buffer (pH... 

In linear development of a chromatogram, unidirectional or bidirectional developments of the chromatogram are possible. Similarly, as in liquid column chromatography, there are possible, in this case, either on-line or off-line techniques of sample application, separation, and detection, as well as various modifications (e.g., partly off-line method). Bidirectional development can also be vertical. Using vertical bidimensional development, applying different eluents, components of complex, difficult mixtures can be separated. The separation of such mixtures is also possible by means of this technique using multiple automatic development of chromatogram. 

The fundamental parameter used to characterize the position of a sample zone in a thin-layer chromatogram is the retardation factor, or Rp value. It represents the ratio of the distance migrated by the sample compared to the distance traveled by the solvent front, and for linear development is given by Eq. (6.1)... 

For linear development, samples are applied along one edge of the plate and separated in the direction of the opposite edge. Viewed in the direction of development, the chromatogram consists of a series of compact symmetrical spots of increasing diameter, or if samples are applied as bands, as rectangular zones of increasing width. 

In linear development of a chromatogram, unidirectional or bidirectional developments of the chromatogram... 

Finally, an officially updated definition of the retardation factor, R, issued by lUPAC is important to the whole field of planar chromatography (the linear and the nonlinear TLC mode included). The importance of such a definition has two reasons. First, it is promoted by the growing access of planar chromatography users for densitometric evaluation of their chromatograms and second, by the vagueness of the present definition in the case of skewed concentration profiles with the samples developed under mass overload conditions. 

Qualitatively equation (7.15) is adequate to describe tiM f influence of layer quality, selectivity, and zone position in the 1 chromatogram upon resolution for a single unidimensional development under capillary flow controlled conditions. The variation of R, with Rf is not a simple function as can be seen from Figure 7.6. The resolution increases with the layer efficiency in a manner that depends linearly on the R, value. — Relatively small changes in selectivity have an enormous impact on... 

A simple and rapid RP-HPLC method was developed for the determination of retinoid in galenicals. Commercial preparations were diluted, filered and used for separation. Measurements were carried out in an ODS column (150 X 4.6 mm i.d. particle size 3 /xm). Solvents A and B were methanol-10 mM ammonium acetate (75 25, v/v) and methanol-THF (84 16, v/v), respectively. The flow rate was 0.8ml/min. Gradient conditions were 0-25 min, 0 per cent B 35 min, 100 per cent B, isocratic for 10 min. Typical chromatograms are shown in Fig. 2.37. The repeatability of peak area ranged between 0.48 -3.2 per cent for UV-DAD and 0.57 - 3.1 per cent for fluorescence detection. The reproducibility varied between 0.26 - 4.6 per cent. It was found that the method is precise, selective, sensitive and linear, therefore, it can be employed for the routine quality control of this class of drags [85],... 

A typical chromatogram obtained using this method is shown in Figure 12, and the retention times (Rt) and the relative retention time (Rrt) for pantoprazole sodium and some of its related compounds are shown in Table 6. The calibration curve for the assay determination, obtained over a concentration range of228-670 pg/mL, was found to be linear with a correlation coefficient of0.999. The recovery and relative standard deviation for various assays were 97.3-101.5 and 1.1, respectively. A calibration curve was also developed for pantoprazole sodium related compounds, covering a concentration range of 1 to 3 pg/mL, and which was found to be linear with a correlation coefficient of more than 0.999. The limits of detection and limits of quantitation were calculated as 0.15 pg/mL and 0.49 pg/mL, respectively. 

---